NADPH provides the reducing power for decomposition of reactive oxygen species (ROS), making it an indispensable part during ROS defense. It remains uncertain, however, if living cells respond to the ROS challenge with an elevated intracellular NADPH level or a more complex NADPH-mediated manner. Herein, we employed a model fungus Aspergillus nidulans to probe this issue. A conditional expression of glucose-6-phosphate dehydrogenase (G6PD)-strain was constructed to manipulate intracellular NADPH levels. As expected, turning down the cellular NADPH concentration drastically lowered the ROS response of the strain; it was interesting to note that increasing NADPH levels also impaired fungal H2O2 resistance. Further analysis showed that excess NADPH promoted the assembly of the CCAAT-binding factor AnCF, which in turn suppressed NapA, a transcriptional activator of PrxA (the key NADPH-dependent ROS scavenger), leading to low antioxidant ability. In natural cell response to oxidative stress, we noticed that the intracellular NADPH level fluctuated “down then up” in the presence of H2O2. This might be the result of a co-action of the PrxA-dependent NADPH consumption and NADPH-dependent feedback of G6PD. The fluctuation of NADPH is well correlated to the formation of AnCF assembly and expression of NapA, thus modulating the ROS defense. Our research elucidated how A. nidulans precisely controls NADPH levels for ROS defense.
Biochar ozonization was previously shown to dramatically increase its cation exchange capacity, thus improving its nutrient retention capacity. The potential soil application of ozonized biochar warrants the need for a toxicity study that investigates its effects on microorganisms.
In the study presented here, we found that the filtrates collected from ozonized pine 400 biochar and ozonized rogue biochar did not have any inhibitory effects on the soil environmental bacteria Pseudomonas putida, even at high dissolved organic carbon (DOC) concentrations of 300 ppm. However, the growth of Synechococcus elongatus PCC 7942 was inhibited by the ozonized biochar filtrates at DOC concentrations greater than 75 ppm. Further tests showed the presence of some potential inhibitory compounds (terephthalic acid and p-toluic acid) in the filtrate of non-ozonized pine 400 biochar; these compounds were greatly reduced upon wet-ozonization of the biochar material. Nutrient detection tests also showed that dry-ozonization of rogue biochar enhanced the availability of nitrate and phosphate in its filtrate, a property that may be desirable for soil application.
Ozonized biochar substances can support soil environmental bacterium Pseudomonas putida growth, since ozonization detoxifies the potential inhibitory aromatic molecules.
By way of broadening the use of diverse sustainable bioethanol feedstocks, the potentials of Paper mulberry fruit juice (PMFJ), as a non-food, sugar-based substrate, were evaluated for fuel ethanol production. The suitability of PMFJ was proven, as maximum ethanol concentration (56.4 g/L) and yield (0.39 g/g) were achieved within half a day of the start of fermentation, corresponding to very high ethanol productivity of 4.7 g/L/hr. The established potentials were further optimally maximized through the response surface methodology (RSM). At the optimal temperature of 30 °C, yeast concentration of 0.55 g/L, and pH of 5, ethanol concentration, productivity, and yield obtained were 73.69 g/L, 4.61 g/L/hr, and 0.48 g/g, respectively. Under these ideal conditions, diverse metal salts were afterward screened for their effects on PMFJ fermentation. Based on a two-level fractional factorial design, nutrient addition had no positive impact on ethanol production. Thus, under the optimal process conditions, and without any external nutrient supplementation, bioethanol from PMFJ compared favorably with typical sugar-based energy crops, highlighting its resourcefulness as a high-value biomass resource for fuel ethanol production.
Dunaliella salina is a green microalga with the great potential to generate natural β-carotene. However, the corresponding mathematical models to guide optimized production of β-carotene in Dunaliella salina (D. salina) are not yet available. In this study, dynamic models were proposed to simulate effects of environmental factors on cell growth and β-carotene production in D. salina using online monitoring system. Moreover, the identification model of the parameter variables was established, and an adaptive particle swarm optimization algorithm based on parameter sensitivity analysis was constructed to solve the premature problem of particle swarm algorithm. The proposed kinetic model is characterized by high accuracy and predictability through experimental verification, which indicates its competence for future process design, control, and optimization. Based on the model established in this study, the optimal environmental factors for both β-carotene production and microalgae growth were identified. The approaches created are potentially useful for microalga Dunaliella salina cultivation and high-value β-carotene production.
Cold-adapted organisms, such as fishes, insects, plants and bacteria produce a group of proteins known as antifreeze proteins (AFPs). The specific functions of AFPs, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), dynamic ice shaping (DIS) and interaction with membranes, attracted significant interest for their incorporation into commercial products. AFPs represent their effects by lowering the water freezing point as well as preventing the growth of ice crystals and recrystallization during frozen storage. The potential of AFPs to modify ice growth results in ice crystal stabilizing over a defined temperature range and inhibiting ice recrystallization, which could minimize drip loss during thawing, improve the quality and increase the shelf-life of frozen products. Most cryopreservation studies using marine-derived AFPs have shown that the addition of AFPs can increase post-thaw viability. Nevertheless, the reduced availability of bulk proteins and the need of biotechnological techniques for industrial production, limit the possible usage in foods. Despite all these drawbacks, relatively small concentrations are enough to show activity, which suggests AFPs as potential food additives in the future. The present work aims to review the results of numerous investigations on marine-derived AFPs and discuss their structure, function, physicochemical properties, purification and potential applications.
Terpenoids form the most diversified class of natural products, which have gained application in the pharmaceutical, food, transportation, and fine and bulk chemical industries. Extraction from naturally occurring sources does not meet industrial demands, whereas chemical synthesis is often associated with poor enantio-selectivity, harsh working conditions, and environmental pollutions. Microbial cell factories come as a suitable replacement. However, designing efficient microbial platforms for isoprenoid synthesis is often a challenging task. This has to do with the cytotoxic effects of pathway intermediates and some end products, instability of expressed pathways, as well as high enzyme promiscuity. Also, the low enzymatic activity of some terpene synthases and prenyltransferases, and the lack of an efficient throughput system to screen improved high-performing strains are bottlenecks in strain development. Metabolic engineering and synthetic biology seek to overcome these issues through the provision of effective synthetic tools. This review sought to provide an in-depth description of novel strategies for improving cell factory performance. We focused on improving transcriptional and translational efficiencies through static and dynamic regulatory elements, enzyme engineering and high-throughput screening strategies, cellular function enhancement through chromosomal integration, metabolite tolerance, and modularization of pathways.
The hydrodynamic cavitation comes out as a promising route to lignocellulosic biomass pretreatment releasing huge amounts of energy and inducing physical and chemical transformations, which favor lignin–carbohydrate matrix disruption. The hydrodynamic cavitation process combined with other pretreatment processes has shown an attractive alternative with high pretreatment efficiency, low energy consumption, and easy setup for large-scale applications compared to conventional pretreatment methods. This present review includes an overview of this promising technology and a detailed discussion on the process of parameters that affect the phenomena and future perspectives of development of this area.
In current years, natural pigments are facing a fast-growing global market due to the increase of people’s awareness of health and the discovery of novel pharmacological effects of various natural pigments, e.g., carotenoids, flavonoids, and curcuminoids. However, the traditional production approaches are source-dependent and generally subject to the low contents of target pigment compounds. In order to scale-up industrial production, many efforts have been devoted to increasing pigment production from natural producers, via development of both in vitro plant cell/tissue culture systems, as well as optimization of microbial cultivation approaches. Moreover, synthetic biology has opened the door for heterologous biosynthesis of pigments via design and re-construction of novel biological modules as well as biological systems in bio-platforms. In this review, the innovative methods and strategies for optimization and engineering of both native and heterologous producers of natural pigments are comprehensively summarized. Current progress in the production of several representative high-value natural pigments is also presented; and the remaining challenges and future perspectives are discussed.
Use of green agronomic techniques for plant development and crop protection is essential for environmental sustainability. The current research investigates a more efficient and long-term technique of manufacturing silica nanoparticles (SiO2 NPs) from agricultural waste (sugarcane bagasse and corn cob). SiO2 NPs were synthesized by calcinations of waste residues in muffle furnace with varying temperatures (400–1000 °C)/2 h in the present of static air. Field emission scanning electron microscopy (FESEM), Fourier transmission infrared spectroscopy (FTIR), X-ray diffraction (XRD), and energy dispersive X-ray spectroscopy (EDX) were used to characterize SiO2 NPs and assessed for their antifungal activity simultaneously investigated the effects of various concentrations of produced SiO2 NPs on Eruca sativa (E. sativa) physiological and biochemical. With SiO2 NPs treatment at 1000 µg L−1 concentration, the seed germination rate was found to be up to 95.5%, and growth characteristics were enhanced compared to control. Accordingly, the ones treated with SiO2 NPs grew better than the control ones. The treatment of plant with SiO2 NPs (500 μg L−1) increased the protein content by 14.8 mg g−1, and chlorophyll level was also increased by 4.08 mg g−1 in leaves compared to untreated plant. Disc diffusion experiment was conducted to test the efficiency of SiO2 NPs against Fusarium oxysporum and Aspergillus niger for antifungal activities. Highest mycelia growth inhibition was obtained with 73.42% and 58.92% for F. oxysporum and A. niger, respectively. The result shows that the SiO2 NPs have a favorable effect on E. sativa growth and germination, enhancing plant production which helps to improve the sustainable agriculture farming and acting as a possible antifungal agent against plant pathogenic fungi.
Rasburicase is an expensive treatment used to control hyperuricemia caused by tumour lysis syndrome (TLS). In this study, a non-chromatographic method was designed based on nano-oil bodies for convenient and economical purification of the recombinant uricase. For this purpose, two chimaeras were synthesized with a different arrangement of the uricase, caleosin and intein fragments. After confirming the protein expression by measuring the uricase activity at 293 nm, purification was conducted through oil-body construction. The results were resolved on the 12% SDS-PAGE gel. Finally, the stability of the oil bodies was examined against different salts, surfactants, temperatures, and pH values. According to our results, the overexpression of uricase–caleosin chimaera under the T7 promoter in Escherichia coli led to the production of soluble protein, which was successfully purified by artificial oil bodies. The active uricase was subsequently released through the self-splicing of intein. Further investigations highlighted the importance of the free C-terminus of caleosin in constructing artificial oil bodies. Moreover, surfactants and low temperature, in contrast to salts, improved the stability of oil bodies. In conclusion, caleosins are an efficient purification tag reducing the cost of purification compared to conventional chromatography methods.
l-Ornithine, an important non-essential amino acid, has considerable medicinal value in the treatment of complex liver diseases. Microbial fermentation strategies using robust engineered strains have remarkable potential for producing l-ornithine. We showed that glucose and sucrose co-utilization accumulate more l-ornithine in Corynebacterium glutamicum than glucose alone. Further manipulating the expression of intracellular fructose-1-phosphate kinase through the deletion of pfkB1resulted in the engineered strain C. glutamicum SO30 that produced 47.6 g/L of l-ornithine, which represents a 32.8% increase than the original strain C. glutamicum SO26 using glucose as substrate (35.88 g/L). Moreover, fed-batch cultivation of C. glutamicum SO30 in 5-L fermenters produced 78.0 g/L of l-ornithine, which was a 78.9% increase in yield compared with that produced by C. glutamicum SO26. These results showed that manipulating the fructose metabolic pathway increases l-ornithine accumulation and provides a reference for developing C. glutamicum to produce valuable metabolites.
The current energy crisis has prompted the development and utilization of renewable energy and energy storage material. In this study, levulinic acid (LA) and 1,4-butanediol (BDO) were used to synthesize a novel levulinic acid 1,4-butanediol ester (LBE) by both enzymatic and chemical methods. The enzymatic method exhibited excellent performance during the synthesis process, and resulted in 87.33% of LBE yield, while the chemical method caused more by-products and higher energy consumption. What’s more, the thermal properties of the obtained LBE as a phase change material (PCM) were evaluated. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) showed that the melting temperature, latent heat of melting, and pyrolysis temperature were 50.51 °C, 156.1 J/g, and 150–160 °C, respectively. Compared with the traditional paraffin, the prepared PCM has a superior phase transition temperature, a higher latent heat of melting, and better thermal stability. The thermal conductivity could be increased to 0.34 W/m/k after adding expanded graphite (EG). In summary, LBE has great potential in the application of energy storage as a low-temperature phase change energy storage material.
| • | Report of tissue distribution of chitobiose and chitotriose after intragastric administration in rats. |
| • | A trace detection method for the determination of chitobiose and chitotriose in rat serum and organs using UPLC–MS was developed and validated. |
| • | Our results reveal that chitobiose and chitotriose could be quickly absorbed into the blood, with an oral bioavailability of 0.32%–0.52% in rats. |
The by-products produced from fruit processing industries could be a potential hazard to environmental pollution. However, these by-products contain several biologically active molecules (essential fatty acid, phenolic compounds, flavonoids, coloring pigments, pectin, proteins, dietary fibers, and vitamins), which can be utilized for various applications in the food, pharmaceutical, cosmetic and textile industries. Nevertheless, during extraction, these bioactive compounds' recovery must be maximized using proper extraction technologies, keeping both economy and environment under consideration. In addition, the characteristics of the extract obtained from those by-products depend mainly on the parameters considered during the extraction process. In this review, an overview of different technologies used to extract bioactive compounds from fruit industry by-products such as seeds and peels has been briefly discussed, along with their mechanisms, process, advantages, disadvantages, and process parameters. In addition, the characteristics of the extracted bioactive compounds have also been briefly discussed in this review.
Autotrophic syngas fermentation with clostridia enables the conversion of CO, CO2, and H2 into organic acids and alcohols. The batch process performance of Clostridium ragsdalei was studied in fully controlled and continuously gassed (600 mbar CO, 200 mbar H2, 200 mbar CO2) stirred-tank bioreactors. The final ethanol concentration varied as function of the reaction conditions. Decreasing the pH from pH 6.0–5.5 at a temperature of 37 °C increased the ethanol concentration from 2.33 g L−1 to 3.95 g L−1, whereas lowering the temperature from 37 to 32 °C at constant pH 6.0 resulted in a final ethanol concentration of 5.34 g L−1 after 5 days of batch operation. The sulphur availability was monitored by measuring the cysteine concentration in the medium and the H2S fraction in the exhaust gas. It was found that most of the initially added sulphur was stripped out within the first day of the batch process (first half of the exponential growth phase). A continuous sodium sulfide feed allowed ethanol concentrations to increase more than threefold to 7.67 g L−1 and the alcohol-to-acetate ratio to increase 43-fold to 17.71 g g−1.
Biohydrogen production through dark fermentation is very attractive as a solution to help mitigate the effects of climate change, via cleaner bioenergy production. Dark fermentation is a process where organic substrates are converted into bioenergy, driven by a complex community of microorganisms of different functional guilds. Understanding of the microbiomes underpinning the fermentation of organic matter and conversion to hydrogen, and the interactions among various distinct trophic groups during the process, is critical in order to assist in the process optimisations. Research in biohydrogen production via dark fermentation is currently advancing rapidly, and various microbiology and molecular biology tools have been used to investigate the microbiomes. We reviewed here the different systems used and the production capacity, together with the diversity of the microbiomes used in the dark fermentation of industrial wastes, with a special emphasis on palm oil mill effluent (POME). The current challenges associated with biohydrogen production were also included. Then, we summarised and discussed the different molecular biology tools employed to investigate the intricacy of the microbial ecology associated with biohydrogen production. Finally, we included a section on the future outlook of how microbiome-based technologies and knowledge can be used effectively in biohydrogen production systems, in order to maximise the production output.
Endo-β-mannanases are important enzymes for degrading lignocellulosic biomass to generate mannan, which has significant health effects as a prebiotic that promotes the development of gut microbiota. Here, a novel endo-β-mannanase belonging to glycoside hydrolase (GH) family 113 from Paenibacillus cineris (PcMan113) was cloned, expressed and characterized, as one of only a few reported GH113 family β-mannanases. Compared to other functionally and structurally characterized GH113 mannanases, recombinant PcMan113 showed a broader substrate spectrum and a better performance. Based on a structural homology model, the highly active mutant PcMT3 (F110E/N246Y) was obtained, with 4.60- and 5.53-fold increases of enzyme activity (towards KG) and catalytic efficiency (kcat/Km, against M5) compared with the WT enzyme, respectively. Furthermore, molecular dynamics (MD) simulations were conducted to precisely explore the differences of catalytic activity between WT and PcMT3, which revealed that PcMT3 has a less flexible conformation, as well as an enlarged substrate-binding channel with decreased steric hindrance and increased binding energy in substrate recognition. In conclusion, we obtained a highly active variant of PcMan113 with potential for commercial application in the manufacture of manno-oligosaccharides.
We investigated the dye-removal potential of a collection of 61 cold-adapted yeasts from the King George Island, Antarctica, on agar plates supplemented with 100 mg L–1 of several textile dyes; among which isolates 81% decolorized Reactive Black 5 (RB-5), with 56% decolorizing Reactive Orange 16, but only 26% doing so with Reactive Blue 19 and Acid Blue 74. Furthermore, we evaluated the ligninolytic potential using 2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulfonic-acid) diammonium salt-, 3,5-dimethoxy-4-hydroxybenzaldehydazine-, or manganese-supplemented plates but detected no activity, possibly due to a dye-removal mechanism involving reductases. The removal kinetics were studied in liquid medium supplemented with 100 mg L–1 of RB-5 in a selection of 9 yeasts. The highest volumetric-removal rates (η) were found for Candida sake 41E (4.14 mg L–1 h–1), Leucosporidium muscorum F20A (3.90 mg L–1 h–1), and Cystofilobasidium infirmominiatum F13E (3.90 mg L–1 h–1). Different UV–Vis spectra were obtained if the dye removal occurred by biodegradation or biosorption/bioaccumulation. L. muscorum F20A was selected to study the dye-removal mechanism of RB-5 and the effect of different chemical and environmental parameters on the process. Optimum dye-removal conditions were obtained with 10 g L–1 of glucose within an initial medium pH range of 5.0 to 6.0. Up to 700 mg L–1 of dye could be removed in 45 h. High-performance liquid chromatography profiles obtained were consistent with a biodegradation of the dye. Phytotoxicity was estimated by calculating the 50%-inhibition concentration (IC50) with Lactuca sativa L. seeds. These findings propose psychrophilic yeasts as a novel environmentally suitable alternative for the treatment of dye-industry wastewaters.
The efficient asymmetric bio-synthesis of chiral β-hydroxy esters is of great importance for industrial production. In this work, a simple and productive engineered E.coli cell-immobilized strategy was applied for the asymmetric reduction of MAA to (R)-HBME with high enantioselectivity. Compared with the corresponding inactivated free cells, the alginate-immobilized cells remained 45% of initial activity at 50 ℃ and 65% after reuse of 10 times. After 60 days of storage at 4 ℃, the immobilized cells maintained more than 80% relative activity. Immobilization contributed significantly to the improvement of thermal stability, pH tolerance, storage stability and operation stability without affecting the yield of product. The immobilized recombinant E. coli cell had absolute enantioselectivity for the asymmetric reduction of MAA to (R)-HBME with e.e. > 99.9%. Therefore, microbial cell immobilization is a perspective approach in asymmetric synthesis of chiral β-hydroxy esters for industrial applications.
3D printing is revolutioning many industrial sectors and has the potential to enhance also the biotechnology and bioprocessing fields. Here, we propose a new flexible material formulation to 3D print support matrices with complex, perfectly ordered morphology and with tuneable properties to suit a range of applications in bioprocess engineering.
Supports were fabricated using functional monomers as the key ingredients, enabling matrices with bespoke chemistry, such as charged groups, chemical moieties for further functionalization, and hydrophobic/hydrophilic groups. Other ingredients, e.g. crosslinkers and porogens, can be employed to fabricate supports with diverse characteristics of their porous network, providing an opportunity to further regulate the mechanical and mass transfer properties of the supports. Through this approach, we fabricated and demonstrated the operation of Schoen gyroid columns with (I) positive and negative charges for ion exchange chromatography, (II) enzyme bioreactors with immobilized trypsin to catalyse hydrolysis, and (III) bacterial biofilm bioreactors for fuel desulphurization.
This study demonstrates a simple, cost-effective, and flexible fabrication of customized 3D printed supports for different biotechnology and bioengineering applications.
Supercritical carbon dioxide (Sc-CO2) is an alternative tool to extract lipid for the production of fish oil and enzyme from fish by-products (FBPs). In the application of Sc-CO2, this review covers sample preparation, lipid extraction operation, and characterization of fish oil and enzyme as final products. Generally, the fish samples with moisture content less than 20% and particle size less than 5 mm are considered before lipid extraction with Sc-CO2. Sc-CO2 parameters, such as pressure (P), temperature (T), extraction time (text), and flow rate (F), for simultaneous recovery of fish oil, protein, and enzyme were found to be less severe (P: 10.3–25 MPa; T: 25–45 °C, text: 20–150 min; F: 3–50 g/min) than the extraction of fish oil alone (P: 10–40 Mpa; T: 35–80 °C; text: 30–360 min; F: 1–3000 g/min). The enzyme from the Sc-CO2 defatted sample showed higher activity up to 45 U/mg due to lower denaturation of protein as compared to the organic solvent treated sample albeit both samples having similar pH (6–10) and temperature stability (20–60 °C). Overall, mild extraction of lipid from FBPs using Sc-CO2 is effective for the production of enzymes suitable in various industrial applications. Also, fish oil as a result of extraction can be produced as a health product with high polyunsaturated fatty acids (PUFAs) and low contamination of heavy metals.
The vitamin A component retinol has become an increasingly sought-after cosmetic ingredient. In previous efforts for microbial biosynthesis of vitamin A, a mixture of retinoids was produced. In order to efficiently produce retinol at high purity, the precursor and NADPH supply was first enhanced to improve retinoids accumulation in the S. cerevisiae strain constructed from a β-carotene producer by introducing β-carotene 15,15ʹ-dioxygenase, following by screening of heterologous and endogenous oxidoreductases for retinal reduction. Env9 was found as an endogenous retinal reductase and its activity was verified in vitro. By co-expressing Env9 with the E. coli ybbO, as much as 443.43 mg/L of retinol was produced at 98.76% purity in bi-phasic shake-flask culture when the antioxidant butylated hydroxytoluene was added to prevent retinoids degradation. The retinol titer reached 2479.34 mg/L in fed-batch fermentation. The success in selective biosynthesis of retinol would lay a solid foundation for its biotechnological production.
Natural free fatty acids show inhibitory effects on α-glucosidase and can hence have potential applications in diabetes treatment. This study indicated that the inhibitory effect of fatty acids showed a significant negative correlation with affinity energy (− 0.87) and melting point (− 0.88). Guided by this relationship, two promotion strategies of hydration and esterification were put forward to increase the inhibitory effect of fatty acids on α-glucosidase. The hydration can import an extra hydroxy group into the C=C bond of fatty acids, that will enhance the interaction with α-glucosidase, while the esterification will lower the melting point of fatty acids, and promote the inhibitory effect. Hydroxy fatty acids and fatty acid isopropyl esters possessed higher inhibitory effects than the natural fatty acids. Then, rubber seed oil was modified into novel fatty acid derivatives with higher inhibitory effect on α-glucosidase. The inhibitory IC50 of hydroxy products and isopropanol esters was 0.42 ± 0.01 μM and 0.57 ± 0.01 μM, respectively. The result reveals a feasible route to construct fatty acid derivatives from natural oil with α-glucosidase inhibitory effect.
As an important monomer for bio-based nylons PA5X, cadaverine is mainly produced by enzymatic decarboxylation of L-lysine. A key issue with this process is the instability of L-lysine decarboxylase (CadA) during the reaction due to the dissociation of CadA subunits with the accumulation of alkaline cadaverine. In this work, we attempted to improve the thermal and alkaline stability of CadA by combining directed evolution and computation-guided virtual screening. Interestingly, site 477 residue located at the protein surface and not the decamer interface was found as a hotspot in directed evolution. By combinatorial mutagenesis of the positive mutations obtained by directed evolution and virtual screening with the previously reported T88S mutation, K477R/E445Q/T88S/F102V was generated as the best mutant, delivering 37% improvement of cadaverine yield at 50 ºC and pH 8.0. Molecular dynamics simulations suggested the improved rigidity of regional structures, increased number of salt bridges, and enhancement of hydrogen bonds at the multimeric interface as possible origins of the improved stability of the mutant. Using this four-point mutant, 160.7 g/L of cadaverine was produced from 2.0 M Lysine hydrochloride at 50 °C without pH regulation, with a conversion of 78.5%, whereas the wild type produced 143.7 g/L cadaverine, corresponding to 70% conversion. This work shows the combination of directed evolution and virtual screening as an efficient protein engineering strategy.
Pullulanase is a well-known debranching enzyme that can specifically hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides, however, it suffers from low stability and catalytic efficiency under industrial conditions. In the present study, four residues (A365, V401, H499, and T504) lining the catalytic pocket of Anoxybacillus sp. AR-29 pullulanase (PulAR) were selected for site-directed mutagenesis (SDM) by using a structure-guided consensus approach. Five beneficial mutants (PulAR-A365V, PulAR-V401C, PulAR-A365/V401C, PulAR-A365V/V401C/T504V, and PulAR-A365V/V401C/T504V/H499A) were created, which showed enhanced thermostability, pH stability, and catalytic efficiency. Among them, the quadruple mutant PulAR-A365V/V401C/T504V/H499A displayed 6.6- and 9.6-fold higher catalytic efficiency toward pullulan at 60 ℃, pH 6.0 and 5.0, respectively. In addition, its thermostabilities at 60 ℃ and 65 ℃ were improved by 2.6- and 3.1-fold, respectively, compared to those of the wild-type (WT). Meanwhile, its pH stabilities at pH 4.5 and 5.0 were 1.6- and 1.8-fold higher than those of WT, respectively. In summary, the catalytic performance of PulAR was significantly enhanced by a structure-guided consensus approach. The resultant quadruple mutant PulAR-A365V/V401C/T504V/H499A demonstrated potential applications in the starch industry.
β-Nicotinamide mononucleotide (NMN) is the direct precursor of nicotinamide coenzymes such as NAD+ and NADP+, which are widely applied in industrial biocatalysis especially involving cofactor-dependent oxidoreductases. Moreover, NMN is a promising candidate for medical uses since it is considered to be beneficial for improving health of aged people who usually suffer from an insufficient level of NAD+. To date, various methods have been developed for the synthesis of NMN. Chemical phosphorylation of nicotinamide riboside (NR) to NMN depends on excessive phosphine oxychloride and delicate temperature control, while fermentation of NMN is limited by low product titers, making it unsuitable for industrial-scale NMN production. As a result, the more efficient synthesis process of NMN is still challenging.
This work attempted to construct an eco-friendly and cost-effective biocatalytic process for transforming the chemically synthesized NR into the highly value-added NMN.
A new nicotinamide riboside kinase (Klm-NRK) was identified from Kluyveromyces marxianus. The specific activity of purified Klm-NRK was 7.9 U·mg–1 protein, ranking the highest record among the reported NRKs. The optimal pH of Klm-NRK was 7.0 in potassium phosphate buffer. The purified Klm-NRK retained a half activity after 7.29 h at 50 °C. The catalytic efficiencies (kcat/KM) toward ATP and nicotinamide riboside (NR) were 57.4 s−1·mM−1 and 84.4 s−1·mM−1, respectively. In the presence of an external ATP regeneration system (AcK/AcP), as much as 100 g·L–1 of NR could be completely phosphorylated to NMN in 8 h with Klm-NRK, achieving a molar isolation yield of 84.2% and a space–time yield of 281 g·L−1·day−1. These inspiring results indicated that Klm-NRK is a promising biocatalyst which provides an efficient approach for the bio-manufacturing of NMN in a high titer.
Improved understanding of cellulose swelling mechanism is beneficial for increasing the hydrolysis efficiency of cellulosic substrates. Here, we report a family 5 glycoside hydrolase ArCel5 isolated from the cellulose-gelatinizing fungus Arthrobotrys sp. CX1. ArCel5 exhibited low specific hydrolysis activity and high cellulose swelling capability, which suggested that this protein might function as an accessory protein. Homology modeling glycosylation detection revealed that ArCel5 is a multi-domain protein including a family 1 carbohydrate-binding module, a glycosylation linker, and a catalytic domain. The adsorption capacity, structural changes and hydrature index of filter paper treated by different ArCel5 mutants demonstrated that CBM1 and linker played an essential role in recognizing, binding and decrystallizing cellulosic substrates, which further encouraged the synergistic action between ArCel5 and cellulases. Notably, glycosylation modification further strengthened the function of the linker region. Overall, our study provides insight into the cellulose decrystallization mechanism by a novel accessory protein ArCel5 that will benefit future applications.
Protein-based biomaterials have the characteristics of stability and biocompatibility. Based on these advantages, various bionic materials have been manufactured and used in different fields. However, current protein-based biomaterials generally need to form monomers in cells and be purified before being assembled in vitro. The preparation process takes a long time, and the complex cellular environment is challenging to be optimized for producing the target protein product. Here this study proposed technology for in situ synthesis and assembly of the target protein, namely the cell-free protein synthesis (CFPS), which allowed to shorten the synthesis time and increase the flexibility of adding or removing natural or synthetic components. In this study, successful expression and self-assembly of the dihedral symmetric proteins proved the applicability of the CFPS system for biomaterials production. Furthermore, the fusion of different functional proteins to these six scaffold proteins could form active polymers in the CFPS system. Given the flexibility, CFPS is expected to become a powerful tool as the prototyping and manufacturing technology for protein-based biomaterials in the future.
τ-Cadinol is a sesquiterpene that is widely used in perfume, fine chemicals and medicines industry. In this study, we established a biosynthetic pathway for the first time in engineered Escherichia coli for production of τ-cadinol from simple carbon sources. Subsequently, we further improved the τ-cadinol production to 35.9 ± 4.3 mg/L by optimizing biosynthetic pathway and overproduction of rate-limiting enzyme IdI. Finally, the titer was increased to 133.5 ± 11.2 mg/L with a two-phase organic overlay-culture medium system. This study shows an efficient method for the biosynthesis of τ-cadinol in E. coli with the heterologous hybrid MVA pathway.
The meat industry generates large amounts of by-products that are costly to be treated and discarded ecologically; moreover, they could be used to extract high added-value compounds. In this work, we present an innovative combined process which allowed the parallel extraction of both organic and mineral compounds; more specifically protein hydrolysates and single-phase hydroxyapatite were obtained. The protein hydrolysates, extracted through an enzymatic hydrolysis with alcalase, showed a degree of hydrolysis of 53.3 ± 5.1%; moreover, they had a high protein content with peptides with molecular weight lower than 1.2 kDa. Their antioxidant activities, measured with ABTS and ORAC tests, were 21.1 ± 0.5 mg ascorbic acid equivalent/g of dry extract and 87.7 ± 6.3 mg Trolox equivalent/g of dry extract, respectively. Single-phase hydroxyapatite, obtained with a simple calcination at 700 °C on the residues of the hydrolysis process, showed a Ca/P ratio close to the stoichiometric one (1.65 vs. 1.67) and presented a nanometric structure. This study reports a simple and feasible process for the valorization of porcine by-products in a large-scale up generating products with potential applications for environment remediation, biomedicine, nutrition and catalysis/bioenergy.
Organophosphates (OPs) are hazardous pesticides, but an indispensable part of modern agriculture; collaterally contaminating agricultural soil and surrounding water. They have raised serious food safety and environmental toxicity that adversely affect the terrestrial and aquatic ecosystems and therefore, it become essential to develop a rapid bioremediation technique for restoring the pristine environment. A newly OPs degrading Arthrobacter sp. HM01 was isolated from pesticide-contaminated soil and identified by a ribotyping (16S rRNA) method. Genus Arthrobacter has not been previously reported in chlorpyrifos (CP) degradation, which shows 99% CP (100 mg L−1) degradation within 10 h in mMSM medium and also shows tolerance to a high concentration (1000 mg L−1) of CP. HM01 utilized a broad range of OPs pesticides and other aromatic pollutants including intermediates of CP degradation as sole carbon sources. The maximum CP degradation was obtained at pH 7 and 32 °C. During the degradation, a newly identified intermediate 2,6-dihydroxypyridine was detected through TLC/HPLC/LCMS analysis and a putative pathway was proposed for its degradation. The study also revealed that the organophosphate hydrolase (opdH) gene was responsible for CP degradation, and the opdH-enzyme was located intracellularly. The opdH enzyme was characterized from cell free extract for its optimum pH and temperature requirement, which was 7.0 and 50 °C, respectively. Thus, the results revealed the true potential of HM01 for OPs-bioremediation. Moreover, the strain HM01 showed the fastest rate of CP degradation, among the reported Arthrobacter sp.
Bear bile powder is a precious natural material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a ratio of 1.00–1.50 to taurochenodeoxycholic acid (TCDCA).
In this study, we use the crude enzymes from engineered Saccharomyces cerevisiae to directionally convert TCDCA from chicken bile powder to TUDCA at the committed ratio in vitro. This S. cerevisiae strain was modified with heterologous 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH) genes. S. cerevisiae host and HSDH gene combinatorial optimization and response surface methodology was applied to get the best engineered strain and the optimal biotransformation condition, respectively, under which 10.99 ± 0.16 g/L of powder products containing 36.73 ± 6.68% of TUDCA and 28.22 ± 6.05% of TCDCA were obtained using 12.00 g/L of chicken bile powder as substrate.
This study provides a healthy and environmentally friendly way to produce potential alternative resource for bear bile powder from cheap and readily available chicken bile powder, and also gives a reference for the green manufacturing of other rare and endangered animal-derived valuable resource.
NADH-dependent phenylalanine amine dehydrogenase (F-AmDH) engineered from phenylalanine dehydrogenase (PheDH) catalyzes the synthesis of aromatic chiral amines from prochiral ketone substrates. However, its low coenzyme affinity and catalytic efficiency limit its industrial application. Here, we developed a chimeric amine dehydrogenase, cFLF-AmDH, based on the relative independence of the structure at the domain level, combined with a substrate-binding domain from F-AmDH and a high-affinity cofactor-binding domain from leucine amine dehydrogenase (L-AmDH). The kinetic parameters indicated that cFLF-AmDH showed a twofold improvement in affinity for NADH and a 4.4-fold increase in catalytic efficiency (kcat/Km) compared with the parent F-AmDH. Meanwhile, cFLF-AmDH also showed higher thermal stability, with the half-life increased by 60% at 55 °C and a broader substrate spectrum, than the parent F-AmDH. Molecular dynamics simulations suggested that the constructed cFLF-AmDH had a more stable structure than the parent F-AmDH, thereby improving the affinity of the coenzyme. The reaction rate increased by 150% in the reductive amination reaction catalyzed by cFLF-AmDH. When the NAD+ concentration was 0.05 mM, the conversion rate was increased by 150%. These results suggest that the chimeric protein by domain shuffling from different domain donors not only increased the cofactor affinity and catalytic efficiency, but also changed the specificity and thermal stability. Our study highlights that domain engineering is another effective method for creating biodiversity with different catalytic properties.
Abundant seawater resources can replace the shortage of freshwater resources. The co-production of xylo-oligosaccharides and glucose from sugarcane bagasse by subcritical CO2-assisted seawater pretreatment was studied in this paper. We investigated the effects of pretreatment conditions of temperature, CO2 pressure and reaction time on the yield of xylo-oligosaccharides in subcritical CO2-assisted seawater systems. The maximum xylo-oligosaccharide yield of 68.23% was obtained at 165 °C/2 MPa/5 min. After further enzymatic hydrolysis of the solid residue, the highest glucose yield of 94.45% was obtained. In this system, there is a synergistic effect of mixed ions in seawater and CO2 to depolymerize xylan into xylo-oligosaccharides with a lower degree of polymerization. At the same time, the addition of CO2 increased the pore size and porosity of sugarcane bagasse, improved the efficiency of enzymatic hydrolysis and increased the yield of glucose. Therefore, this study provides a more environmentally friendly and sustainable process for the co-production of xylo-oligosaccharides and glucose from sugarcane bagasse, and improves the utilization of seawater resources.
| • | Efficiency of bioreduction of sulfate and biomass can be increased by CdS NPs. |
| • | It is important for the EPS of RSB to improve the sulfate reduction efficiency. |
| • | Utilization efficiency of extracellular electrons by RSB can be enhanced through EPS. |
| • | Humic acid can alleviate the oxidative stress induced by CdS NPs to SRB. |
Epigallocatechin-3-gallate (EGCG) is a plant-derived flavonoid compound with the ability to promote the differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) into osteoblasts. However, the effect of EGCG on the osteogenic differentiation of the human umbilical cord-derived mesenchymal stem cells (HUMSCs) is rarely studied. Therefore, in this study, the osteogenic effects of EGCG are studied in the HUMSCs by detecting cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition and the expression of relevant osteogenic markers. The results showed that EGCG can promote the proliferation and osteogenic differentiation of the HUMSCs in vitro at a concentration of 2.5–5.0 μM. Unfortunately, the EGCG is easily metabolized by cells during cell culture, which reduces its bioavailability. Therefore, in this paper, EGCG-loaded microspheres (ECM) were prepared and embedded in chitosan/carboxymethyl cellulose/montmorillonite (CS/CMC/MMT) scaffolds to form CS/CMC/MMT-ECM scaffolds for improving the bioavailability of EGCG. The HUMSCs were cultured on CS/CMC/MMT-ECM scaffolds to induce osteogenic differentiation. The results showed that the CS/CMC/MMT-ECM scaffold continuously released EGCG for up to 22 days. In addition, CS/CMC/MMT-ECM scaffolds can promote osteoblast differentiation. Taken together, the present study suggested that entrainment of ECM into CS/CMC/MMT scaffolds was a prospective scheme for promotion osteogenic differentiation of the HUMSCs.
Using parameters of optimal conditions from laboratory experiments often results in the loss of significant time and resources when trying to scale up the process. In this study, the comparison of results of laboratory and semi-industrial experiments of enzymatic hydrolysis of soy protein isolate is considered. The kinetics of peptides accumulation was investigated by colorimetric method in both microtube (volume reaction is 0.7 ml, 7.14 mg/ml of substrate, incubation in solid state thermostat) and industrial homogenizer (volume reaction is 4,000 ml, 100 mg/ml of substrate, rotor–stator type mixer). The enzyme preparation Protosubtilin G3x (main component is subtilisin) was used as an analogue of the Alcalase preparation, which is already widely used in the food industry. It was found that the pH and the number of proteolytic units in the reaction mixture of both scales had slightly different results of the kinetics, while the temperature showed significantly one. The laboratory scale of the reaction had a wide range of optimal temperature (40–60
Keratinases can specifically degrade keratins, which widely exist in hair, horns, claws and human skin. There is a great interest in developing keratinase to manage keratin waste generated by the poultry industry and reusing keratin products in agriculture, medical treatment and feed industries. Degradation of keratin waste by keratinase is more environmentally friendly and more sustainable compared with chemical and physical methods. However, the wild-type keratinase-producing strains usually cannot meet the requirements of industrial production, and some are pathogenic, limiting their development and utilization. The main purpose of this study is to improve the catalytic performance of keratinase via directed evolution technology for the degradation of feathers. We first constructed a mutant library through error-prone PCR and screened variants with enhanced enzyme activity. The keratinase activity was further improved through fermentation conditions optimization and fed-batch strategies in a 7-L bioreactor. As a result, nine mutants with enhanced activity were identified and the highest enzyme activity was improved from 1150 to 8448 U/mL finally. The mutant achieved efficient biodegradation of feathers, increasing the degradation rate from 49 to 88%. Moreover, a large number of amino acids and soluble peptides were obtained as degradation products, which were excellent protein resources to feed. Therefore, the study provided a keratinase mutant with application potential in the management of feather waste and preparation of protein feed additive.
To obtain immunomodulatory peptides from isolated soy protein (ISP), pepsin was selected to prepare hydrolysates and 4-h treatment (Pepsin-ISPH4h) showed the highest yield and immunomodulatory activities. The Pepsin-ISPH4h was sequentially fractionated by 30, 10 and 1-kDa molecular weight cut-off (MWCO) membranes, in which 1-kDa MWCO permeate (1P) exhibited the most significant enhancement of phagocytosis activity without causing excessive inflammation as compared with Pepsin-ISPH4h. To further purify and enhance the immunomodulatory activity, 1P was distinct by high-performance liquid chromatography equipped with a reverse-phase column and in vivo immunomodulatory activity of fractions was examined in mice. Fraction 1 (F1) significantly elevated phagocytosis activity of mice spleen macrophages and neutrophils. However, increase of phagocytosis activity did not result from the induction of macrophages M1 or M2 polarization. The immunomodulatory peptide sequence, EKPQQQSSRRGS, from F1 was identified by LC–MS/MS. Phagocytosis activity and macrophage M1 polarization were elevated by synthetic peptide treatment. Hence, our results indicated that isolated soy protein hydrolysates prepared by pepsin could provide a source of peptides with immunomodulatory effects.
The biomass pretreatment strategies using organic acids facilitate lignin removal and enhance the enzymatic digestion of cellulose. However, lignin always suffers a severe and irreversible condensation. The newly generated C–C bonds dramatically affect its further upgrading. In this study, we used a recyclable hydrotrope (p-Toluenessulfonic acid, p-TsOH) to dissolve lignin under mild condition and stabilized lignin with a quenching agent (formaldehyde, FA) during extraction, achieving both value-added lignin extraction and efficient enzymatic saccharification of cellulose. Approximately 63.7% of lignin was dissolved by 80% (wt. %) p-TsOH with 1.5% FA addition at 80 °C, 30 min. The obtained lignin was characterized by FTIR spectroscopy, TGA, 2D HSQC NMR spectroscopy, and GPC. The results indicated that the extracted lignin exhibited excellent properties, such as light color, a low molecular weight (Mw, 5371 g/mol), and a narrow polydispersity (Mw/Mn, 1.63). The pretreated substrate was converted to ethanol via a quasi-simultaneous saccharification and fermentation process (Q-SSF). After fermentation of 60 h, the ethanol concentration reached 38.7 ± 3.3 g/L which was equivalent to a theoretical ethanol yield of 82.9 ± 2.2% based on the glucan content, while the residual glucose concentration was only 4.69 ± 1.4 g/L. In short, this pretreatment strategy protected lignin to form new C–C linkages and improved the enzymatic saccharification of glucan for high-titer ethanol production.
Atom-transfer radical polymerization (ATRP) is a well-known technique for controlled polymer synthesis. However, the ATRP usually employed toxic heavy metal ionas as the catalyst and was susceptible to molecular oxygen, which made it should be conducted under strictly anoxic condition. Conducting ATRP under ambient and biocompatible conditions is the major challenge. In this study, cytochrome C was explored as an efficient biocatalyst for ATRP under biocompatible conditions. The cytochrome C catalyzed ATRP showed a relatively low polymer dispersity index of 1.19. More interestingly, the cytochrome C catalyzed ATRP showed superior oxygen resistance as it could be performed under aerobic conditions with high dissolved oxygen level. Further analysis suggested that the Fe(II) embed in the cytochrome C might serve as the catalytic center and methyl radical was responsible for the ATRP catalysis. This work explored new biocompatible catalyst for aerobic ATRP, which might open new dimension for practical ATRP and application of cytochrome C protein.
Plastic polymers are non-degradable solid wastes that have become a great threat to the whole world and degradation of these plastics would take a few decades. Compared with other degradation processes, the biodegradation process is the most effective and best way for plastic degradation due to its non-polluting mechanism, eco-friendly nature, and cost-effectiveness. Biodegradation of synthetic plastics is a very slow process that also involves environmental factors and the action of wild microbial species. In this plastic biodegradation, fungi play a pivotal role, it acts on plastics by secreting some degrading enzymes, i.e., cutinase`, lipase, and proteases, lignocellulolytic enzymes, and also the presence of some pro-oxidant ions can cause effective degradation. The oxidation or hydrolysis by the enzyme creates functional groups that improve the hydrophilicity of polymers, and consequently degrade the high molecular weight polymer into low molecular weight. This leads to the degradation of plastics within a few days. Some well-known species which show effective degradation on plastics are Aspergillus nidulans, Aspergillus flavus, Aspergillus glaucus, Aspergillus oryzae, Aspergillus nomius, Penicillium griseofulvum, Bjerkandera adusta, Phanerochaete chrysosporium, Cladosporium cladosporioides, etc., and some other saprotrophic fungi, such as Pleurotus abalones, Pleurotus ostreatus, Agaricus bisporus and Pleurotus eryngii which also helps in degradation of plastics by growing on them. Some studies say that the degradation of plastics was more effective when photodegradation and thermo-oxidative mechanisms involved with the biodegradation simultaneously can make the degradation faster and easier. This present review gives current knowledge regarding different species of fungi that are involved in the degradation of plastics by their different enzymatic mechanisms to degrade different forms of plastic polymers.
Spent hen are egg-laying hens reaching the end of their laying cycles; billions of spent hens are produced globally each year. Differences in people’s attitudes towards spent hen as foods lead to their different fates among countries. While spent hens are consumed as raw or processed meat products in Asian countries such as China, India, Korea, and Thailand, they are treated as a byproduct or waste, not a food product, in the western society; they are instead disposed by burial, incineration, composting (as fertilizers), or rendering into animal feed and pet food, which either create little market value or cause animal welfare and environmental concerns. Despite being a waste, spent hen is a rich source of animal proteins and lipids, which are suitable starting materials for developing valorized products. This review discussed the conventional uses of spent hens, including food, animal feed, pet food, and compost, and the emerging uses, including biomaterials and functional food ingredients. These recent advances enable more sustainable utilization of spent hen, contributing to alternative solutions to its disposal while yielding residual value to the egg industry. Future research will continue to focus on the conversion of spent hen biomass into value-added products.
The cellulase cocktail of marine Aspergillus niger exhibited salt-tolerant and thermostable properties, which is of great potential in industrial application. In order to excavate the single tolerant cellulase components from complex cellulase cocktail, constitutive homologous expression was employed for direct obtainment of the endoglucanase (AnEGL). Enzymatic property study revealed that AnEGL exhibited a property of salt tolerance and a strong thermostability in high salinity environment. Significantly, its activity increased to 129% and the half-life at 65 °C increased to 27.7-fold with the presence of 4.5 M NaCl. Molecular dynamics simulation revealed that Na+ and Cl− could form salt bridges with charged residues, and then influenced the activity of loops and the stability of substrate binding pocket, which accounted for the salt tolerance and thermostability. Further, site-specific mutagenesis study proved that the residues Asp95 and Asp99 in the pocket were of great concern for the tolerant properties. The salt-tolerant and thermostable AnEGL was of great value in lignocellulosic utilization and the conjectural mechanisms were of referential significance for other tolerant enzymes.
In recent years the use of organic matter soil amendments, such as agricultural by-products, has been implemented with the aim of increasing soil fertility, while minimizing the environmental impact of agriculture. Sheep wool residues (SWR) have shown beneficial effects on plant nutrition and soil properties, while only few works assessed their impact on soil microbial communities. The main aim of this work was to investigate the possible valorization of two SWR types (scoured residues, white wool, WW, and carbonized scoured residues, black wool, BW) as organic soil amendments, in pot-grown olive trees, by evaluating their impact on soil bacterial communities and mycorrhizal symbionts. The two SWR types did not negatively impact on the diversity and composition of soil bacterial communities, as revealed by PCR-denaturating gradient gel electrophoresis (PCR-DGGE) of partial 16S rRNA gene, and on the activity of native arbuscular mycorrhizal fungi (AMF), while positively affecting plant growth. Only the highest doses of one SWR type (2% BW) caused a decrease in bacterial diversity and native AMF ability to colonize olive roots. DGGE bands sequencing allowed the identification of the major bacterial taxa. Sequences corresponding to Ohtaekwangia spp., Beta proteobacterium, Blastocatella sp., Ramlibacter monticola and Massilia frigida/rubra, Dongia sp. and Chloroflexi were mainly represented in SWR-amended soils, while those represented by Chryseolinea soli and Acidobacteria were abundant in control soil. Overall, this work showed that SWR may be valorized as organic soil amendments, as soil bacteria and AMF, representing key factors of biological soil fertility, were not negatively affected, while the activity of bacterial genera and species known for their ability to decompose complex compounds was boosted. Further studies will investigate the biodegradation efficiency of the diverse bacterial taxa developing in SWR-amended soils.
• Biodiesel from oleaginous bacteria and its importance are summarized.
• Biochemical pathways for fatty acid synthesis are described.
• Critical biotechnological approaches for bacterial strain improvement have been discussed.
• Sustainable biodiesel production, challenges, and future possibilities have been discussed.
Quercetin is an essential ingredient in functional foods and nutritional supplements, as well as a promising therapeutic reagent. Also, the green technique to produce quercetin via rutin biotransformation is attractive. Genes encoding two thermostable glycosidases from Dictyoglomus thermophilum were cloned and expressed in Escherichia coli, which were applied in rutin biotransformation to produce highly pure quercetin at a high temperature. The production of biocatalysts were scaled up in a 5-L bioreactor, yielding a several-fold increase in total enzyme activity and a quercetin production of 14.22 ± 0.26 g/L from 30 g/L of rutin. Feeding strategies were optimized to boost biomass and enzyme production, achieving an activity of 104,801.80 ± 161.99 U/L for rhamnosidase and 12,637.23 ± 17.94 U/L for glucosidase, and a quercetin yield of 20.24 ± 0.27 g/L from the complete conversion of rutin. This study proposes a promising approach for producing high-quality quercetin in an industrial setting.
This study investigated the combustion kinetics and spontaneous ignition of sweet sorghum using thermogravimetric analysis and the Frank-Kamenetskii theory. The aim was to determine the proper operating conditions for a direct combustion reactor and predict the safe ambient temperature limits for given silo designs. Oxidative heating rates of 2, 5, and 10 °C/min were set up. Graphical observation shows that combustion was composed of two different stages representing the overlapping processes of pyrolysis and char oxidation, at 131–336 °C and 336–475 °C, respectively. Samples were found to ignite at 215 °C and were extinguished at 433 °C. Different heating rates shifted combustion characteristics to higher temperatures and increased reactivity for ignition and combustion indices up to 12 and 10 times higher. The Friedman method determined the apparent activation energies representing the combustion reaction by 132.91 kJ/mol. Regarding spontaneous ignition, the temperature safe limits were predicted to be 83–84 °C and 84–87 °C for cylindrical and box silos with diameter and height of 15 and 10 m, respectively. Calculations of silos were designed within the limits of certain dimension ratios.
The application of natural killer (NK) cells as potential antitumor effector cells appears to be valuable for immunotherapies. However, the clinical use of NK cells is limited because the technical difficulties associated with mass production NK cells at sufficiently high numbers represents a great challenge. Ex vivo expansion of NK cells is a key technology for cell therapy. Bioreactor systems can generate homogeneous culture condition and modulate the environmental and biochemical cues. In this study, a novel magnetically controlled bioreactor was developed for supporting NK cells ex vivo expansion. Using synthetic magnetic beads, the stirring device of the magnetically controlled bioreactor generated reduced shearing force. The intermittent magnetic field was applied for magnetic beads movement to homogenize the culture system. NK-92 cells were cultured in the magnetically controlled bioreactor and the expansion and function of expanded cells were investigated on day 8. The results showed that the expansion of NK-92 cells in the bioreactor was 67.71 ± 10.60-fold, which was significantly higher than that of the T25 culture flask (P < 0.05). Moreover, the proportions of CD3−CD56+ cells and cell killing activity of expanded cells in the bioreactor did not reveal any differences compared to T25 flasks. Taken together, this study demonstrated the possibility of magnetically controlled bioreactor as a potent strategy in NK cells production for facilitating cancer immunotherapy.
Solid-state fermentation (SSF) may be a suitable bioprocess to produce protein-vegetal ingredients with increased nutritional and functional value. This study assessed changes in phenol content, antinutrient content, biomass production and protein production resulting from the metabolic activity of Pleurotus ostreatus, an edible fungus, in lentils and quinoa over 14 days of SSF. The impact of particle size on these parameters was also assessed because the process was conducted in both seeds and flours. Fungus biomass increased during fermentation, reaching 30.0 ± 1.4 mg/g dry basis and 32 ± 3 mg/g dry basis in lentil grain and flour and 52.01 ± 1.08 mg/g dry basis and 45 ± 2 mg/g dry basis in quinoa seeds and flour after 14 days of SSF. Total protein content also increased by 20% to 25% during fermentation, in all cases except lentil flour. However, the soluble protein fraction remained constant. Regarding phytic acid, SSF had a positive impact, with a progressive decrease being higher in flours than in seeds. Regarding antioxidant properties, autoclaving of the substrates promoted the release of polyphenols, together with antioxidant activity (ABTS, DPPH and FRAP), in all substrates. However, these parameters drastically decreased as fermentation progressed. These results provide scientific knowledge for producing lentil- or quinoa-based ingredients with low antinutrient content enriched with protein fungal biomass.
Microalgal starch is considered as renewable and sustainable feedstock for biofuels and biorefinery. High cell density culture is favourable for photoautotrophic starch production in microalgae in the aspects of productivity and economy, but it often encounters low starch content or extra stress exposure that limits the production. This study aimed to economically enhance photosynthetic starch production from CO2 fixation in a green microalga Tetraselmis subcordiformis by regulating photosynthetic stress status with a signalling molecule γ-aminobutyric acid (GABA) combined with the application of high initial cell density culture. By increasing initial cell density (ICD) from the normal of 1.1 g L−1 (NICD) to as high as 2.8 g L−1 (HICD), the starch content, yield, and theoretical productivity were improved by 7%, 63%, and 42%, respectively. The addition of GABA under HICD resulted in 14%, 19%, and 26% of further enhancement in starch content, yield, and theoretical productivity, respectively. GABA exhibited distinct regulatory mechanisms on photosynthesis and stress status under HICD relative to NICD. GABA augmented excessive light energy absorption and electron transfer through photosystem II that reinforced the photoinhibition under NICD, while alleviated the stress reversely under HICD, both of which facilitated starch production by enabling a suitable stress status while simultaneously maintaining a sufficient photosynthetic activity. The increase of ICD and/or GABA supply particularly boosted amylopectin accumulation, leading to the changes in starch composition and was more favourable for fermentation-based biofuels production. Preliminary techno-economic analysis showed that the highest net extra benefit of 9.64 $ m−3 culture could be obtained under HICD with 2.5 mM GABA supply where high starch content (62%DW) and yield (2.5 g L−1) were achieved. The combined HICD-GABA regulation was a promising strategy for economic starch production from CO2 by microalgae for sustainable biomanufacturing.
Although current computational biology software is available and has prompted the development of enzyme–substrates simulation, they are difficult to install and inconvenient to use. This makes the time-consuming and error-prone process. By far there is still a lack of a complete tool which can provide a one-stop service for the enzyme–substrates simulation process. Hence, in this study, several computational biology software was extended development and integrated as a website toolbox named Atomevo. The Atomevo is a free web server providing a user-friendly interface for enzyme–substrates simulation: (1) protein homologous modeling; (2) parallel docking module of Autodock Vina 1.2; (3) automatic modeling builder for Gromacs molecular dynamics simulation package; and (4) Molecular Mechanics/Poisson–Boltzmann Surface Area (MMPBSA) analysis module for receptor–ligand binding affinity analysis. We officially launched the web server and provided instructions through a case for the design and simulation of Candida antarctica lipase B (CalB) fusion protein called Maltose Binding Protein—Thioredoxin A—Candida antarctica lipase B (MBP-TrxA-CalB).
Chitin is abundant in nature and its degradation products are highly valuable for numerous applications. Thermophilic chitinases are increasingly appreciated for their capacity to biodegrade chitin at high temperatures and prolonged enzyme stability. Here, using deep learning approaches, we developed a prediction tool, Preoptem, to screen thermophilic proteins. A novel thermophilic chitinase, Chi304, was mined directly from the marine metagenome. Chi304 showed maximum activity at 85 ℃, its Tm reached 89.65 ± 0.22℃, and exhibited excellent thermal stability at 80 and 90 °C. Chi304 had both endo- and exo-chitinase activities, and the (GlcNAc)2 was the main hydrolysis product of chitin-related substrates. The product yields of colloidal chitin degradation reached 97% within 80 min, and 20% over 4 days of reaction with crude chitin powder. This study thus provides a method to mine the novel thermophilic chitinase for efficient chitin biodegradation.
With the increase in population growth and environmental pollution, the daily protein supply is facing great challenges. Single-cell protein (SCP) produced by microorganism fermentation is a good alternative for substituting plant- and animal-derived proteins. In this study, Paracoccus communis MA5 isolated from soil previously demonstrated an excellent ability to synthesize SCP directly from sodium formate. To investigate the central metabolic network of formic acid assimilation and protein synthesis, genome-scale analyses were performed. Genomic analysis showed that complete tetrahydrofolate cycle-, serine cycle-, glycolytic pathway-, tricarboxylic acid (TCA) cycle- and nitrogen metabolism-relevant genes were annotated in the genome. These pathways play key roles in the conversion of formic acid into proteins. Transcriptional analysis showed that sodium formate stress could stimulate the metabolic pathway in response to environmental stress, but weaken the sulfur metabolic pathway to inhibit amino acid synthesis, resulting in a decrease in protein content (30% vs 44%). However, under culture conditions with ammonium sulfate, metabolic pathways associated with protein synthesis were accelerated, causing an increase in protein content (53% vs 44%); while the tetrahydrofolate cycle associated with formic acid assimilation was inhibited, causing a 62.5% decrease in growth rate (OD600: 0.21 vs 0.56). These results provide evidence of protein synthesis from sodium formate in strain MA5 at the gene level and lay a theoretical foundation for the optimization of fermentation systems using formic acid as a carbon source.
To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis. This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation.
Prokaryotic Argonaute (pAgo) proteins are well-known oligonucleotide-directed endonucleases, which contain a conserved PIWI domain required for endonuclease activity. Distantly related to pAgos, PIWI-RE family, which is defined as PIWI with conserved R and E residues, has been suggested to exhibit divergent activities. The distinctive biochemical properties and physiological functions of PIWI-RE family members need to be elucidated to explore their applications in gene editing.
Here, we describe the catalytic performance and cellular functions of a PIWI-RE family protein from Pseudomonas stutzeri (PsPIWI-RE). Structural modelling suggests that the protein possesses a PIWI structure similar to that of pAgo, but with different PAZ-like and N-terminal domains. Unlike previously reported pAgos, recombinant PsPIWI-RE acts as an RNA-guided DNA nuclease, as well as a DNA-guided RNA nuclease. It cleaves single-stranded DNA at temperatures ranging from 20 to 65 °C, with an optimum temperature of 45 °C. Mutation at D525 or D610 significantly reduced its endonuclease activity, confirming that both residues are key for catalysis. Comparing with wild-type, mutant with PIWI-RE knockout is more sensitive to ciprofloxacin as DNA replication inhibitor, suggesting PIWI-RE may potentially be involved in DNA replication.
Our study provides the first insights into the programmable nuclease activity and biological function of the unknown PIWI-RE family of proteins, emphasizing their important role in vivo and potential application in genomic DNA modification.
α-Alkenes (terminal alkenes) are important fuel and platform chemicals that are mainly produced from petroleum. Microbial synthesis might provide a sustainable approach for α-alkenes. In this work, we engineered the methylotrophic yeast Pichia pastoris to produce long-chain (C15:1, C17:1 and C17:2) α-alkenes via a decarboxylation of fatty acids. Combinatorial engineering, including enzyme selection, expression optimization and peroxisomal compartmentalization, enabled the production of 1.6 mg/L α-alkenes from sole methanol. This study represents the first case of α-alkene biosynthesis from methanol and also provides a reference for the construction of methanol microbial cell factories of other high-value chemicals.
There are many options for the utilization of biogas in different energy sectors (power, heat, mobility). The technical possibilities of using biogas are more diverse than the actual business models applied in the biogas industry. This paper shows the possible utilization pathways of biogas, divided into coupled power and heat generation, direct utilization and upgrading to a gas of a higher value. Subsequently, an overview of the business models discussed is given by a systematic literature review. The latter shows that the investigation of biogas business models is focused mainly on the last decade and has increased slightly over time. The regions of investigation can be found worldwide, with a clear focus on Europe. Direct use is studied mainly in the Asian and African regions. In the European context, a shift from investigating combined heat and power use to upgrading the biogas produced is evident.
A new approach was used for the immobilization of Thermomyces lanuginosus lipase (TLL), Candida antarctica lipase B (CALB), and Rhizomucor miehei lipase (RML) on amine-functionalized magnetic nanoparticles (Fe3O4@SiO2-NH2) via a multi-component reaction route (using cyclohexyl isocyanide). The used method offered a single-step and very fast process for covalent attachment of the lipases under extremely mild reaction conditions (25 °C, water, and pH 7.0). Rapid and simple immobilization of 20 mg of RML, TLL, and CALB on 1 g of the support produced 100%, 98.5%, and 99.2% immobilization yields, respectively, after 2 h of incubation. The immobilized derivatives were then used for biodiesel production from waste cooking oil. Response surface methodology (RSM) in combination with central composite rotatable design (CCRD) was employed to evaluate and optimize the biodiesel production. The effect of some parameters such as catalyst amount, reaction temperature, methanol concentration, water content for TLL or water-adsorbent for RML and CALB, and ratio of t-butanol (wt%) were investigated on the fatty acid methyl esters (FAME) yield.
Lignin is a renewable bioresource that can be used for a variety of value-added applications. However, the effective separation of lignin from lignocellulosic biomass remains an ongoing challenge. In this study, lignin was extracted from waste palm fiber and successfully converted into a dehumidifying material. The following four process parameters of lignin extraction from palm fiber were optimized systematically and comprehensively using the response surface methodology: reaction time, extraction temperature, ethanol concentration and solid/liquid ratio. The results revealed that under the optimum processing conditions (111 min of extraction at 174 °C using 73% ethanol at 1/16 g/mL solid/liquid ratio), the extraction yield of lignin was 56.2%. The recovery of ethanol solvent was as high as 91.8%. Further, the lignin could be directly used without purification to produce lignin-based activated carbon fibers (LACFs) with specific surface area and total pore volume of 1375 m2/g and 0.881 cm3/g, respectively. Compared with the commercial pitch-based activated carbon fiber, the LACF has a higher specific area and superior pore structure parameters. This work provides a feasible route for extracting lignin from natural palm fiber and demonstrates its use in the preparation of activated carbon fiber with a remarkable performance as a solid dehumidification agent.
Synthetic biology has boosted the rapid development on using non-methylotrophy as chassis for value added chemicals production from one-carbon feedstocks, such as methanol and formic acid. The one-carbon dissimilation pathway can provide more NADH than monosaccharides including glucose, which is conducive for reductive chemicals production, such as succinic acid. In this study, the one-carbon dissimilation pathway was introduced in E. coli Suc260 to enhance the succinic acid production capability. Through the rational construction of methanol dissimilation pathway, the succinic acid yield was increased from 0.91 to 0.95 g/g with methanol and sodium formate as auxiliary substrates in anaerobic fed-batch fermentation. Furthermore, the metabolic flux of by-product pyruvate was redirected to succinic acid together with the CO2 fixation. Finally, through the immobilization on a specially designed glycosylated membrane, E. coli cells are more resistant to adverse environments, and the final yield of succinic acid was improved to 0.98 g/g. This study proved the feasibility of endowing producers with methanol dissimilation pathway to enhance the production of reductive metabolites.
Microbial weathering processes can significantly promote soil properties and reduce rock-to-soil ratio. Some soil-inhabiting bacteria exhibit efficient rock-dissolution abilities by releasing organic acids and other chemical elements from the silicate rocks. However, our understanding of the molecular mechanisms involved during bacterial rock-dissolution is still limited. In this study, we performed silicate rock-dissolution experiments on a Pseudomonas sp. NLX-4 strain isolated from an over-exploited mining site. The results revealed that Pseudomonas sp. NLX-4 strain efficiently accelerates the dissolution of silicate rocks by secreting amino acids, exopolysaccharides, and organic acids. Through employing genome and transcriptome sequencing (RNA-seq), we identified the major regulatory genes. Specifically, 15 differentially expressed genes (DEGs) encoding for siderophore transport, EPS and amino acids synthesis, organic acids metabolism, and bacterial resistance to adverse environmental conditions were highly up-regulated in silicate rock cultures of NLX-4 strain. Our study reports a potential bacterial based approach for improving the ecological restoration of over-exploited rock mining sites.
Microfluidic devices have shown promising applications in the bioprocessing industry. However, the lack of modularity and high cost of testing and error limit their implementation in the industry. Advances in 3D printing technologies have facilitated the conversion of microfluidic devices from research output to applicable industrial systems. Here, for the first time, we presented a 3D printed modular microfluidic system consisting of two micromixers, one spiral microfluidic separator, and one microfluidic concentrator. We showed that this system can detach and separate mesenchymal stem cells (MSCs) from microcarriers (MCs) in a short time while maintaining the cell’s viability and functionality. The system can be multiplexed and scaled up to process large volumes of the industry. Importantly, this system is a closed system with no human intervention and is promising for current good manufacturing practices.
Although celebrating its golden jubilee, rapamycin’s importance keeps increasing by the day. Starting as a promising antifungal agent, then as a potent immunosuppressant, strong anticancer drug, and now rapamycin is attracting serious attention as a rejuvenative agent and a possible contributor in treating this era pandemic, COVID-19. Due to its diverse biological activities and promising medical applications, we aimed in this review to put rapamycin under the spot and highlight its discovery, famous microbial producers, reported biological activities, chemical structure, famous analogues, and biosynthesis. Moreover, discuss some rapamycin production approaches including solid-state fermentation, and stressing out producing strain. On the other hand, describe its action mechanism and trials to use it in treatment of COVID-19. Additionally, we highlighted some of the side effects accompanying its use, and describe some approaches reported to minimize these undesired effects. Finally, we report the current status of rapamycin and its analogues in global market, and discuss future prospects of this potent drug.
The goal of this study was to determine the capacity of Pleurotus spp. lignocellulosome to transform frequent pomiculture residues (grapevine-, plum-, and raspberry sawdust) into raw materials for biotechnological processes. All three lignocellulosics induced the synthesis of ligninolytic and cellulolytic enzymes in the tested species. Laccase was dominant in the ligninolytic cocktail, with a maximum activity of 40,494.88 U L−1 observed after the cultivation of P. pulmonarius on grapevine sawdust. Grapevine sawdust also proved to be the optimal substrate for the synthesis of versatile peroxidases especially in P. eryngii (1010.10 U L−1), while raspberry sawdust favored the production of Mn-dependent peroxidase in P. pulmonarius (479.17 U L−1). P. pulmonarius was the dominant cellulolytic agent and raspberry sawdust was optimal for the synthesis of xylanases, and endo- and exo-cellulases (15,746.35 U L−1, 9741.56 U L−1, and 836.62 U L−1), while grapevine sawdust mostly induced β-glucosidase activity (166.11 U L−1). The degree of residues delignification was more substrate- than species-dependent, ranging between 6.44 and 23.72% after the fermentation of grapevine and raspberry sawdust with P. pulmonarius. On the other hand, the lowest level of cellulose consumption was also observed on raspberry sawdust after the cultivation of P. eryngii, which together with high delignification also induced the highest selectivity index (1.27). The obtained results show the exceptional lignocellulolytic potential of Pleurotus spp. enzyme cocktails which opens up many possibilities for their application in numerous biotechnological processes.
Biocorrosion, also called microbiologically influenced corrosion (MIC), is a common operational threat to many industrial processes. It threatens carbon steel, stainless steel and many other metals. In the bioprocessing industry, reactor vessels in biomass processing and bioleaching are prone to MIC. MIC is caused by biofilms. The formation and morphology of biofilms can be impacted by fluid flow. Fluid velocity affects biocide distribution and MIC. Thus, assessing the efficacy of a biocide for the mitigation of MIC under flow condition is desired before a field trial. In this work, a benchtop closed flow loop bioreactor design was used to investigate the biocide mitigation of MIC of C1018 carbon steel at 25 °C for 7 days using enriched artificial seawater. An oilfield biofilm consortium was analyzed using metagenomics. The biofilm consortium was grown anaerobically in the flow loop which had a holding vessel for the culture medium and a chamber to hold C1018 carbon steel coupons. Peptide A (codename) was a chemically synthesized cyclic 14-mer (cys-ser-val-pro-tyr-asp-tyr-asn-trp-tyr-ser-asn-trp-cys) with its core 12-mer sequence originated from a biofilm dispersing protein secreted by a sea anemone which possesses a biofilm-free exterior. It was used as a biocide enhancer. The combination of 50 ppm (w/w) THPS (tetrakis hydroxymethyl phosphonium sulfate) biocide + 100 nM (180 ppb by mass) Peptide A resulted in extra 1-log reduction in the sulfate reducing bacteria (SRB) sessile cell count and the acid producing bacteria (APB) sessile cell count compared to 50 ppm THPS alone treatment. Furthermore, with the enhancement of 100 nM Peptide A, extra 44% reduction in weight loss and 36% abatement in corrosion pit depth were achieved compared to 50 ppm THPS alone treatment.
5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid which has involved in heme metabolism of organisms, and has been widely applied in agriculture, and medical fields nowadays. 5-ALA is used in the elimination of pathogens or cancer cells by photodynamic therapy (PDT) owing to the photosensitizer reaction which releases the reactive oxygen species (ROS). Currently, biofabrication of 5-ALA is regarded as the most efficient and eco-friendly approach, but the complicated ingredient of medium causes the nuisance process of purification, resulting in low recovery and high producing cost. In this study, hydrogen chloride, sodium acetate, and ammonia were examined to maximize the recovery of 5-ALA from ion-exchange chromatography (IEC), thus a 92% recovery in 1 M ammonia at pH 9.5 was obtained. Afterward, the activated carbon was used for decolorization to further remove the pigments from the eluent. Four organic solvents, i.e., diethyl ether, methanol, ethanol, and acetone were compared to extract and form 5-ALA precipitation. The purified 5-ALA was verified to eliminate 74% of A549 human lung cancer and 83% of A375 melanoma skin cancer cell. Moreover, Proteus hauseri, Aeromonas hydrophila, Bacillus cereus, and Staphylococcus aureus were killed via anti-microbial PDT with 1% 5-ALA and reached 100% killing rate at optimal condition. With the addition of 0.05% 5-ALA during the culture, the growth of microalgae Chlorella sorokiniana was improved to against a common aquatic pathogen, A. hydrophila. The broad application of 5-ALA was demonstrated in this study for the first time.
| 1. | Effects of different operating conditions on CH4 emission. |
| 2. | The competitive relationship between electricigens and methanogens was analysed. |
| 3. | The morphology and content of C element in different phases were discussed. |
| 4. | The bacterial population structure under different conditions was analysed. |
| 5. | The mechanism of CH4 emission from CW–MFC was described in detail. |
Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (kcat/Km) due to enhancing π–π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.
Sulphite addition during steam pretreatment of softwoods under acidic, neutral and alkaline conditions was assessed to try to minimize lignin condensation. Although pretreatment under neutral/alkaline conditions resulted in effective lignin sulphonation, non-uniform size reduction was observed. In contrast, acidic sulphite steam treatment at 210 °C for 10 min resulted in homogenous particle size reduction and water-insoluble component that was 62% carbohydrate and 33% lignin. This carbohydrate-rich substrate was readily hydrolyzed and fermented which indicated the lack of fermentation inhibitors in the steam-pretreated whole slurry. The use of high solid loading (25% w/v) resulted in a hydrolysis yield of 58% at an enzyme loading of 40 mg protein/g glucan and efficient fermentation (46.6 g/L of ethanol). This indicated that the addition of acidic sulphite at the steam pretreatment of softwoods improved both the enzymatic hydrolysis and fermentation of steam-pretreated whole slurries.
Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.
The combination of metal-catalyzed reactions and enzyme catalysis has been an essential tool for synthesizing chiral pharmaceutical intermediates in the field of drug synthesis. Metal catalysis commonly enables the highly efficient synthesis of molecular scaffolds under harsh organic conditions, whereas enzymes usually catalyze reactions in mild aqueous medium to obtain high selectivity. Since the incompatibility between metal and enzyme catalysis, there are limitations on the compatibility of reaction conditions that must be overcome.
We report a chemoenzymatic cascade reaction involved Palladium (Pd) catalyzed Suzuki–Miyaura coupling and whole-cell catalyzed C = O asymmetric reduction for enantioselective synthesis of value-added chiral alcohol. The cell membrane serves as a natural barrier can protect intracellular enzymes from organic solvents.
With dual advantages of cascade catalysis and biocompatibility, our work provides a rational strategy to harvest chiral alcohols in high yield and excellent enantioselectivity, as a channel to establish chemoenzymatic catalysis.
Plant growth-promoting rhizobacteria (PGPR) or Biocontrol strains inevitably encounter heavy metal excess stress during the product’s processing and application. Bacillus amyloliquefaciens Bam1 was a potential biocontrol strain with strong heavy metal resistant ability. To understand its heavy metal resistance mechanism, the complete genome of Bam1 had been sequenced, and the comparative genomic analysis of Bam1 and FZB42, an industrialized PGPR and biocontrol strain with relatively lower heavy metal tolerance, was conducted. The comparative genomic analysis of Bam1 and the other nine B. amyloliquefaciens strains as well as one Bacillus velezensis (genetically and physiologically very close to B. amyloliquefaciens) was also performed. Our results showed that the complete genome size of Bam1 was 3.95 Mb, 4219 coding sequences were predicted, and it possessed the highest number of unique genes among the eleven analyzed strains. Nine genes related to heavy metal resistance were detected within the twelve DNA islands of Bam1, while only two of them were detected within the seventeen DNA islands of FZB42. When compared with B. amyloliquefaciens type strain DSM7, Bam1 lacked contig L, whereas FZB42 lacked contig D and I, as well as just possessed contig B with a very small size. Our results could also deduce that Bam1 promoted its essential heavy metal resistance mainly by decreasing the import and increasing the export of heavy metals with the corresponding homeostasis systems, which are regulated by different metalloregulators. While Bam1 promoted its non-essential heavy metal resistance mainly by the activation of some specific or non-specific exporters responding to different heavy metals. The variation of the genes related to heavy metal resistance and the other differences of the genomes, including the different number and arrangement of contigs, as well as the number of the heavy metal resistant genes in Prophages and Genomic islands, led to the significant different resistance of Bam1 and FZB42 to heavy metals.
Catalytic valorization of raw glycerol derived from biodiesel into high-value chemicals has attracted great attention. Here, we report chemoenzymatic cascade reactions that convert glycerol to lactic acid and glycolic acid. In the enzymatic step, a coenzyme recycling system was developed to convert glycerol into 1,3-dihydroxyacetone (DHA) with a yield of 92.3% in potassium phosphate buffer (300 mM, pH 7.1) containing 100 mM glycerol, 2 mM NAD+, 242 U/mL glycerol dehydrogenase-GldA and NADH oxidase-SpNoxK184R at 30 °C. Subsequently, NaOH or NaClO2 catalyzes the formation of lactic acid and glycolic acid from DHA. The high yield of lactic acid (72.3%) and glycolic acid (78.2%) verify the benefit of the chemoenzymatic approaches.
Gallic acid is a natural phenolic acid that has a stress inhibition effect on Escherichia coli. This study by integrates fermentation characteristics and transcriptional analyses to elucidate the physiological mechanism of E. coli 3110 response to gallic acid. Compared with the control (without stress), the cell growth was severely retarded, and irregular cell morphology appeared in the case of high levels of gallic acid stress. The glucose consumption of E. coli was reduced successively with the increase of gallic acid content in the fermentation medium. After 20 h of gallic acid stress, cofactor levels (ATP, NAD+ and NADH) of E. coli 3110 were similarly decreased, indicating a more potent inhibitory effect of gallic acid on E. coli. The transcriptional analysis revealed that gallic acid altered the gene expression profiles related to five notable differentially regulated pathways. The genes related to the two-component system were up-regulated, while the genes associated with ABC-transporter, energy metabolism, carbon metabolism, and fatty acid biosynthesis were down-regulated. This is the first report to comprehensively assess the toxicity of gallic acid on E. coli. This study has implications for the efficient production of phenolic compounds by E. coli and provides new ideas for the study of microbial tolerance to environmental stress and the identification of associated tolerance targets.
The present study aimed to investigate the functional properties of soybean protein isolate (SPI) treated with alkaline protease and high-speed shearing homogenization. Alkaline protease-hydrolyzed SPIs that were characterized by varying degrees of hydrolysis between 0 and 6% were treated with high-speed shearing homogenization to obtain different micro-particulate proteins. The results showed that this combined treatment could significantly reduce the particle size of SPI by markedly degrading the structure of both the 7S and 11S subunits, thereby resulting in a significantly reduced content of β-sheet and β-turn structures. The surface hydrophobicity increased considerably for samples with hydrolysis below the threshold of 2% and then declined gradually above this threshold. Furthermore, the combination of hydrolysis and homogenization significantly improved the emulsion stability of SPI hydrolysates. It also significantly improved the foaming properties of SPI. These results demonstrated that alkaline protease hydrolysis combined with high-speed shearing homogenization represents a promising approach for improving the functional and structural properties of SPI.
Energy shortage and environmental concern urgently require establishing the feasible bio-refinery process from various feedstocks. The methylotrophic yeast Ogataea polymorpha is thermo-tolerant and can utilize various carbon sources, such as glucose, xylose and methanol, which makes it a promising host for bio-manufacturing. Here, we explored the capacity of O. polymorpha for overproduction of free fatty acids (FFAs) from multiple substrates. The engineered yeast produced 674 mg/L FFA from 20 g/L glucose in shake flask and could sequentially utilize the mixture of glucose and xylose. However, the FFA producing strain failed to survive in sole methanol and supplementing co-substrate xylose promoted methanol metabolism. A synergistic utilization of xylose and methanol was observed in the FFA producing strain. Finally, a mixture of glucose, xylose and methanol was evaluated for FFA production (1.2 g/L). This study showed that O. polymorpha is an ideal host for chemical production from various carbon sources.
In this study, a high frequency monitoring method was used to assess how semi-continuous feeding affects H2S production in high solid anaerobic digestion. The results showed that H2S characteristics at a monitoring frequency of 1 point/3 h were different to that of 1 point/24 h, its concentration decreased from 3449 ± 227 mg/m3 at 0 h to 298 ± 45 mg/m3 at 3 h. H2S concentration was negatively correlated with volatile fatty acids (VFAs), and oxidation reduction potential (ORP). 72–82% of H2S reduction in the first 3 h resulted from the introduction of O2 during feeding, and 18–28% of that was closely related to the production of a large quantity of soluble acidic matter, such as VFAs. A more accurate H2S release model was established according to the content of VFAs. Totally, this study implies that feed carrying air is a promising method for in situ control of H2S production in anaerobic digestion.
In this study, we selected and engineered a flavin adenine dinucleotide (FAD)-dependent alcohol oxidase (AOX) to produce 1,4-cyclohexanedicarboxaldehyde (CHDA), an initial raw material for spiral compounds, from 1,4-cyclohexanedimethanol (CHDM). First, the structure of alcohol oxidase from Arthrobacter cholorphenolicus (AcCO) was analyzed, and the mechanism of AcCO-catalyzed primary alcohol oxidation was elucidated, demonstrating that the energy barrier of the hydride (H−) transfer (13.4 kcal·mol−1 and 20.4 kcal·mol−1) decreases the catalytic efficiency of the primary alcohol oxidation reaction. Therefore, we designed a protein engineering strategy to adjust the catalytically active conformation to shorten the distance of hydride (H−) transfer and further decreased the core energy barrier. Following this strategy, variant W4 (S101A/H351V/N378S/Q329N) was obtained with 112.5-fold increased catalytic efficiency to produce CHDA compared to that of the wild-type strain. The 3 L scale preparation of CHDA reached a titer up to 29.6 g·L−1 with a 42.2% yield by an Escherichia coli whole-cell catalyst, which demonstrates the potential of this system for industrial application.
Corn fiber, a by-product from the corn processing industry, mainly composed of residual starch, cellulose, and hemicelluloses, is a promising raw material for producing cellulosic ethanol and value-added products due to its abundant reserves and low costs of collection and transportation. Now, several technologies for the production of cellulosic ethanol from corn fiber have been reported, such as the D3MAX process, Cellerate™ process, etc., and part of the technologies have also been used in industrial production in the United States. The ethanol yields range from 64 to 91% of the theoretical maximum, depending on different production processes. Because of the multicomponent of corn fiber and the complex structures highly substituted by a variety of side chains in hemicelluloses of corn fiber, however, there are many challenges in cellulosic ethanol production from corn fiber, such as the low conversion of hemicelluloses to fermentable sugars in enzymatic hydrolysis, high production of inhibitors during pretreatment, etc. Some technologies, including an effective pretreatment process for minimizing inhibitors production and maximizing fermentable sugars recovery, production of enzyme preparations with suitable protein compositions, and the engineering of microorganisms capable of fermenting hexose and pentose in hydrolysates and inhibitors tolerance, etc., need to be further developed. The process integration of cellulosic ethanol and value-added products also needs to be developed to improve the economic benefits of the whole process. This review summarizes the status and progresses of cellulosic ethanol production and potential value-added products from corn fiber and presents some challenges in this field at present.
| • | We report a recombinant Escherichia coli BL21(DE3) strain for de novo production of taxadien-5α-ol, the key precursor of paclitaxel. |
| • | Through screening of key enzymes and the fermentation condition optimization, the final production of total oxygenated taxanes was raised up to 27 mg L−1 in 50-mL flask cultivation, of which the yield of taxadien-5α-ol was 7.0 mg L−1, representing approximately a 12-fold and 23-fold improvements, respectively. |
| • | It is believed that the strategy used in this study will guide in the synthesis of terpenoids. |
Microalgae are cosmopolitan organisms in nature with short life cycles, playing a tremendous role in reducing the pressure of industrial carbon emissions. Besides, microalgae have the unique advantages of being photoautotrophic and harboring both prokaryotic and eukaryotic expression systems, becoming a popular host for recombinant proteins. Currently, numerous advanced molecular tools related to microalgal transgenesis have been explored and established, especially for the model species Chlamydomonas reinhardtii (C. reinhardtii hereafter). The development of genetic tools and the emergence of new strategies further increase the feasibility of developing C. reinhardtii chloroplasts as green factories, and the strong genetic operability of C. reinhardtii endows it with enormous potential as a synthetic biology platform. At present, C. reinhardtii chloroplasts could successfully produce plenty of recombinant proteins, including antigens, antibodies, antimicrobial peptides, protein hormones and enzymes. However, additional techniques and toolkits for chloroplasts need to be developed to achieve efficient and markerless editing of plastid genomes. Mining novel genetic elements and selectable markers will be more intensively studied in the future, and more factors affecting protein expression are urged to be explored. This review focuses on the latest technological progress of selectable markers for Chlamydomonas chloroplast genetic engineering and the factors that affect the efficiency of chloroplast protein expression. Furthermore, urgent challenges and prospects for future development are pointed out.
| 1. | Biochemical and molecular structural characterization of a novel GH10 xylanase (Xyl10B) from a termite gut microbiome. |
| 2. | Xyl10B is a candidate biocatalyst in the bleaching process of pulp and the paper industries because of its inactivity on carboxymethyl cellulose. |
| 3. | The shorter xylooligosaccharides generated from the hydrolysis of xylan would be suitable in different applications, including the food industry as prebiotics. |
This research aims to modify ultrafine fully vulcanized powdered natural rubber (UFPNR) prepared by emulsion graft-copolymerization with styrene (St) and acrylonitrile (AN) monomers onto deproteinized natural rubber (DPNR). The effects of monomers content and St/AN weight ratio on grafting efficiency and thermal stability of the developed DPNR-g-(PS-co-PAN) were investigated. The results showed that grafting efficiency was enhanced up to 86% with monomers content 15 phr and weight ratio St:AN 80:20. The obtained DPNR-g-(PS-co-PAN) was radiated by an electron beam at various doses, followed by a spray drying process to produce UFPNR. The obtained modified UFPNR particles irradiated at dose up to 300 kGy were relatively spherical with a particle size of approximately 4.4 µm. Furthermore, the degradation temperature of 5wt% loss (Td5) of UFPNR was found in the range of 349–356 °C. The results revealed that the modified UFPNR is suitable as a toughening filler for a broader spectrum of polymers.
Marine Vibrio species are natural degraders of chitin and usually secrete high levels of chitinolytic enzymes to digest recalcitrant chitin to chitooligosaccharides. This study used an endochitinase (VhChiA) from Vibrio campbellii to produce high-quality chitobiose from crustacean chitins. The enzyme was shown to be fully active and stable over 24 h when BSA was used as an additive. When different chitin sources were tested, VhChiA preferentially digested shrimp and squid (α) chitins compared to crab (β) chitin and did not utilize non-chitin substrates. The overall yields of chitobiose obtained from small-scale production using a single-step reaction was 96% from shrimp, and 91% from squid pen and crab-shell chitins. Larger-scale production yielded 200 mg of chitobiose, with > 99% purity after a desalting and purification step using preparative HPLC. In conclusion, we report the employment of an in-house produced chitinase as an effective biocatalyst to rapidly convert chitin food wastes to chitobiose, in a quantity and quality suitable for use in research and commercial purposes. Chitobiose production by this economical and eco-friendly approach can be easily scaled up to obtain multi-gram quantities of chitobiose for chemo-enzymic synthesis of rare chitooligosaccharide derivatives and long chain chitooligosaccharides, as well as preparation of sugar-based functionalized nanomaterials.
Robust ex vivo expansion of NK-92 cells is essential for clinical immunotherapy. The vitamin B group is critical for the expansion and function of immune cells. This study optimized a vitamin combination by response surface methodology based on an in-house designed chemically defined serum-free medium EM. The serum-free medium EM-V4 with an optimal vitamin combination favoured ex vivo expansion of NK-92 cells. The characteristics of glucose metabolism of NK-92 cells in EM-V4 and the relationships between cell expansion and metabolism were investigated. NK-92 cells in EM-V4 underwent metabolic reprogramming. An elevated ratio of glucose-6-phosphate dehydrogenase/phosphofructokinase (G6PDH/PFK) indicated that NK-92 cells shifted towards the pentose phosphate pathway (PPP). An increase in the ratio of pyruvate dehydrogenase/lactate dehydrogenase (PDH/LDH) suggested that the cells shifted towards the Krebs (TCA) cycle, i.e., from glycolysis to aerobic metabolism. The enhanced ratio of oxygen consumption rate/extracellular acidification rate (OCR/ECAR) indicated that NK-92 cells were more reliant on mitochondrial respiration than on glycolysis. This shift provided more intermediate metabolites and energy for biosynthesis. Thus, EM-V4 accelerated biomass accumulation and energy production to promote NK-92 cell expansion by regulating the metabolic distribution. Our results provide valuable insight for the large-scale ex vivo expansion of clinically available NK-92 cells.
(−)-Limonene, one of cyclic monoterpenes, is an important renewable compound used widely as a key building block for the synthesis of new biologically active molecules and fine chemicals. (−)-Perillamine, as derived from (−)-limonene, is a highly useful synthon for constructing more complicated and functionally relevant chemicals.
We aimed to report a more sustainable and more efficient method for the regiospecific C–H amination of (−)-limonene into (−)-perillamine.
Here, we report an artificial penta-enzymatic cascade system for the transformation of the cheap and easily available (−)-limonene into (−)-perillamine for the first time. This system is composed of cytochrome P450 monooxygenase, alcohol dehydrogenase and w-transaminase for the main reactions, as well as formate dehydrogenase and NADH oxidase for cofactor recycling. After optimization of the multi-enzymatic cascade system, 10 mM (−)-limonene was smoothly converted into 5.4 mM (−)-perillamine in a one-pot two-step biotransformation, indicating the feasibility of multi-enzymatic C7-regiospecific amination of the inert C–H bond of (−)-limonene. This method represents a concise and efficient route for the biocatalytic synthesis of derivatives from similar natural products.
pH-sensitive hydrogels prepared by gamma irradiation find promising biological applications, partially, in the field of localized drug liberation. Herein, optimal conditions for fabricating high-molecular-weight chitosan/polyvinyl alcohol hybrid hydrogels using gamma irradiation at 10, 25, and 30 kGy were investigated by studying the water uptake behavior, the pore size on the surface, and thermal stability. Furthermore, the crosslinking mechanism of irradiated hydrogels was examined via solid-state 13C NMR spectrum. The swelling ratio of the gamma-irradiated CS/PVA hydrogel was pH-dependent; particularly, the hybrid hydrogel exhibited high swelling ratios under acidic conditions than those under basic conditions due to the protonation of amino groups on CS-backbone in acidic environments. In addition, amoxicillin was used as a model drug in the in vitro drug release investigations in pH-simulated gastric fluid and deionized water at 37 °C. To identify the drug release mechanism, several kinetic models composing zero-order, first-order, Higuchi, Hixson–Crowell, and Korsmeyer–Peppas models were used. The findings suggested that drug release is mediated by a non-Fickian transport mechanism.
Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is a potent antitumor agent. However, lack of efficient genome editing tools greatly hinders the AP-3 overproduction in A. pretiosum. To solve this problem, a tailor-made pCRISPR–Cas9apre system was developed from pCRISPR–Cas9 for increasing the accessibility of A. pretiosum to genetic engineering, by optimizing cas9 for the host codon preference and replacing pSG5 with pIJ101 replicon. Using pCRISPR–Cas9apre, five large-size gene clusters for putative competition pathway were individually deleted with homology-directed repair (HDR) and their effects on AP-3 yield were investigated. Especially, inactivation of T1PKS-15 increased AP-3 production by 27%, which was most likely due to the improved intracellular triacylglycerol (TAG) pool for essential precursor supply of AP-3 biosynthesis. To enhance a “glycolate” extender unit, two combined bidirectional promoters (BDPs) ermEp-kasOp and j23119p-kasOp were knocked into asm12-asm13 spacer in the center region of gene cluster, respectively, by pCRISPR–Cas9apre. It is shown that in the two engineered strains BDP-ek and BDP-jk, the gene transcription levels of asm13-17 were significantly upregulated to improve the methoxymalonyl-acyl carrier protein (MM-ACP) biosynthetic pathway and part of the post-PKS pathway. The AP-3 yields of BDP-ek and BDP-jk were finally increased by 30% and 50% compared to the parent strain L40. Both CRISPR–Cas9-mediated engineering strategies employed in this study contributed to the availability of AP-3 PKS extender units and paved the way for further metabolic engineering of ansamitocin overproduction.
Efficient biodegradation of lignocellulosic biomass needs a battery of enzymes targeting cellulose, hemicellulose, and lignin. In this study, recombinant Trichoderma reesei ZJ-09 with Pycnoporus sanguineus laccase gene was used to degrade rice straw by in situ production of laccase, xylanase, and cellulases under solid-state fermentation (SSF). Effects of parameters on key enzymes (cellulase, xylanase, and laccase) in biodegradation during SSF were investigated. Under the optimized SSF conditions, the FPA, xylanase activity, and laccase activity reached 110.47 FPU/g, 5787.59 IU/g, and 24.45 IU/g, respectively, on day 12. The obtained recombinant T. reesei SSF system achieved efficient degradation of rice straw with the final mass loss up to 51.16% which was 1.4-fold higher than the host strain. Further, bioconversion of rice straw into a novel laccase-enriched koji for persistent organic pollutants bioremediation (LKPB) was conducted by the optimized SSF system. LKPB was found to degrade persistent organic pollutants (POPs) effectively without the addition of mediators. 4-h removal rates of three POPs mediated by LKPB (87.21% for 2,4,5-trichlorophenol, 92.45% for nonylphenol, and 90.73% for oxytetracycline) were comparable to those achieved by laccase-co-mediator system. The newly established recombinant T. reesei SSF system could be potential to effectively degrade lignocellulosic wastes as well as organic pollutants.
Bacterial cis-epoxysuccinic acid hydrolases (CESHs) are intracellular enzymes used in the industrial production of enantiomeric tartaric acids. The enzymes are mainly used as whole-cell catalysts because of the low stability of purified CESHs. However, the low cell permeability is the major drawback of the whole-cell catalyst. To overcome this problem, we developed whole-cell catalysts using various surface display systems for CESH[L] which produces L(+)-tartaric acid. Considering that the display efficiency depends on both the carrier and the passenger, we screened five different anchoring motifs in Escherichia coli. Display efficiencies are significantly different among these five systems and the InaPbN-CESH[L] system has the highest whole-cell enzymatic activity. Conditions for InaPbN-CESH[L] production were optimized and a maturation step was discovered which can increase the whole-cell activity several times. After optimization, the total activity of the InaPbN-CESH[L] surface display system is higher than the total lysate activity of an intracellular CESH[L] overexpression system, indicating a very high CESH[L] display level. Furthermore, the whole-cell InaPbN-CESH[L] biocatalyst exhibited good storage stability at 4 °C and considerable reusability. Thereby, an efficient whole-cell CESH[L] biocatalyst was developed in this study, which solves the cell permeability problem and provides a valuable system for industrial L(+)-tartaric acid production.
Progesterone is one of the classical hormone drugs used in medicine for maintaining pregnancy. However, its manufacturing process, coupled with organic reagents and poisonous catalysts, causes irreversible environmental pollution. Recent advances in synthetic biology have demonstrated that the microbial biosynthesis of natural products, especially difficult-to-synthesize compounds, from building blocks is a promising strategy. Herein, overcoming the heterologous cytochrome P450 enzyme interdependency in Mycolicibacterium neoaurum successfully constructed the CYP11A1 running module to realize metabolic conversion from waste phytosterols to progesterone. Subsequently, progesterone yield was improved through strategies involving electron transfer and NADPH regeneration. Mutant CYP11A1 (mCYP11A1) and adrenodoxin reductase (ADR) were connected by a flexible linker (L) to form the chimera mCYP11A1-L-ADR to enhance electron transfer. The chimera mCYP11A1-L-ADR, adrenodoxin (ADX), and ADR-related homolog ARH1 were expressed in M. neoaurum, showed positive activity and produced 45 mg/L progesterone. This electron transfer strategy increased progesterone production by 3.95-fold compared with M. neoaurum expressing mCYP11A1, ADR, and ADX. Significantly, a novel inorganic–biological hybrid system was assembled by combining engineered M. neoaurum and InP nanoparticles to regenerate NADPH, which was increased 84-fold from the initial progesterone titer to 235 ± 50 mg/L. In summary, this work highlights the green and sustainable potential of obtaining synthetic progesterone from sterols in M. neoaurum.
Chitooligosaccharides (COS) are found naturally in the ocean and present a variety of physiological activities, of which hypoglycemic action has attracted considerable research attention. This study aimed to assess the therapeutic effect of COS on mice suffering from type 2 diabetes mellitus (T2DM). COS effectively reduced blood glucose and blood lipid levels and improved glucose tolerance. Furthermore, COS revealed strong inhibitory activity against α-glucosidase, reducing postprandial blood glucose levels. Molecular docking data showed that COS might interact with surrounding amino acids to form a complex and decrease α-glucosidase activity. Additionally, COS enhanced insulin signal transduction and glycogen synthesis while restricting gluconeogenesis in the liver and muscles, reducing insulin resistance (IR) as a result. Moreover, COS effectively protected and restored islet cell function to increase insulin secretion. These results indicated that COS exhibited a significant hypoglycemic effect with multi-target participation. Therefore, COS may serve as a new preventive or therapeutic drug for diabetes to alleviate metabolic syndrome.
Nowadays renewable energy with low prices is a global target that has taken the attention to compare alternatives energy sources with fossil fuels. Therefore, this study was established to find suitable and sustainable alternative low-cost fuels source. Cooking oil waste (COW) was mixed with non-pretreated citrus tree fibers (CTF) (0.5 mL to 1 g ratio) and pressed to formulate coal (CTF/COW). Otherwise, this mixture was subjected to in situ fungal pretreated using Aspergillus flavus isolate to simplify the mixture composition and pressed to offer in a usable form with enhancing their heating value for the first time. CTF/COW was characterized using attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), scanning electron microscope (SEM) and thermal analysis (TGA) before and after treatment. The fungal isolate was observed with enzyme productivity and activity of CMCase, avicelase, xylanase, mannanase, α-glucosidase, β-glucosidase, lignin peroxidase and lipase according to enzyme assays and the chemical compositions of CTF before and after fungal treatment, where the best PH for enzymes extraction was between 5 and 7. The fungal enzymes increased the heating value by about two and half folds in comparison with non-pretreated coal. Moreover, the calorific value of tCTF/COW was 43,422 kJ/kg, which was higher than CTF recorded 18,214 kJ/kg and COW recorded 39,823 kJ/kg. Our result suggests that fungal treatment of the mixture of citrus trees and cooking oil waste presents as a promising low-cost and eco-friendly coal.
Trichoderma reesei RUT-C30 is a well-known high-yielding cellulase-producing fungal strain that converts lignocellulose into cellulosic sugar for resource regeneration. Calcium is a ubiquitous secondary messenger that regulates growth and cellulase production in T. reesei. We serendipitously found that adding Sr2+ to the medium significantly increased cellulase activity in the T. reesei RUT-C30 strain and upregulated the expression of cellulase-related genes. Further studies showed that Sr2+ supplementation increased the cytosolic calcium concentration and activated the calcium-responsive signal transduction pathway of Ca2+–calcineurin-responsive zinc finger transcription factor 1 (CRZ1). Using the plasma membrane Ca2+ channel blocker, LaCl3, we demonstrated that Sr2+ induces cellulase production via the calcium signaling pathway. Supplementation with the corresponding concentrations of Sr2+ also inhibited colony growth. Sr2+ supplementation led to an increase in intracellular reactive oxygen species (ROS) and upregulated the transcriptional levels of intracellular superoxide dismutase (sod1) and catalase (cat1). We further demonstrated that ROS content was detrimental to cellulase production, which was alleviated by the ROS scavenger N-acetyl cysteine (NAC). This study demonstrated for the first time that Sr2+ supplementation stimulates cellulase production and upregulates cellulase genes via the calcium signaling transduction pathway. Sr2+ leads to an increase in intracellular ROS, which is detrimental to cellulase production and can be alleviated by the ROS scavenger NAC. Our results provide insights into the mechanistic study of cellulase synthesis and the discovery of novel inducers of cellulase.
An imminent change in the world energy matrix makes it necessary to increase the production of renewable fuels. The United States and Brazil are the world's largest producers, but their production methods are very different, using different raw materials, ground corn and sugarcane juice, respectively. In recent years, strong investments have been made to expand the use of corn in Brazilian ethanol production. The combination of the sugar cane and corn ethanol industries has generated innovations in the sector, such as the "flex" mills, which are traditional sugar cane mills adapted to produce corn ethanol in the sugar cane off-season. Brazil has a portfolio of robust industrial yeasts for sugarcane ethanol production, naturally evolved and selected over the past 50 years. In this work, we analyze for the first time the performance of Brazilian industrial strains (BG-1, CAT-1, PE-2 and SA-1, widely used in sugarcane ethanol production) in corn ethanol production using different stress conditions. Ethanol Red yeast, traditionally used in corn ethanol plants around the world, was used as a control. In terms of tolerance to temperature (35 °C), strains BG-1 and SA-1 stood out. In fermentations with high solids concentration (35%), strain BG-1 reached ethanol contents higher than 19% w/v and had a productivity gain of 5.8% compared to fermentation at 30%. This was the first time that these industrial strains were evaluated using the high solids concentration of 35% and the results point to ways to improve the corn ethanol production process.
Bispecific antibodies (bsAbs), though possessing great therapeutic potential, are extremely challenging to obtain at high purity within a limited number of scalable downstream processing steps. Complementary to Protein A chromatography, polishing strategies play a critical role at removing the remaining high molecular weight (HMW) and low molecular weight (LMW) species, as well as host cell proteins (HCP) in order to achieve a final product of high purity. Here, we demonstrate using two knob-into-hole (KiH) bsAb constructs that two flow-through polishing steps utilising Capto Butyl ImpRes and Capto adhere resins, performed after an optimal Protein A affinity chromatography step can further reduce the HCP by 17- to 35-fold as well as HMW and LMW species with respect to monomer by ~ 4–6% and ~ 1%, respectively, to meet therapeutical requirement at 30–60 mg/mL-resin (R) load. This complete flow-through polishing strategy, guided by Design of Experiments (DoE), eliminates undesirable aggregation problems associated with the higher aggregation propensity of scFv containing bsAbs that may occur in the bind and elute mode, offering an improved ease of overall process operation without additional elution buffer preparation and consumption, thus aligning well with process intensification efforts. Overall, we demonstrate that through the employment of (1) Protein A chromatography step and (2) flow-through polishing steps, a final product containing < 1% HMW species, < 1% LMW species and < 100 ppm HCP can be obtained with an overall process recovery of 56–87%.
Infectious disease is one of the major threats to humans and it is the second leading cause of death worldwide. Edible mushrooms have many nutritional and medicinal values to human health. The medicinal properties of edible mushroom extract in inhibiting pathogenic microorganisms had advantages over the use of chemically synthetic antimicrobial compounds due to less unwanted side effects and can combat microbial resistance. This study hypothesized that the polarity affects the extraction quality of Hericium erinaceus fruiting bodies which was prepared and subsequently affects its activity as an antimicrobial against six tested microorganisms, including MRSA, and Streptococcus mutans, Enterobacter cloaca, Salmonella typhimurium, and Candida lipolytica; antiviral against Hepatitis A virus (HAV) virus; antioxidant using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay; and anti-inflammatory potential. So, the mushroom was quantitatively evaluated to assess its content of flavonoids, alkaloids, tannins, saponins, carbohydrates, protein, nitrogen, and oil. The current research clarified here that aquatic extract has a significant activity as an antioxidant (IC50 = 53.7 µg/mL) and antiviral (IC50 = 24.97 µg/mL), while ethyl acetate extract showed a reasonable antimicrobial activity rather than all tested extract against tested microorganisms. Unfortunately, all extracts under investigation possess low anti-inflammatory action according to the adopting protocol. The superior results of both water and ethyl acetate extracts were later investigated by HPTLC and GC–MS for preliminary prediction of the chemical constitution of those extracts. H. erinaceus mushroom succeeded to establish promising antimicrobial, antiviral, and antioxidant activities while it has low anti-inflammatory activity. Both HPTLC and GC–MS could identify the chemical constituents of the mushroom crude extract.
Gazania rigens (L.) Gaertn. (Asteraceae) is a medicinal plant with high ornamental potential and use in landscaping. The therapeutic potential of sesquiterpene lactones (SLs) as plant natural products for pharmaceutical development has gained extensive interest with costunolide (chemical name: 6E,10E,11aR-6,10-dimethyl-3-methylidene-3a,4,5,8,9,11a-hexahydrocyclodeca[b]furan-2-one) used as a popular herbal remedy due to its anti-cancer, antioxidant, anti-inflammatory, anti-microbial, anti-allergic, and anti-diabetic activities, among others. In the present study, two explant types (leaf, stem) and four 2,4-dichlorophenoxy acetic acid (2,4-D) concentrations (0, 0.5, 1 and 2 mg/L) were tested for callusing potential. The results showed that stem explants treated with 1.5 mg/L 2,4-D exhibited higher callus induction percentage (90%) followed by leaf explants (80%) with 1 mg/L 2,4-D, after a 4-week period. Cell suspension cultures were established from friable callus obtained from stem explants following a sigmoid pattern of growth curve with a maximum fresh weight at 20 days of subculture and a minimum one at 5 days of subculture. In the following stage, the effects of elicitation of cell suspension cultures with either yeast extract (YE) or methyl jasmonate (MeJA), each applied in five concentrations (0, 100, 150, 200 and 250 mg/L) on cell growth (fresh and dry biomass) and costunolide accumulation were tested. After 20 days of culture, YE or MeJA suppressed cell growth as compared to the non-elicited cells, while costunolide accumulation was better enhanced under the effect of 150 mg/L MeJA followed by 200 mg/L YE, respectively. In the subsequent experiment conducted, the optimal concentration of the two elicitors (200 mg/L YE, 150 mg/L MeJA) was selected to investigate further elicitation time (0, 5, 10, 15 and 20 days). The results revealed that YE biotic elicitation stimulated cell growth and costunolide production, being maximum on day 20 for fresh biomass, on day 5 for dry biomass and on day 15 for the bioactive compound. Accordingly, cell growth parameters were maximized under the effect of abiotic elicitation with MeJA for 15 days, while highest costunolide content was achieved after 10 days. Overall, MeJA served as a better elicitor type than YE for biomass and costunolide production. Irrespective of elicitor type, elicitor concentration and elicitation time, maximal response was obtained with 150 mg/L MeJA for 10 days regarding costunolide accumulation (18.47 ppm) and 15 days for cell growth (fresh weight: 954 mg and dry weight: 76.3 mg). The application of elicitors can lead the large quantity of costunolide to encounter extensive range demand through marketable production without endangering of G. rigens.
| 1. | A replicative and integrative Di-CRISPR platform was promising in generating an efficient xylose-utilizing Saccharomyces cerevisiae strain. |
| 2. | The functional activity of chimeric xylanases was demonstrated in direct hemicellulose-to-ethanol conversion. |
| 3. | The feasibility of an accessory enzymatic system of cellulase for simultaneous saccharification and co-fermentation from pretreated C4 grass was evaluated. |
Silicon (Si) and its nanomaterials could help plants cope with different negative effects of abiotic and/or biotic stresses. In this study, the antifungal role of silver/silicon dioxide nanocomposite (Ag/SiO2NC) biosynthesized using a free-cell supernatant of Escherichia coli D8 was investigated for controlling the growth parameters and yield of faba bean (Vicia faba L.) infected by Botrytis cinerea. This nanocomposite was characterized using UV–Vis spectroscopy, Fourier transform-infrared (FTIR), transmission electron microscopy (TEM), zeta analysis, and X-ray diffraction pattern (XRD). Positively charged Ag/SiO2NC (+ 31.0 mV) with spherical-shaped silver nanoparticles (AgNPs) showed strong in vitro antifungal activity with minimal inhibition concentration (MIC) value equal to 40 ppm. In vivo experiments revealed the good resistance of Ag/SiO2NC-treated plants against the B. cinerea infection due to the increase of total phenolic content, peroxidase, and polyphenol oxidase activity. The ultrastructure of Ag/SiO2NC-treated plants showed normal morphology of cells including cell membranes and ellipsoidal-shaped chloroplasts with big starch grains. The concentration of silver content in Ag/SiO2NC-treated plants was similar to the untreated control plant indicating the low realizability of AgNPs. All of these results are promising outcomes for the application of the biosynthesized Ag/SiO2NC as a safe and effective antifungal agent against B. cinerea.
Rapeseed meal (RSM) is an agro-industrial residue of increased functional biological value that contains high-quality proteins for animal feed. Due to the presence of antinutritional factors and immature development technology, RSM is currently used as a limited feed additive and in other relatively low-value applications. With increasing emphasis on green and sustainable industrial development and the added value of agro-industrial residues, considerable attention has been directed to the removal of antinutritional factors from RSM using high-efficiency, environment-friendly, and cost-effective biotechnology. Similarly, the high-value biotransformations of RSM have been the focus of research programmes to improve utilization rate. In this review, we introduce the sources, the nutrient and antinutrient content of RSM, and emphasize improvements on RSM feed quality using biological methods and its biotransformation applications.
| 1. | Effects of salinity, alkalinity and their combined impact were analysed with transcriptome and and proteomics methods in G. przewalskii. |
| 2. | The validity of transcriptome and proteome results in G. przewalskii was compared. |
| 3. | Hub genes in the adaptive responses to the combined impact of salinity and alkalinity were isolated. |
| 4. | Physiological regulation mechanisms regarding salinity, alkalinity, and salinity-alkalinity interactions were discussed. |
Xylose is an abundant bioresource for obtaining diverse chemicals and added-value products. The production of xylose from green alternatives like enzymatic hydrolysis is an important step in a biorefinery context. This research evaluated the synergism among four classes of hydrolytic purified enzymes—endo-1,4-β-xylanase, α-l-arabinofuranosidase, β-xylosidase, and α-d-glucuronidase—over hydrolysis of glucuronoarabinoxylan (GAX) obtained from brewers’ spent grain (BSG) after alkaline extraction and ethanol precipitation. First, monosaccharides, uronic acids and glycosidic-linkages of alkaline extracted GAX fraction from BSG were characterized, after that different strategies based on the addition of one or two families of enzymes—endo-1,4-β-xylanase (GH10 and GH11) and α-l-arabinofuranosidase (GH43 and GH51)—cooperating with one β-xylosidase (GH43) and one α-d-glucuronidase (GH67) into enzymatic hydrolysis were assessed to obtain the best yield of xylose. The xylose release was monitored over time in the first 90 min and after a prolonged reaction up to 48 h of reaction. The highest yield of xylose was 63.6% (48 h, 40 ℃, pH 5.5), using a mixture of all enzymes devoid of α-l-arabinofuranosidase (GH43) family. These results highlight the importance of GH51 arabinofuranosidase debranching enzyme to allow a higher cleavage of the xylan backbone of GAX from BSG and their synergy with 2 endo-1,4-β-xylanase (GH10 and GH11), one β-xylosidase (GH43) and the inclusion of one α-d-glucuronidase (GH67) in the reaction system. Therefore, this study provides an environmentally friendly process to produce xylose from BSG through utilization of enzymes as catalysts.
Gibberellic acid (GA3) is a plant growth hormone that plays an important role in the production of crops, fruits, and vegetables with a wide market share. Due to intrinsic advantages, liquid fermentation of Fusarium fujikuroi has become the sole method for industrial GA3 production, but the broader application of GA3 is hindered by low titer. In this study, we combined atmospheric and room-temperature plasma (ARTP) with ketoconazole-based screening to obtain the mutant strain 3-6-1 with high yield of GA3. Subsequently, the medium composition and fermentation parameters were systematically optimized to increase the titer of GA3, resulting in a 2.5-fold increase compared with the titer obtained under the initial conditions. Finally, considering that the strain is prone to substrate inhibition and glucose repression, a new strategy of fed-batch fermentation was adopted to increase the titer of GA3 to 575.13 mg/L, which was 13.86% higher than the control. The strategy of random mutagenesis combined with selection and fermentation optimization developed in this study provides a basis for subsequent research on the industrial production of GA3.
RNA interference (RNAi) represents one of the most conserved pathways evolved by eukaryotic cells for regulating gene expression. RNAi utilises non-translatable double-stranded RNA (dsRNA) molecules to sequester or degrade mRNA molecules gene. In RNAi, specifically designed exogenous dsRNA delivered to the cell can silence a target gene, a phenomenon that has been exploited in many functional studies and explored in biopesticide applications. The search for safe and sustainable crop pest management options drives the need to offset the effect of inorganic pesticides on biodiversity. The prospect of replacing inorganic pesticides with dsRNA crop spray is gaining popularity, enhanced by its high-target specificity and low environmental impact. However, for dsRNA to reach the pesticide market, it must be produced cost-effectively and sustainably. In this paper, we develop a high-yield expression media that generates up to 15-fold dsRNA yield compared to existing expression media utilising 1 mM IPTG. We also optimise a low-cost purification method that generates high-quality and purified dsRNA. The developed method circumvents the need for hazardous chemical reagents often found in commercial kits or commercial nucleases to eliminate contaminating DNA or single-stranded RNA (ssRNA) species. We also demonstrate that the production platform is scalable, generating 6.29 mg dsRNA from 259 mg wet E. coli cell pellet. The results also provide structural insights into the heterogeneous dsRNA species within the microbial-derived dsRNA pool. Finally, we also show that the purified ‘naked’ dsRNA, without prior formulation, can induce insect toxicity under field conditions. This study provides a novel, complete, low-cost process dsRNA platform with potential for application in industrial dsRNA production.
Terminators serve as the regulatory role in gene transcription termination; however, few researches about terminator optimization have been conducted, which leads to the lack of available and universal terminator for gene expression regulation in Bacillus. To solve this problem and expand synthetic biology toolbox of Bacillus licheniformis, the terminator T1 of endogenous α-amylase gene (amyL) was characterized in this research, with a termination efficiency of 87.81%. Then, we explored and optimized the termination strength of terminator T1 from four aspects: the distance between stop codon and terminator, GC content at the bottom of stem structure, loop size, and U-tract length, and the best terminator T24 was attained by combination optimization strategy, which termination efficiency was increased to 97.97%, better than the commonly used terminator T7 (T7P) from Escherichia coli. Finally, terminator T24 was applied to protein expression, which, respectively, led to 33.00%, 25.93%, and 11.78% increases of green fluorescence intensity, red fluorescence intensity, and keratinase activity, indicating its universality in protein expression. Taken together, this research not only expands a plug-and-play synthetic biology toolbox in B. licheniformis but also provides a reference for the artificial design of versatile intrinsic terminator.
Pyruvic acid is an important organic acid and a key industrial raw material. It is widely used in the chemical, agricultural, and food fields. Candida glabrata is the preferred strain for pyruvic acid production. The waste yeast cell for pyruvic acid fermentation with C. glabrata are rich in protein, amino acid, nucleic acid, and vitamins, as potential and cost-effective nitrogen source raw material. In this study, the potential of C. glabrata to produce pyruvic acid using spent yeast cell dry powder was evaluated. When 30 g/L of spray-dried spent yeast cell powder was used as the seed nitrogen source, a high titer of pyruvic acid was obtained. The pyruvic acid production reached 63.4 g/L with a yield of 0.59 g/g in a 5 L bioreactor. After scale-up to a 50 L bioreactor using the fermented spent yeast cell dry powder as a seed nitrogen source, 65.1 g/L of pyruvic acid was harvested, with a yield of 0.61 g/g. This study proposes a promising
approach for increasing the pyruvic acid titer and reducing the costs.
Bacterial consortium is an important source of lignocellulolytic strains, but it is still a challenge to distinguish the direct decomposers of lignocellulose from other bacteria in such a complex community. This study aims at addressing this issue by focusing on the dynamic changes in community structure and degradation activity of MMBC-1, an established and stable lignocellulolytic bacterial consortium, during its subculturing revival. MMBC-1 was cryopreserved with glycerol as a protective agent and then inoculated for revival. Its enzyme activities for degradation recovered to the maximum level after two rounds of subculturing. Correspondingly, the cellulose and hemicellulose in lignocellulosic carbon source were gradually decomposed during the revival. Meanwhile, the initial dominant bacteria represented by genus Clostridium were replaced by the bacteria belonging to Lachnospira, Enterococcus, Bacillus, Haloimpatiens genera and family Lachnospiraceae. However, only three high-abundance (> 1%) operational taxonomic units (OTUs) (Lachnospira, Enterococcus and Haloimpatiens genera) were suggested to directly engage in lignocellulose degradation according to correlation analysis. By comparison, many low-abundance OTUs, such as the ones belonging to Flavonifractor and Anaerotruncus genera, may play an important role in degradation. These findings showed the dramatic changes in community structure that occurred during the subculturing revival, and paved the way for the discovery of direct decomposers in a stable consortium.
Ilamycins E1/E2 are novel cyclic heptapeptides from Streptomyces atratus SCSIO ZH16, which have the MIC value of 9.8 nM against Mycobacterium tuberculosis H37Rv. However, the lower fermentative titer of ilamycins E1/E2 cut off further development for novel anti-TB lead drugs. In order to break the obstacle, the combinatorial strategy of medium optimization, fermentative parameters optimization, exogenous addition of metal ions, precursors, and surfactants was developed to promoted the production of ilamycins E1/E2. Addition of 1 mM ZnCl2 at 0 h, 1 g/L tyrosine at 96 h, and 2 g/L shikimic acid at 48 h increased the production of ilamycins E1/E2 from 13.51 to 762.50 ± 23.15, 721.39 ± 19.13, and 693.83 ± 16.86 mg/L, respectively. qRT-PCR results showed that the transcription levels of key genes in Embden–Meyerhof–Parnas pathway, hexose phosphate shunt pathway, and shikimic acid pathway were upregulated. In addition, the production of ilamycins E1/E2 reached 790.34 mg/L in a 5-L bioreactor by combinatorial strategy. Combinatorial strategies were used for improving ilamycins E1/E2 production in S. atratus ΔilaR and provided a sufficient basis on further clinic development.
In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography methods are labor intensive, time consuming and not compatible with real-time analysis. Here, we developed an inexpensive, high-throughput, and real-time detection method for the monitoring of in vitro RNA synthesis called iSpinach aptamer-based monitoring of Transcription Activity in Real-time (STAR). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the green fluorescent protein gene and the 3T4T variants demonstrated significantly enhanced transcription output, with at least 1.7-fold and 2.8-fold greater output than the wild-type DNA template and common transcription template, respectively. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA-related enzymes and new drugs.
The fungus Trichoderma reesei is a powerful host for secreted production of proteins. The promoter of cdna1 gene, which encodes a small basic protein of unknown function and high expression, is commonly used for constitutive protein production in T. reesei. Nevertheless, the production level of proteins driven by this promoter still needs to be improved. Here, we identified that the region 600- to 700-bp upstream of the start codon is critical for the efficiency of the cdna1 promoter. Increasing the copy number of this region to three improved the production of a heterologous β-mannanase by 37.5%. Screening of several stressful conditions revealed that the cdna1 promoter is heat inducible. Cultivation at 37 °C significantly enhanced the production of β-mannanase as well as a polygalacturonase with the cdna1 promoter compared with those at 30 °C. Combing the strategies of promoter engineering, multi-copy gene insertion, and control of cultivation temperature, β-mannanase of 199.85 U/mL and relatively high purity was produced in shake flask, which was 6.6 times higher than that before optimization. Taken together, the results advance the understanding of the widely used cdna1 promoter and provide effective strategies for enhancing the production of recombinant proteins in T. reesei.
Recovery of bioactive compounds from biowaste is gaining more and more interest in circular economy models. The oilseed cakes are usually insufficiently exploited by most technologies since they represent valuable matrices abundant in proteins, minerals, and phytochemicals, but their use is mostly limited to feed ingredients, fertilizers or biofuel production. This study was thus focused on the exploration of new valorization pathways of oilseed cakes by subcritical water, representing a safe and economic alternative in the creation of value chains. Pumpkin, hemp, and flax seed cakes were treated with subcritical water in nitrogen and carbon-dioxide atmospheres, as well as in nitrogen atmosphere with the addition of acid catalyst. The degradation of carbohydrate fraction was studied by quantifying sugars and sugar degradation products in the obtained extracts. The extracts obtained under different conditions were further compared chemically with respect to total phenols and flavonoids, as well as to the content of individual phenolic compounds. Furthermore, the effects of subcritical water treatment conditions on antioxidant, antiradical and cytotoxic properties of thus obtained extracts were defined and discussed.
It is found that the osteon is composed of thin and thick lamellae which are periodic and approximately concentric, every 5 lamellae is a cycle, the periodic helix angle of mineralized collagen fibers in two adjacent sub-lamellae is 30°. Four bionic composite models with different fiber helix angles were established and fabricated according to the microstructure of mineralized collagen fibers in osteon. Based on the impact analysis of four kinds of bionic composite models, the effects of the fiber periodic spiral structure on the impact resistance and energy dissipation of multi-layer bionic composite were investigated. The analysis results show that the fiber helix angle affects the impact damage resistance and energy dissipation of multi-layer fiber reinforced composites. Among the 4 kinds of multi-layer composite models, the composite model with helix angle of 30° has better comprehensive ability to resist impact damage. The test results show that the impact damage area of the specimen with 30° helix angle is smallest among the 4 types of bionic specimens, which is consistent with the results of finite-element impact analysis. Furthermore, in the case of without impact damage, the smaller the fiber helix angle is, the more uniform the stress distribution is and more energy is dissipated in the impact process. The periodic spiral structure of mineralized collagen fibers in osteon are the result of natural selection of biological evolution. This structure can effectively improve the ability of cortical bone to resist external impact. The research results can provide useful guidance for the design and manufacture of high-performance and strong impact resistant bionic composites.
The main male hormone, testosterone is obtained from cheap and readily available phytosterol using the strains of Mycolicibacterium neoaurum VKM Ac-1815D, or Ac-1816D. During the first “oxidative” stage, phytosterol (5–10 g/L) was aerobically converted by Ac-1815D, or Ac-1816D to form 17-ketoandrostanes: androstenedione, or androstadienedione, respectively. At the same bioreactor, the 17-ketoandrostanes were further transformed to testosterone due to the presence of 17β-hydroxysteroid dehydrogenase activity in the strains (“reductive” mode). The conditions favorable for “oxidative” and “reductive” stages have been revealed to increase the final testosterone yield. Glucose supplement and microaerophilic conditions during the “reductive” mode ensured increased testosterone production by mycolicibacteria cells. Both strains effectively produced testosterone from phytosterol, but highest ever reported testosterone yield was achieved using M. neoaurum VKM Ac-1815D: 4.59 g/l testosterone was reached from 10 g/l phytosterol thus corresponding to the molar yield of over 66%. The results contribute to the knowledge on phytosterol bioconversion by mycolicibacteria, and are of significance for one-pot testosterone bioproduction from phytosterol bypassing the intermediate isolation of the 17-ketoandrostanes.
Collagen, the highest content protein in the body, has irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding the recombinant type III human-like collagen α1 chain fragment was integrated into P. pastoris genome after partial amino acids were substituted. Combined with promoter engineering and high-density fermentation technology, soluble secretory expression with the highest yield of 1.05 g L−1 was achieved using two-stage feeding method, and the purity could reach 96% after affinity purification. The determination of N/C-terminal protein sequence were consistent with the theoretical expectation and showed the characteristics of Gly-X-Y repeated short peptide sequence. In amino acid analysis, glycine shared 27.02% and proline 23.92%, which were in accordance with the characteristics of collagen. Ultraviolet spectrum combined with Fourier transform infrared spectroscopy as well as mass spectrometry demonstrated that the target product conformed to the characteristics of collagen spectrums and existed as homologous dimer and trimer in the broth. This work provided a sustainable and economically viable source of the recombinant type III human-like collagen.
The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD520 of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD520 of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.
Collagen is a biofunctional protein that has been widely used in many fields, including but not limited to biomedical, cosmetics and skin care, food, and novel materials. Recombinant collagen has great potential as an alternative to collagen extracted from animals because it avoids the immune response, and the yield and properties are stable. However, challenges remain in the industrial application of recombinant collagen, including improving the expression yield, reducing the cost of purification for industry and expanding applications. In this study, a cloning and recombination method was used to heterologously express the recombinant human-like collagen (RHLC) in Pichia pastoris GS115 using the pPIC9k expression vector. The RHLC expression titre was 2.33 g/L via a 5-L fermenter, and the purification was completed within 48 h and was 98% pure. The characteristics of RHLC were investigated. Furthermore, potential applications for RHLC were explored, such as basal collagen sponge preparation, forming films with chitosan and production of collagen hydrolysed peptides. RHLC has various potential applications due to its triple helical structure, thermostability, good biocompatibility and film-forming ability.
Menaquinone-7 (MK-7) is a kind of vitamin K2 playing an important role in the treatment and prevention of cardiovascular disease, osteoporosis and arterial calcification. The purpose of this study is to establish an adaptive evolution strategy based on a chemical modulator to improve MK-7 biosynthesis in Bacillus natto. The inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), glyphosate, was chosen as the chemical modulator to perform the experiments. The final strain ALE-25–40, which was obtained after 40 cycles in 25 mmol/L glyphosate, showed a maximal MK-7 titer of 62 mg/L and MK-7 productivity of 0.42 mg/(L h), representing 2.5 and 3 times the original strain, respectively. Moreover, ALE-25–40 generated fewer spores and showed a higher NADH and redox potential. Furthermore, the mechanism related to the improved performance of ALE-25–40 was investigated by comparative transcriptomics analysis. Genes related to the sporation formation were down-regulated. In addition, several genes related to NADH formation were also up-regulated. This strategy proposed here may provide a new and alternative directive for the industrial production of vitamin K2.
1,3-Dihydroxyacetone (DHA) is a commercially important chemical and widely used in cosmetics, pharmaceuticals, and food industries as it prevents excessive water evaporation, and provides anti-ultraviolet radiation protection and antioxidant activity. Currently, the industrial production of DHA is based on a biotechnological synthetic route using Gluconobacter oxydans. However, achieving higher production requires more improvements in the synthetic process. In this study, we compared DHA synthesis levels in five industrial wild-type Gluconobacter strains, after which the G. oxydans WSH-003 strain was selected. Then, 16 dehydrogenase genes, unrelated to DHA synthesis, were individually knocked out, with one strain significantly enhancing DHA production, reaching 89.49 g L−1 and 42.27% higher than the wild-type strain. By optimizing the culture media, including seed culture and fermentation media, DHA production was further enhanced. Finally, using an established fed-batch fermentation system, DHA production reached 198.81 g L−1 in a 5 L bioreactor, with a glycerol conversion rate of 82.84%.
In recent years, the need for clean, viable and sustainable source of alternative fuel is on the rampage in the global space due to the challenges posed by human factors including fossil induced emissions, fuel shortage and its ever-rising prices. These challenges are the major reason to utilize alternative source of energy such as lignocellulosic biomass as domestic and industrial feedstock. However, biomass in their raw form is problematic for application, hence, a dire need for torrefaction pre-treatment is required. The torrefaction option could ameliorate biomass limitations such as low heating value, high volatile matter, low bulk density, hygroscopic and combustion behaviour, low energy density and its fibrous nature. The torrefied product in powder form could cause air pollution and make utilization, handling, transportation, and storage challenging, hence, densification into product of higher density briquettes. This paper therefore provides an overview on the performance of torrefied briquettes from agricultural wastes. The review discusses biomass and their constituents, torrefaction pre-treatment, briquetting of torrefied biomass, the parameters influencing the quality, behaviour and applications of torrefied briquettes, and way forward in the briquetting sector in the developing world.
Neomycin, an aminoglycoside antibiotic with broad-spectrum antibacterial resistance, is widely used in pharmaceutical and agricultural fields. However, separation and purification of neomycin B as an active substance from Streptomyces fradiae are complicated. Although NeoN can catalyze conversion of neomycin C to neomycin B, the underlying catalytic mechanism is still unclear. In this study, the genomic information of high-yielding mutant S. fradiae SF-2 was elucidated using whole-genome sequencing. Subsequently, the mechanism of NeoN in catalyzing conversion of neomycin C to neomycin B was resolved based on NeoN–SAM–neomycin C ternary complex. Mutant NeoNV252A showed improved NeoN activity, and the recombinant strain SF-2-NeoNV252A accumulated 16,766.6 U/mL neomycin B, with a decrease in neomycin C ratio from 16.1% to 6.28%, when compared with the parental strain SF-2. In summary, this study analyzed the catalytic mechanism of NeoN, providing significant reference for rational design of NeoN to improve neomycin B production and weaken the proportion of neomycin C.
Environmental problems such as greenhouse effect, the consumption of fossil energy, and the increase of human demand for energy are becoming more and more serious, which force researcher to turn their attention to the reduction of CO2 and the development of renewable energy. Unsafety, easy to lead to secondary environmental pollution, cost inefficiency, and other problems limit the development of conventional CO2 capture technology. In recent years, many microorganisms have attracted much attention to capture CO2 and synthesize valuable products directly. Fatty acid derivatives (e.g., fatty acid esters, fatty alcohols, and aliphatic hydrocarbons), which can be used as a kind of environmentally friendly and renewable biofuels, are sustainable substitutes for fossil energy. In this review, conventional CO2 capture techniques pathways, microbial CO2 concentration mechanisms and fixation pathways were introduced. Then, the metabolic pathway and progress of direct production of fatty acid derivatives from CO2 in microbial cell factories were discussed. The synthetic biology means used to design engineering microorganisms and optimize their metabolic pathways were depicted, with final discussion on the potential of optoelectronic–microbial integrated capture and production systems.
The objective of the present study was an optimization of operating parameters and the performance of the methanogenesis reactor in phased anaerobic digestion (AD) of slaughterhouse wastewater at 37.5°C. Accordingly, the feedstock of the methanogenic reactor was effluent from the hydrolytic-acidogenic reactor operating at HRT of 3-days and OLR of 1789 mg/L. The methanogenesis phase was also investigated at different hydraulic retention time (HRT) values ranging from 12 to 3 days at 3-day intervals, and organic loading rates (OLR) of 149, 199, 298, and 596 mg of COD/L. The methanogenesis reactor effluent concentrations of TN, TP, PO4 − 3, SO4 − 2, and S2 − 2 were ranging between 424–464, 83–117, 63–86, 130–197, and 0.98–1.02 mg/L, respectively. The removal efficiencies of TN and TP were vary from 10–17% to 17–21%, respectively. The average biogas production was 125 ± 16, 150 ± 10, 185 ± 4, and 154 ± 17 mL at HRT of 12, 9, 6, and 3 days, respectively. Methane quality (%) and yield (mg/L of COD) were 55–67% and 0.02–0.03, respectively. Furthermore, the average stability indicator parameter values of (total volatile fatty acid (TVFA) = 520 ± 19 mg/L, total alkalinity (TotA) = 1424 ± 10 mg/L, TVFA:TotA. Ratio = 0.36, salinity = 1172 mg/L, pH = 6.92) and performance indicator parameters removal efficiency (RE) for (chemical oxygen demand (COD) = 81%, volatile solid (VS) RE = 95%, biogas production = 185 ± 4 mL, methane yield = 0.03 per mg COD consumed) were achieved at HRT of 6 days and OLR of 298 mg of COD/L. Low removal efficiencies of TP and TN at all HRT/OLR were observed for the methanogenic reactor signifying further treatment system.
As a promising industrial microorganism, methylotroph is capable of using methane or methanol as the sole carbon source natively, which has been utilized in the biosynthesis of various bioproducts. However, the relatively low efficiency of carbon conversion has become a limiting factor throughout the development of methanotrophic cell factories due to the unclear genetic background. To better highlight their advantages in methane or methanol-based biomanufacturing, some metabolic engineering strategies, including upstream transcription regulation projects, are being popularized in methylotrophs. In this review, several strategies of transcription regulations applied in methylotrophs are summarized and their applications are discussed and prospected.
Ultraviolet rays in sunlight can cause skin damage and premature aging. This study demonstrates that Lactobacillus reuteri SJ-47 strain exopolysaccharides (EPS) protect human skin fibroblasts (HSF) under UVA radiation. During the course of the experiments, we investigate the oxidative stress protection and antiaging effects of exopolysaccharides on HSF at the biochemical, cellular, and molecular levels. The results show that EPS can increase the antioxidant capacity of cells, decrease the amount of reactive-oxygen species (ROS) and malondialdehyde (MDA), while improve the expression of antioxidant enzymes. At the same time, EPS can increase collagen content, which can effectively regulate the expression of genes in the senescence and apoptosis pathways, and delay skin photoaging caused by UVA irradiation.
As a widely consumed fermented milk product, yogurt undergoes constant development to increase its functional properties. Monascus purpureus-fermented durian seed, which has been proven to possess antioxidative properties, has the potential to improve yogurt properties. This study aimed to analyze the use of Monascus-fermented durian seed (MFDS) as a functional ingredient in yogurt and its effect on physicochemical properties, lactic acid bacteria (LAB) count, antioxidative properties, and consumer acceptability of set-type yogurt during refrigeration. Changes in physicochemical properties, including color, pH, titratable acidity, syneresis, LAB count, total phenolic content (TPC), and antioxidant activity were evaluated at 7-day intervals during 14 days of refrigerated storage (4 °C). Sensory evaluations were carried out for freshly made samples after 7 days of storage. The results showed that the addition of MFDS to yogurt gave significant effects on some of the parameters measured. Yogurt with added MFDS powder produced a more red color (L = 88.55 ± 1.28, a* = 2.63 ± 0.17, b* = 11.45 ± 1.15, c = 11.75 ± 1.15, H = 77.00 ± 0.64), reached the highest TPC (2.21 ± 0.46 mg/GAE g), antioxidant activity (0.0125 ± 0.0032 mg GAE/g), and syneresis (5.24 ± 0.51%) throughout 14 days of storage. The addition of MFDS only gave a slight difference to pH and titratable acidity, while no significant difference was made for LAB count. For sensory evaluation, the addition of MFDS, particularly the ethanol extract, to yogurt was well-liked by panelists. Citrinin content in MFDS yogurt can be decreased under the limits set. Overall, the addition of MFDS has a high potential of improving yogurt properties, particularly its antioxidative properties.
Modified xanthan produced by xanthan lyase has broad application prospects in the food industry. However, the catalytic performance of xanthan lyase still needs to be improved through rational design. To address this problem, in this work, the glycosylation and its influences on the catalytic performance of a xanthan lyase (EcXly), which was heterologously expressed in Escherichia coli, were reported. Liquid chromatography coupled to tandem mass spectrometry analysis revealed that the N599 site of EcXly was modified by a single N-glycan chain. Based on sequence alignment and three-dimensional structure prediction, it could be deduced that the N599 site was located in the catalytic domain of EcXly and in close proximity to the catalytic residues. After site-directed mutagenesis of N599 with alanine, aspartic acid and glycine, respectively, the EcXly and its mutants were characterized and compared. The results demonstrated that elimination of the N-glycosylation had diminished the specific activity, pH stability, and substrate affinity of EcXly. Fluorescence spectra further revealed that the glycosylation could significantly affect the overall tertiary structure of EcXly. Therefore, in prokaryotic hosts, the N-glycosylation could influence the catalytic performance of the enzyme by changing its structure. To the best of our knowledge, this is the first report about the post-translational modification of xanthan lyase in prokaryotes. Overall, our work enriched research on the role of glycan chains in the functional performance of proteins expressed in prokaryotes and should be valuable for the rational design of xanthan lyase to produce modified xanthan for industrial application.
Marine microalgae have received much attention as a sustainable source of the two health beneficial omega-3-fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, photoautotrophic monocultures of microalgae can only produce either DHA or EPA enriched biomass. An alternative may be the photoautotrophic co-cultivation of Tisochrysis lutea as DHA-producer with Microchloropsis salina for simultaneous EPA production to obtain EPA- and DHA-rich microalgae biomass in a nutritionally balanced ratio. Photoautotrophic co-cultivation processes of T. lutea and M. salina were studied, applying scalable and fully controlled lab-scale gas-lift flat-plate photobioreactors with LED illumination for dynamic climate simulation of a repeated sunny summer day in Australia [day–night cycles of incident light (PAR) and temperature]. Monocultures of both marine microalgae were used as reference batch processes. Differences in the autofluorescence of both microalgae enabled the individual measurement, of cell distributions in co-culture, by flow cytometry. The co-cultivation of T. lutea and M. salina in artificial sea water with an inoculation ratio of 1:3 resulted in a balanced biomass production of both microalgae simultaneously with a DHA:EPA ratio of almost 1:1 (26 mgDHA gCDW −1, and 23 mgEPA gCDW −1, respectively) at harvest after depletion of the initially added fertilizer. Surprisingly, more microalgae biomass was produced within 8 days in co-cultivation with an increase in the cell dry weight (CDW) concentration by 31%, compared to the monocultures with the same amount of light and fertilizer. What is more, DHA-content of the microalgae biomass was enhanced by 33% in the co-culture, whereas EPA-content remained unchanged compared to the monocultures.
This work aimed to investigate the effect of pyrolysis temperature on the yield and properties of biochars synthesized from herbaceous and woody plants. Four typical materials, including two herbaceous plants (rice straw, corn straw) and two woody plants (camellia oleifera shells, garden waste), were used in the experiments under five operating temperatures (from 300 °C to 700 °C, with an interval of 100 °C). The results showed biochar derived from herbaceous plants had a significantly higher pH (from 7.68 to 11.29 for RS), electrical conductivity (EC, from 6.5 Ms cm−1 to 13.2 mS cm−1 for RS), cation exchange conductivity (CEC, from 27.81 cmol kg−1 to 21.69 cmol kg−1 for RS), and ash content (from 21.79% to 32.71% for RS) than the biochar from woody plants, but the volatile matter (VM, from 42.23% to 11.77% for OT) and specific surface area (BET, from 2.88 m2 g−1 to 301.67 m2 g−1 for OT) in the woody plant-derived biochar were higher. Except for CEC and VM, all the previously referred physicochemical characteristics in the as-prepared biochars increased with the increasing pyrolysis temperature, the H/C and O/C values of herbaceous and woody plant-derived biochar were lower than 0.9 and 0.3, respectively, confirming their potential as the material for carbon sequestration. The results revealed that biochar made from herbaceous plants was more suitable for acidic soil amendments. In contrast, woody plant-derived biochar were recommended to remove heavy metals in environmental remediation and water treatment.
