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.

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.

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.

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 (k_cat/K_m, 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.

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.

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 (k_cat/K_M) 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 (k_cat/K_m) 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.

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 $^{\circ }$ C, 30 $^{\circ }$ C showed slowest rate of kinetics reaction), whereas the semi-industrial scale had 50 $^{\circ }$ C of optimal temperature (30, 40, 60 $^{\circ }$ C had the same kinetics). It also was found that maintaining the pH value of the reaction mixture was not mandatory. The obtained results indicate the need to refine the process conditions using semi-industrial experiments before attracting industrial-scale resources. In the case of selection of conditions for the hydrolysis of soy protein isolate in production, it is necessary first of all to take into account the reaction temperature as the most irreproducible parameter when scaling.

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 (M_w/M_n, 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.

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.

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.