Ansamitocin P-3 (AP-3), produced by Actinosynnema pretiosum, has extraordinary antitumor activity and has been used as toxic “warhead” in antibody-conjugated drugs. However, AP-3 production is limited by its low yield and thus the high cost of fermentation. In this study, we aimed to reduce fermentation costs by utilizing the low-cost substrates and improve the AP-3 production, simultaneously.
In this study, low-cost substrates used for ansamitocins production were selected via single-factor experiments and then the concentrations of these suitable carbon and nitrogen sources were optimized by response surface method (RSM). Cane molasses, glycerol, and cold-pressed soybean powder were determined to be the most suitable economical carbon and nitrogen sources for AP-3 accumulation. The AP-3 titer in shaking flasks reached 111.9 mg/L using the optimized medium containing cane molasses (63.22 g/L), glycerol (22.91 g/L), and cold-pressed soybean powder (3.29 g/L). In addition, appropriate supplementation with isobutanol, soybean oil, and vitamin B1 resulted in a marked enhancement in AP-3 production, to 141 mg/L. Meanwhile, the cost of this optimized fermentation medium was around 50% lower than those of two other high-yielding media.
Our findings provided practical and economically competitive cultivation conditions for the production of a value-added antitumor agent and may thus extend the applications of ansamitocin P-3.
Discovery of deoxyribonucleic acid (DNA) solved out the mystery of cellular functioning and abnormality in cellular metabolism. Understanding the coding of DNA resulted in enormous medical growth and helps the researchers in finding the genes which trigger major chronic diseases in humans. Further, the structural and sequential analysis brought humans into a new era of medical industry. The advancement in understanding of DNA could be a boon for agricultural sector as it allowed the farmers/breeders to choose better varieties with disease resistant features. In developing nations where the staple foods suffers with micronutrient deficiencies and stress conditions, DNA modifications and repair mechanism could solve out their problems. The role of DNA damaging factors and repair mechanism plays a crucial role in occurrence of certain disorders. Extracts prepared from various natural resources could either stop or slow down the process of DNA damage. This will help to eradicate major disorders related to DNA from human race. Further on the basis of type and dose of natural extracts, it would ease the planning of diets for patients suffering from chronic disorders.
Biosynthesis of secondary metabolites in actinobacteria is regulated by complex regulatory mechanisms on responding environmental changes. In this study, we have identified a two-component system (TCS) designated as RimA1A2 in the genome of Streptomyces rimosus M4018, with high homology to the TCS RapA1A2 from Streptomyces coelicolor, known for its positive regulatory function towards actinorhodin (ACT) biosynthesis. Using RT-PCR analysis, we demonstrate that rimA1 encodes response regulator (RR) and rimA2 encoding histidine kinase (HK) from S. rimosus that are co-transcribed as a single-polycistronic mRNA. When S. rimosus was cultivated on standard media, no significant difference in culture growth or morphological properties was observed between the rimA1-disrupted mutant and parent strain M4018. However, the rimA1-disrupted strain displayed significant increase in oxytetracycline (OTC) titer when cultivated in minimal medium (MM) containing glycine as sole nitrogen source, and the transcription of selected genes involved in OTC biosynthesis was increased, supporting the hypothesis that RimA1A2 has a negative regulatory role in OTC biosynthesis via global regulation. We observed an increased resistance of the rimA1-disrupted mutant strain to selected antibiotics. Interestingly, in the rimA1-disrupted strain, OTC biosynthesis was affected under different environmental stress conditions such as osmotic and oxidative stress. Accordingly, this phenotype was observed in a medium-dependent manner. Considering complexity of regulatory networks in antibiotic-producing organisms, this study demonstrates the importance of cultivation conditions, which is often neglected.
Aspergillus niger, as an important industrial strain, is widely used in the production of a variety of organic acids and industrial enzymes. To excavate the greater potential of A. niger as a cell factory, the development of highly efficient genome editing techniques is crucial. Here, we developed a modified CRISPR/Cas9 system for A. niger highlighted in two aspects: (1) construction of a single and easy-to-use CRISPR/Cas9 tool plasmid derived from pAN7-1 which is widely used in filamentous fungi; (2) redesign of the easy-to-switch “ribozyme–gRNA–ribozyme (RGR)” element in the tool plasmid. We examined the gene inactivation efficiency without repair fragment and the gene replacement efficiency with repair fragment utilizing the modified system, respectively, and both of them reach the efficiency as high as over 90%. Especially, the co-transformation of the tool plasmid and the specific repair fragment can easily realize one-step knock-out/knock-in of target genes, even with the length of homologous arms as only 100 bp. The establishment of this system will lay a solid foundation for the gene function research and rational design of cell factory in A. niger or broader filamentous fungi hosts.
In this research, different active phytochemical constituents present in Cleome heratensis (C. heratensis) from Capparaceae family were investigated. Moreover, the fatty acids present in the seed and aerial parts of the plant were identified by gas chromatography (GC) after esterification of the oil. Antioxidant activity of the aerial parts and seed of C. heratensis methanolic extract over 2,2′-diphenylpicryl-1-hydrazyl (DPPH) was investigated using ultraviolet–visible (UV–Vis) spectrophotometer.
To study total phenolic compounds and flavonoids, the plant was extracted from ethanol by ultrasonic method, then further extracted with other solvents. Amounts of anthocyanins and tannins/condensed tannins were determined by their corresponding ethanolic and acetone extracts. Antioxidant activity of the plant species was studied by a spectrophotometric method using 80% methanolic extract.
The high content of phenolics as 16.915 mg tannic acid equivalents per gram of dry matter (TAE/g DM), tannins (12.231 mg TA/gr DM) and condensed tannins (4.086 mg TA/g DM) was obtained for the C. heratensis extract. The most flavonoids content was found 4.444 rutin equivalents (in mg) per gram of dry matter (mg RE/g DM) in plant’s aerial extract. The most amount of anthocyanin (0.48 mmol/gr WM) was observed in flowering stage. Antioxidant activity of the aerial parts and seed of C. heratensis methanolic extract were 11.92 and 63.54 mg/mL IC50, respectively.
High level of phenolic components including flavonoids, proanthocyanidins and tannins was detected in the extract of aerial parts of the plant. The oil of seed of this plant is a rich source of saturated and unsaturated fatty acids. Finally, C. heratensis aerial part extract was found as an excellent natural antioxidant.
Terpenoids are a group of largest natural products with important biological functions, and their efficient biosynthesis is of particular importance to both academia and industry. As the building blocks for terpenoid biosynthesis, a suitable supply of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) is extremely crucial for efficient terpenoid biosynthesis. With this focus, we first introduce biosynthetic pathways of IPP and DMAPP, and then summarize the current strategies adopted for manipulating IPP and DMAPP supply. At last, how to further manage IPP and DMAPP supply to improve terpenoid biosynthesis is also proposed.
By cultivating a strain of Aspergillus tubingensis on agro-industrial by-products using solid-state fermentation technology, a biocatalyst containing more than 130 different enzymes was obtained. The enzymatic complex was composed mainly of hydrolases, among which a protease, an aspergillopepsin, accounted for more than half of the total proteins. Cell-wall-degrading enzymes such as pectinases, cellulases and hemicellulases were also highly represented. Adding the biocatalyst to corn mash at 1 kg/T corn allowed to significantly improve ethanol production performances. The final ethanol concentration was increased by 6.8% and the kinetics was accelerated by 14 h. The aim of this study was to identify the enzymes implicated in the effect on corn ethanol production. By fractionating the biocatalyst, the particular effect of the major enzymes was investigated. Experiments revealed that, together, the protease and two cellulolytic enzymes (an endoglucanase and a β-glucosidase) were responsible for 80% of the overall effect of the biocatalyst. Nevertheless, the crude extract of the biocatalyst showed greater impact than the combination of up to seven purified enzymes, demonstrating the complementary enzymatic complex obtained by solid-state fermentation. This technology could, therefore, be a relevant natural alternative to the use of GMO-derived enzymes in the ethanol industry.
Modern seafood processing practices result in amassment of a large volume of waste products, i.e., skin, head, tails, shells, scales, backbones, etc. These waste products may often encompass several high-value products which are still untapped due to the dearth of appropriate management. Moreover, inadequate disposal of waste also has negative implications on both environment and human health. This seafood waste often contains a huge amount of chitin, a polysaccharide that exhibits exceptional inherent characteristics including biocompatibility, biodegradability, antimicrobial, antitumor and antioxidant activities. The present review summarizes the existing methods for recovery of chitin and its derivatives from marine waste. The preparation of chitin nanoparticles was discussed along with blending of chitin and chitosan with other biopolymers. The recent trends of the application of chitin and chitosan nanostructures in various sectors were explored. This review is an attempt to highlight the extraction methods of chitin and chitosan from marine waste resources and its transformation into valuable commercial products as a solution to waste management.
(R)- or (S)-2-Hydroxy-4-(methylthio)butanoic acid (HMTBA) is used as a poultry nutritional supplement and to treat renal failure disease. Herein, we report an artificially designed biocatalytic cascade process, which uses l-methionine to synthesize (R)- or (S)-HMTBA. This biocatalysis cascade comprises a basic module and two different extender modules and operates in a modular assembly manner. The basic module responsible for the transformation of l-methionine to α-keto-γ-methylthiobutyric acid (KMTB) is comprised of the l-amino acid deaminase. Two different extender modules responsible for the transformation of KMTB to (R)- or (S)-HMTBA are comprised of the R/S-specific lactate dehydrogenase in combination with the formate dehydrogenase, respectively. Engineered Escherichia coli catalysts, one containing the basic module, the other containing the one of two different extender modules, produced 97.6 g L−1 (R)-HMTBA and 96.4 g L−1 (S)-HMTBA with a yield of 96.9% and 95.8% at the large scale (1 L) using a two-stage strategy in one pot, respectively. Therefore, this biocatalytic process lays the foundation for the industrial-scale conversion of low-cost l-amino acids to corresponding high-value enantiopure chiral 2-hydroxy acids.
Electrochemically active bacteria (EAB) receive considerable attention in sustainable biotechnology, since they are essential components in microbial fuel cells (MFCs) that are able to generate electricity from biomass wastes. EAB are also expected to be applied to the production of valued chemicals in microbial electrosynthesis systems (MESs) with the supply of electric energy from electrodes. It is, therefore, important to deepen our understanding of EAB in terms of their physiology, genetics and genomics. Knowledge obtained in these studies will facilitate the engineering of EAB for developing more efficient biotechnology processes. In this article, we summarize current knowledge on Shewanella oneidensis MR-1, a representative EAB extensively studied in the laboratory. Studies have shown that catabolic activities of S. oneidensis MR-1 are well tuned for efficiently conserving energy under varied growth conditions, e.g., different electrode potentials, which would, however, in some cases, hamper its application to biotechnology processes. We suggest that understanding of molecular mechanisms underlying environmental sensing and catabolic regulation in EAB facilitates their biotechnological applications.
As a natural antibacterial cationic peptide, ε-poly-l-lysine (ε-PL) is applied as a food preservative. However, the mechanism of ε-PL against Staphylococcus aureus (S. aureus) has not been elucidated. Especially, its antimicrobial mechanism at the metabolomics has not been yet thoroughly described.
This work aimed at clarifying the antibacterial activity and mechanism of ε-PL against S. aureus. Effects of ε-PL with different concentration on cell morphology, cell wall, and membrane integrity were investigated. Furthermore, the effect of ε-PL on metabolite properties of S. aureus was also studied. The results revealed that ε-PL disrupted the cell wall and membrane integrity of treated cells. ε-PL induced the structural change of peptidoglycan in cell wall, causing cell wall more fragile. Meanwhile, the permeability of the S. aureus cell membrane was increased by ε-PL. More importantly, ε-PL with different concentration could cause different effects on metabolic pathways of S. aureus. ε-PL with high concentration could directly restrain the central carbon metabolism. However, ε-PL with low concentration could only inhibit the glycolytic pathway.
These results showed that the antimicrobial mechanism of ε-PL against S. aureus was a synergistic action.
The FGF/MEK/ERK and Wnt/β-catenin signaling pathways have previously been proved to regulate mouse embryonic stem cell (mESCs) function. However, the relationships between these two pathways, especially their different functions on proliferation and pluripotency of mESCs, were rarely mentioned. Here, we investigated the effects of FGF/MEK/ERK and Wnt/β-catenin pathway regulators and their combinations on the proliferation and pluripotency of mESCs under serum- and feeder-free conditions. We found that MEK inhibitor PD0325901 and FGFR inhibitor SU5402 has paradoxical function on mESCs; one could promote proliferation along with differentiation and the other one could improve pluripotency while impairing cell proliferation. The combination of these two kinds of inhibitors could better regulate FGF/MEK/ERK pathway. Wnt/β-catenin pathway regulators SB216763 led to differentiation while promoting proliferation of mESCs. When we used FGF/MEK/ERK and Wnt/β-catenin pathway regulators in combination, the total expansion fold of mESCs reached 318.78 ± 47.95 and the proportion of SSEA-1-positive cells reached 82.40 ± 2.74% which were significantly higher than using the regulators alone. This finding indicates that regulators of FGF/MEK/ERK and Wnt/β-catenin pathways play different roles in the regulatory networks of mESCs. Their combination can better maintain the undifferentiated state and promote the proliferation of mESCs under serum- and feeder-free conditions.
Biomethane is an environment-friendly, economic, and alternative energy resource for a clean and green future. In the present study, we have evaluated the biomethanation potential of acetate-utilizing methanogenic culture (AUMC) and gelatin-enriched mixed culture (GEMC) with Clostridium acetobutylicum NCIM 2841 (GEMC-CA.) on gelatin as a sole carbon and nitrogen source.
We conducted experiments for examining the specific-methanogenic activity of these cultures in the metabolic assay media containing 1% gelatin. The produced methane and consumed gelatin were quantified by standard experimental methods. Exchange metabolites produced by these cultures were qualitatively analyzed by mass spectrometry.
Results of our study show that the growth-associated amino acid catabolism partially or completely supported the methanogenesis of these defined cultures. AUMC and GEMC found to be suitable for enhanced methanogenic activity on gelatin but a rapid degradation of amino acid was attributed by GEMC-CA. The ammonia released from these cultures was directly proportional to gelatin degradation. Mass spectral data analysis identified some key exchange metabolites from acidogenic culture and methanogenic culture for confirming the growth-associated methanogenesis.
The biomethanation potential of these cultures on gelatin is coupled with the Stickland reactions-directed methanogenesis in a syntrophic manner. The present study provides the importance for the development of a starter culture for the biomethanation of protein-based industrial wastes in effective ways.
Cell surface display system allows for endowing functional proteins expressed on bacterial surface by fusing different anchor proteins. Among PgsA, Blc, and Omp anchor, the antigen 43 (Ag43)-mediated surface display is a novel system in Escherichia coli. Here, we have demonstrated the red fluorescent protein (RFP) and cellulase (EC 3.2.1.4) on the cell surfaces at two different fusion sites in Ag43.
We introduced two fusion sites which are unstructured domain (52–138 aa) and autochaperone domain (600–700 aa) at N-terminal for passenger proteins. As a result, the surface-displayed RFP expressed in plasmid pET28a, but the intracellular RFP expressed more than the surface-displayed RFP. Improved display efficiency of Ag43 was present when fusing at the site of the 138th amino acid (aa) compared to fusing at the site of the 700th aa. For endoglucanase, whole-cell surface-displayed Ag43-138-BsCel5 showed the highest specific activity which was 4.65-fold of BsCel5. Cell-displayed cellulase preserved residual activity ranging from 78% to 38% at temperatures from 55 °C to 80 °C, respectively.
This study is to demonstrate the novel surface display system of Ag43 in E. coli by targeting two different proteins RFP and BsCel5 that were successfully displayed on the cell surfaces at two different fusion sites. The Ag43 system displays surface heterologous proteins and is a potential whole-cell catalyst in the bioconversion of cellulose.
Biohydrogen technology has drawn much attention due to its many advantages. However, it is still necessary to screen much more strains with stronger hydrogen-producing capacity for future commercialization processes. In this paper, a biohydrogen-producing strain Enterobacter aerogenes EB-06 was isolated, identified, and named. It could convert glycerol to biohydrogen by microorganism fermentation. The effects of oxygen content, initial pH, initial glycerol concentration, and initial nitrogen source content on biohydrogen production process were investigated. The results have shown that biohydrogen generation was more favorable under anaerobic conditions. The optimum specific biohydrogen production rate (QH2) was obtained as 41.47 mmol H2/g DCW h at 40 g/L initial glycerol concentration. The optimum volume H2 yield (CH2) was 83.76 mmol H2/L at initial pH 7.0. It was found that nitrogen source content (0–4 g/L) could promote biohydrogen production and cell growth. The biohydrogen production of Enterobacter aerogenes EB-06 from glycerol was optimized by the orthogonal experimental design. The optimal yield coefficient of biohydrogen from glycerol fermentation (YH2/glycerol) of EB-06 was obtained as 1.07 mmol H2/ mol glycerol at 10 g/L initial glycerol concentration, initial pH 5.0, and initial C/N ratio 5/3.
Microorganisms generally encounter a fluctuating environment in their natural habitat and similar conditions also happen in large-scale bioreactors. In this work, the dynamic response of intracellular and extracellular metabolites of Aspergillus niger was investigated after sudden exposure to high and low excess glucose concentrations in chemostats. It was found that the steady-state pathway turnover time of the carbon flux through the central carbon metabolism (CCM) was PP pathway 50 s, EMP pathway 20 s, and TCA cycle 189 s, and an upper limit for individual metabolite concentrations in the CCM was estimated. Regardless of the glucose pulse size, little changes of amino acids levels were observed except for aspartate, which showed a significant decrease. The ATP paradox, known from other organisms, was also observed in the studied A. niger strain. However, a different response of the NAD+/NADH ratio to the glucose pulses was found in A. niger compared to previously published observations on Penicillium chrysogenum and Saccharomyces cerevisiae. These findings are valuable for better understanding A. niger culture performance in large-scale bioreactors.
A rational and high-performance high-throughput screening system effectively improves the efficiency of strain screening.
A rapid and efficient method was developed to detect carbon sources. And then by using glucose analog as screening pressure and two indexes for sophorolipids (SLs) production and glycerol consumption, a mutant of Candida bombicola L1.3 was successfully selected among 3000 mutants. It produces high SLs titer and co-utilizes glucose and glycerol simultaneously.
Compared to the wild-type strain, L1.3 exhibited 15.06% higher SLs titer and 35.69% higher glycerol consumption capacity in a 5-L bioreactor. We believe that L1.3 can potentially be used for the efficient industrial production of SLs with simple downstream processing for separating SLs and glycerol.
A thermostable alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) has been repurposed to perform asymmetric reduction of a series of prochiral ketones with the formation of enantio-pure secondary alcohols, which are crucial chiral synthons needed in the preparation of various pharmaceuticals. However, it is incapable of asymmetric reduction when applied to bulky ketones. Recently, mutations at two key residues A85 and I86 were shown to be crucial for reshaping the substrate binding pocket. Increased flexibility of the active site loop appears to be beneficial in the directed evolution of TbSADH towards difficult-to-reduce ketones.
Using the reported mutant A85G/I86A as template, double-code saturation mutagenesis (DCSM) was applied at selected residues lining the substrate binding pocket with a 2-membered reduced amino acid alphabet.
The mutant A85G/I86A was first tested for activity in the reaction of the model substrate (4-chlorophenyl)-(pyridin-2-yl)methanone, which showed a total turnover number (TTN) of 3071. In order to further improve the turnovers, a small and smart mutant library covering a set of mutations at Q101, W110, L294, and C295 was created. Eventually, a triple-mutant A85G/I86A/Q101A was identified to be a superior catalyst that gave S-selective product with 99% ee and 6555 TTN. Docking computations explain the source of enhanced activity. Some of the best variants are also excellent catalysts in the reduction of other difficult-to-reduce ketones.
Squalene-type triterpenoids (STs) are a class of linearized triterpenoids with significant bioactivities, including anti-cancer, anti-oxidative, and anti-inflammatory activities. The efficient biosynthesis of STs has gained increasing attention.
Using Saccharomyces cerevisiae as a heterologous host, we discovered that overexpression of CYP505D13 from Ganoderma lucidum, a famous medicinal mushroom capable of producing various triterpenoids as secondary metabolites, enables the engineered S. cerevisiae strain to produce two new STs, 4,8-dihydroxy-22,23-oxidosqualene (ST-1), 8-hydroxy-2,3;22,23-squalene dioxide (ST-2), and a known ST, 2,3; 22,23-squalene dioxide (ST-3), at the respective titers of 3.28 mg/L, 13.77 mg/L, and 12.23 mg/L after 59 h fermentation. Furthermore, our in vitro enzymatic assay implies that CYP505D13 is involved in the formation of ST-3.
This study provides a promising alternative to discover STs and facilitate their efficient bioproduction.
Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15.
After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in kcat/Km) and higher H2O2 tolerance (sevenfold improvement based on IC50).
To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4.
Prokaryotic argonaute proteins (pAgos) play an important role in host defense in vivo. Most importantly, the thermophilic pAgos with endonuclease activity hold great potential for programmable genetic manipulation. Therefore, exploring argonaute proteins with unique enzyme properties is desired for understanding their diverse catalytic mechanisms and promoting their applications in biotechnology.
The argonaute protein from archaeon Methanocaldococcus fervens (MfAgo) was cloned and overexpressed in Escherichia coli BL21 (DE3). The recombinant protein showed the expected molecular weight of ~ 85.8 kDa by SDS-PAGE. The activity assays demonstrate that MfAgo has cleavage activities toward single-stranded DNA (ssDNA) targets specifically at the site complementary to the position between nucleotides 10 and 11 of the guide strand. Interestingly, MfAgo utilizes small 5′-phosphorylated ssDNA (5′-P ssDNA), 5′-hydroxylated ssDNA (5′-OH ssDNA), and 5′-phosphorylated ssRNA (5′-P ssRNA) as the guides for catalysis. The optimal temperatures are highly dependent on the type of guide and have a range of 80–90 °C. The addition of 0.5 mM Mn2+, Mg2+ or Co2+ to the reaction system significantly enhanced the enzyme activity. Meanwhile, MfAgo is quite active at NaCl concentrations less than 500 mM. Furthermore, structural modeling analyses suggested that its unique wide guide-dependent activity might be related to differing multiple interactions between guides and the MID domain of MfAgo.
MfAgo shows efficient endonuclease activity for ssDNA cleavage. In contrast to most known pAgos, which recognize only one type of guide, MfAgo uses diverse guides, including 5′-P ssDNA, 5′-OH ssDNA, and 5′-P ssRNA, to specifically cleave targets. Characterization of MfAgo expands the understanding of catalysis in the Ago family and provides clues for future genetic manipulation.
Haloacid dehalogenase (HAD)-like hydrolases represent the largest superfamily of phosphatases, which release inorganic phosphate from phosphate containing compounds, such as sugar phosphates. The HAD-like phosphatases with highly substrate specificity, which perform irreversible dephosphorylation, are always integrated into in vitro synthetic enzymatic biosystems as the last enzymatic step for the cost-efficient production of biochemicals. Therefore, identification and characterization of substrate specificity of HAD-like phosphatases are important for exploring their application.
In this study, a hyperthermophilic HAD-like phosphatase from Archaeoglobus fulgidus (AfPase) was cloned, expressed, and characterized. AfPase was identified as a type I Mg2+-dependent HAD-like phosphatase with high optimal temperature and thermostability. Among the tested phosphate containing compounds, AfPase exhibited the highest catalytic activity on p-nitrophenyl phosphate, followed by dihydroxyacetone phosphate (DHAP). On the basis of the high catalytic activity of AfPase to generate 1,3-dihydroxyacetone (DHA) from DHAP, an in vitro synthetic enzymatic biosystem containing this phosphatase and other five enzymes was constructed for the biosynthesis of DHA from inexpensive maltodextrin in one pot. About 14 mM (1.26 g/L) DHA was produced from 10 g/L maltodextrin.
A hyperthermophilic HAD-like phosphatase from Archaeoglobus fulgidus was characterized carefully, and the success of an in vitro synthetic enzymatic biosystem containing this phosphatase provided a promising approach for DHA production from maltodextrin.
Feverfew (Tanacetum parthenium) is one of the most important medicinal plants with different pharmacologic properties, such as anti-inflammatory, cardiotonic, antitumor and antiangiogenic activities. Parthenolide (PN) is a main bioactive molecule in feverfew which belongs to sesquiterpene lactone compounds. Currently, the plant cell suspension has been used as a useful method to produce secondary metabolites (SMs) components. Meanwhile, the elicitor application is an effective strategy to induce the production of SMs in plants. The present study was conducted as two different experiments in cell suspension of feverfew. In the first experiment, the effects of explant (shoot and root), hormone (TDZ + NAA and TDZ + 2. 4-D) on cell dry weight for one month were investigated. In the second experiment, the effect of elicitor (namely, MJ, YE and Ag+) and the hormones after 24, 48 and 72 h on PN content was assessed. The result of the first experiment revealed that the simple effects and the interaction of hormone × explant were significant (P < 0.01) for cell dry weight. Growth rate analysis showed that shoot-derived cell suspension in 1 mg L−1 NAA + 0.5 mg L−1 TDZ treatment had the highest amount of cell dry weight 14 days after the culture. According to the second experiment, the highest PN content was obtained in cell suspension containing 0.5 mg L−1 2, 4-D + 0.1 mg L−1 TDZ with application of the YE + MJ elicitor after 48 h. The cell suspension treatment with each of the elicitors had a positive effect on the PN production. In conclusion, the application of combined elicitors in feverfew cell suspension culture can be used as an efficient tool for large-scale PN production.
The efficient removal of toxic inhibitors from pretreated lignocellulose biomass is crucially important for consequent cellulosic ethanol fermentation. A. resinae ZN1 biodetoxifies all toxic inhibitors at the neutral pH of 4–6, and the neutralization of acid catalyst in the pretreated lignocellulose is required. However, aqueous alkaline solutions such as sodium hydroxide solution and calcium hydroxide slurry are used which generate several difficulties.
In this study, a dry biodetoxification method was investigated using dry calcium carbonate (CaCO3) powder as an acid-neutralizing reagent to avoid the use of an aqueous alkaline solution. Dry biodetoxification provides a mild and stable pH without the generation of phenolic compounds. The acid pretreated and dry biodetoxified wheat straw was used as the feedstock of ethanol fermentation and the same performance with the wet biodetoxification using aqueous Ca(OH)2 slurry. The 72 g/L or 9.1% (v/v) of ethanol produced from wheat straw was very close to that of ethanol from corn starch.
Dry biodetoxification provided a practical method to simplify the process of conventional wet biodetoxification to reduce the time, cost and labor.
Xylanases have been successfully used in food, paper, and pulp industries and are considered to be a key player in the biodegradation of xylan to valuable end products. However, most of the natural xylanases present poor activity in high-temperature and high-alkali environment. Therefore, it is necessary to modify the enzymes to meet the increasing demands of industries.
Directed evolution was used to improve the specific activity and pH stability of the xylanase (XynHBN188A) that originated from Bacillus pumilus HBP8. The xylanase XynHBN188A was mutated by error-prone PCR. The mutant, XynHBN188A217, was screened from the mutant library by functional screening. The specific activity of XynHBN188A217 was 3986.7 U/mg, which was 2.8-fold higher than that of wild type. The optimum temperature of XynHBN188A and XynHBN188A217 was 50 °C and 55 °C, respectively. The optimum pH of XynHBN188A and XynHBN188A217 was pH 8.0 and pH 7.5, respectively. The half-life at 60 °C of XynHBN188A217 was 20 min. Moreover, the pH stability of XynHBN188A217 was significantly better than that of XynHBN188A. Finally, homology models and molecular docking were used to identify the location of mutation sites and to explore the mechanism of the improved properties.
The xylanase XynHBN188A has been improved in the specific activity and pH stability by directed evolution. Also, the enlarged catalytic channel of mutant is beneficial for the substrates access and products release. It may contribute to the improved activity. The mutant XynHBN188A217 will be a potential candidate to be used for industrial application.
Fruits and vegetables, a significant segment of food sector, generate large volume of wastes annually. They constitute an excellent source of several valuable components (carotenoids, polyphenols, etc.), also known as bioactive compounds. These bioactive compounds have a positive impact on health and are known to modulate the metabolic processes as well as influence the cellular activities in the human health due to their antioxidant, anti-cancer, anti-inflammation, anti-allergenic and anti-atherogenic properties; depending upon the pathway and their bioavailability in the body. Despite this, some of these compounds are hydrophobic in nature and therefore are less bioavailable in the body. However, with the technological advancements like nanoemulsions, their solubility, stability and functional properties can be enhanced. This review provides the comprehensive information about the green extraction techniques and innovative delivery system such as nanoemulsions for bioactive compounds generated from fruits and vegetables waste.
Microalgal biofilm-based technologies are of strong interest due to their high biomass concentrations and ability to remove nutrients from wastewater, utilize CO2 and produce secondary valuable products. This study investigated the biomass production and nutrient removal efficiency of the microalgae Scenedesmus vacuolatus ACUF_053 and Chlorella vulgaris ACUF_809 from a synthetic wastewater, describing a start-up process in a new biofilm photobioreactor (PBR) configuration. Two sets of experiments were performed. The first one compared the performance of a suspended and attached cell system under batch conditions. The second set of experiments was addressed under semi-batch conditions to study the microalgae biofilm development in the PBR. Five stages in the development of the biofilm were identified for S. vacuolatus: attachment, biofilm formation, maturation I, adaptation and maturation II. The biofilm development phases had a different nutrient removal efficiency. S. vacuolatus biofilm showed a higher phosphate demand during the first attachment and formation phases, while it had a higher nitrate demand during the subsequent phases. C. vulgaris biofilm formation was affected by the pH increase (up to 10.6). The biofilm PBR design using both S. vacuolatus and C. vulgaris showed potential for wastewater treatment due the higher nutrient removal rates.
Photocontrol of protein activity has become a helpful strategy for regulating biological pathways. Herein, a method for the precise and reversible photocontrol of oxidase activity was developed by using the conformational change of the AsLOV2 domain.
The AsLOV2 domain was inserted into the nonconserved sites exposed on the surface of the AdhP protein, and the alov9 fusion was successfully screened for subsequent optical experiments under the assumption that neither of these actions affected the original activity of AdhP protein. The activity of alov9 was noticeably inhibited when the fusion was exposed to 470 nm blue light and recovered within 30 min. As a result, we could precisely and reversibly photocontrol alov9 activity through the optimization of several parameters, including cofactor concentration, light intensity, and illumination time.
An efficient method was developed for the photoinhibition of enzymatic activity based on the insertion of the light-sensitive AsLOV2 domain, providing new ideas for photocontrolling metabolic pathways without carriers in the future.
Microbial electrosynthesis (MES) is potentially useful for the biological conversion of carbon dioxide into value-added chemicals and biofuels. The study evaluated several limiting factors that affect MES performance. Among all these factors, the optimization of the applied cell voltage, electrode spacing, and trace elements in catholytes may significantly improve the MES performance. MES was operated under the optimal condition with an applied cell voltage of 3 V, an electrode spacing of 8 cm, 2× salt solution, and 8× trace element of catholyte for 100 days, and the maximum acetate concentration reached 7.8 g L−1. The microbial community analyses of the cathode chamber over time showed that Acetobacterium, Enterobacteriaceae, Arcobacter, Sulfurospirillum, and Thioclava were the predominant genera during the entire MES process. The abundance of Acetobacterium first increased and then decreased, which was consistent with that of acetate production. These results provided useful hints for replacing the potentiostatic control of the cathodes in the future construction and operation of MES. Such results might also contribute to the practical operation of MES in large-scale systems.
In the last two decades, studies on plant biomass-degrading fungi have remarkably increased to understand and reveal the underlying molecular mechanisms responsible for their life cycle and wood-decaying abilities. Most of the plant biomass-degrading fungi reported till date belong to basidiomycota or ascomycota phyla. Thus, very few studies were conducted on fungi belonging to other divisions. Recent sequencing studies have revealed complete genomic sequences of various fungi. Our present study is focused on understanding the plant biomass-degrading potentials, by retrieving genome-wide annotations of 56 published fungi belonging to Glomeromycota, Mucoromycota, Zoopagomycota, Blastocladiomycota, Chytridiomycota, Neocallimastigomycota, Microsporidia and Cryptomycota from JGI-MycoCosm repository. We have compared and analyzed the proteomic annotations, especially CAZy, KOG, KEGG and SM clusters by separating the proteomic annotations into lignin-, cellulose-, hemicellulose-, pectin-degrading enzymes and also highlighted the KEGG, KOG molecular mechanisms responsible for the metabolism of carbohydrates (lignocellulolytic pathways of fungi), complex organic pollutants, xenobiotic compounds, biosynthesis of secondary metabolites. However, we strongly agree that studying genome-wide distributions of fungal CAZyme does not completely corresponds to its biomass-degrading ability. Thus, our present study can be used as preliminary materials for selecting ideal fungal candidate for the degradation and conversion of plant biomass components, especially carbohydrates to bioethanol and other commercially valuable products.
Lincomycin A is a clinically important antibiotic produced by Streptomyces lincolnensis that is used against gram-positive bacteria. To increase the yield of lincomycin A, a calcium gluconate feeding strategy was studied in a 15 L bioreactor. The results showed that the addition time of calcium gluconate was optimal during the late fermentation process to ensure a higher yield of lincomycin A. The optimum addition was continuous feeding at a speed of 0.0638 g/L/h from 111 to 158 h, which can increase the lincomycin A titer to 9160 mg/L, 41.3% higher than that without gluconate feeding. Enzyme activities of the central metabolic pathways, accumulation of intermediate metabolites, NADPH and NADH concentrations, and NADPH/NADH ratio were determined to investigate the mechanism of enhanced lincomycin A production by calcium gluconate addition. The activities of key enzymes of the pentose phosphate pathway (PPP) (glucose 6-phosphate dehydrogenase) and TCA cycle (isocitrate dehydrogenase) were enhanced by approximately twofold. A higher ratio of NADPH/NADH was observed in the fermentation process with the optimized feeding strategy providing sufficient reducing power. These data indicated that more flux flows through the PPP and the TCA cycle to provide more precursors, ATP and reducing power to support the synthesis of lincomycin A. The results showed a new strategy to improve the production of lincomycin A by manipulating the flux through the PPP and the TCA cycle.
In quantitative metabolomics studies, the most crucial step was arresting snapshots of all interesting metabolites. However, the procedure customized for Streptomyces was so rare that most studies consulted the procedure from other bacteria even yeast, leading to inaccurate and unreliable metabolomics analysis. In this study, a base solution (acetone: ethanol = 1:1, mol/mol) was added to a quenching solution to keep the integrity of the cell membrane. Based on the molar transition energy (ET) of the organic solvents, five solutions were used to carry out the quenching procedures. These were acetone, isoamylol, propanol, methanol, and 60% (v/v) methanol. To the best of our knowledge, this is the first report which has utilized a quenching solution with ET values. Three procedures were also adopted for extraction. These were boiling, freezing–thawing, and grinding ethanol. Following the analysis of the mass balance, amino acids, organic acids, phosphate sugars, and sugar alcohols were measured using gas chromatography with an isotope dilution mass spectrometry. It was found that using isoamylol with a base solution (5:1, v/v) as a quenching solution and that freezing–thawing in liquid nitrogen within 50% (v/v) methanol as an extracting procedure were the best pairing for the quantitative metabolomics of Streptomyces ZYJ-6, and resulted in average recoveries of close to 100%. The concentration of intracellular metabolites obtained from this new quenching solution was between two and ten times higher than that from 60% (v/v) methanol, which until now has been the most commonly used solution. Our findings are the first systematic quantitative metabolomics tools for Streptomyces ZYJ-6 and, therefore, will be important references for research in fields such as 13C based metabolic flux analysis, multi-omic research and genome-scale metabolic model establishment, as well as for other Streptomyces.
Biochars were produced from softwood chips (spruce–fir mix) and hemp stalk biomasses in an in-house-developed microwave pyrolysis reactor. A kilogram batch raw biomass mixed with 10 wt% microwave absorber was pyrolyzed at 60-min residence time. Microwave power levels were set at 2100, 2400, and 2700 W with optimum heating rates ranging 25–50 °C/min. The proximate analysis indicated a progressive gain in biochar carbon content with power level increase. Both biochars showed a H:C ratio of < 1.2 with a graphite-like structure, which is an important observation for their potential use as a filler in bio-composites structural strength increase. Fourier Transfer Infrared (FT-IR) spectra showed a major loss of functional groups as the power level increased. Brunauer–Emmett–Teller (BET) surface area and porosity distribution contained higher volume of smaller pores in the hemp biochar. The char hardness and Young’s modulus, obtained via nanoindentation technique and load–depth curve analysis, indicated that hemp biochar possessed a higher Young’s modulus and lower hardness than softwood chip biochar.
Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor.
Three homogeneous organosilanes amine and aliphatic primary amine were used as amine catalysts to evaluate their catalytic activity and kinetic towards glucose isomerization. Catalysts structure (primary, secondary, tertiary amine), terminal groups and alkyl chain length were investigated and compared elaborately. Result showed organosilanes tertiary amine behaved the best and amine generated OH− and amine itself contributed the isomerization reaction. The generated acidic by-product not only decreased fructose selectivity but also affected glucose conversion kinetic. The effect of siloxane (–Si–O–CH3) substituent with methyl (–CH3) can be insignificant, but it provided guiding significance for selecting amine-type homogeneous or grafted amine catalysts for glucose isomerization reaction. Longer alkyl chain resulted in lower glucose conversion because of the alkyl chain curls that would weaken the amine catalytic effect and hydration ability. Catalyst loading and initial glucose concentration investigations further showed that amine would effectively catalyze the isomerization reaction under varied operational conditions. This work will provide more details about organic amine catalysts on glucose isomerization into fructose and promote synthesis of platform chemicals in the applications of biorenewable chemicals and fuel.
Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal.
In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in
In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.
Monoamine oxidases (MAOs) use molecular dioxygen as oxidant to catalyze the oxidation of amines to imines. This type of enzyme can be employed for the synthesis of primary, secondary, and tertiary amines by an appropriate deracemization protocol. Consequently, MAOs are an attractive class of enzymes in biocatalysis. However, they also have limitations in enzyme-catalyzed processes due to the often-observed narrow substrate scope, low activity, or poor/wrong stereoselectivity. Therefore, directed evolution was introduced to eliminate these obstacles, which is the subject of this review. The main focus is on recent efforts concerning the directed evolution of four MAOs: monoamine oxidase (MAO-N), cyclohexylamine oxidase (CHAO), D-amino acid oxidase (pkDAO), and 6-hydroxy-D-nicotine oxidase (6-HDNO).
Fermentation is a classic industrial process that can be applied as an efficient strategy to increase the release of bioactive compounds with antioxidant and antidiabetic activities.
This work reported the effects of solid-state fermentation (SSF) performed using strains of Aspergillus oryzae and Aspergillus niger on the antioxidant (DPPH, ABTS and FRAP) and in vitro antidiabetic (inhibition of α-amylase and α-glucosidase activities) potential of lentils.
The results showed that the profiles of the biological activities of the extracts obtained from the fermented samples varied greatly with respect to both the microorganism involved and the fermentation time. The extracts obtained from the fermented lentils by A. oryzae after 72 h and by A. niger after 48 h using the FRAP assay showed the most remarkable changes in the antioxidant activity, increasing by 107 and 81%, respectively, compared to the nonfermented lentils. The lentil extracts produced by fermentation with A. niger after 48 h were able to inhibit the α-glucosidase activity by up to 90%, while a maximal inhibition of amylase (~ 75%) was achieved by the lentil extract obtained after 24 h of fermentation with A. oryzae. The content of the total phenolic compounds (TPCs) and the identification of them in lentil extracts correlated well with the improvement of the biological activities.
These results suggested that SSF was feasible to obtain extracts of fermented lentils with improved antioxidant and antidiabetic properties. Additionally, these results indicated that the proper choice of microorganism is crucial to direct the process for the production of compounds with specific biological activities.
Municipal solid waste management (MSWM) is one of the major environmental issues in Tunisian cities. Rapid growth in urbanization and population rates and the changes in people’s lifestyle have prompted a dramatic increase quantity and a significant shift in the composition of municipal solid waste. There is insufficient data concerning the quantities and the composition of waste streams along with the absence of a comprehensive complete overview and a wider perspective of MSWM potential that provides detailed information at region and city level. As a result, it is still impossible for the scientific community and the authorities to provide synergetic schemes to tie the problems of MSWM with how to integrate economically feasible and environmentally sustainable practices holistically. In the present study, an attempt has been made to provide a comprehensive overview of MSW, through a qualitative (compositional) and quantitative (parametric) characterization of the generated total waste generated in Tunisian cities. A 1-year research survey was conducted in seven regions in Tunisia (Great Tunis, Northeast, Northwest, Midwest, Mideast, Southwest, and Southeast) that cover the 24 provinces of the country. Collected samples revealed that the distribution of waste by region was defined by the region’s demographic, economic, and industrial status. Approaches of possibly more efficient procedures that can be undertaken to improve MSW collection are discussed. At a final stage and based on the potential of biogas calculated in the seven regions, we suggest that the scientific community and the authorities should introduce applicable schemes to valorize MSW through generating biogas as a renewable energy.
Xylan is the second most abundant naturally occurring renewable polysaccharide available on earth. It is a complex heteropolysaccharide consisting of different monosaccharides such as l-arabinose, d-galactose, d-mannoses and organic acids such as acetic acid, ferulic acid, glucuronic acid interwoven together with help of glycosidic and ester bonds. The breakdown of xylan is restricted due to its heterogeneous nature and it can be overcome by xylanases which are capable of cleaving the heterogeneous β-1,4-glycoside linkage. Xylanases are abundantly present in nature (e.g., molluscs, insects and microorganisms) and several microorganisms such as bacteria, fungi, yeast, and algae are used extensively for its production. Microbial xylanases show varying substrate specificities and biochemical properties which makes it suitable for various applications in industrial and biotechnological sectors. The suitability of xylanases for its application in food and feed, paper and pulp, textile, pharmaceuticals, and lignocellulosic biorefinery has led to an increase in demand of xylanases globally. The present review gives an insight of using microbial xylanases as an “Emerging Green Tool” along with its current status and future prospective.
It is becoming imperative to develop renewable fuels such as biodiesel which are sustainable and environmentally friendly. Exploiting non-edible oils is more necessary to reduce dependency of edible oils for biodiesel production. The current study investigated biodiesel production from non-edible Salvadora persica seed oil (SPSO) and crude coconut oil (CCO) by Burkholderia cepacia lipase acting as a biocatalyst in a solvent-free system. The biodiesel yield produced from these feedstocks was compared and the effect of ethanol (acyl acceptor) vs. SPSO and CCO in various ratios on biodiesel production was determined.
The presence of medium-chain fatty acids in majority was confirmed for SPSO and CCO while the average molecular weight was calculated as 749.53 g/mol and 664.57 g/mol, respectively. Thin Layer Chromatography indicated ethyl esters in the produced Salvadora and coconut biodiesel samples. Maximum biodiesel yield (around 70%) was obtained at 1:4 oil-to-ethanol molar ratio from both oils followed by a decline at higher ratios. The gas chromatographic analysis of Salvadora biodiesel at 1:4 molar ratio showed that the yield of individual esters was mostly of medium- and long-chain fatty acids. The analysis of coconut biodiesel at 1:4 molar ratio revealed that it consists mainly of the esters of medium-chain fatty acids. A comparison of estimated properties of biodiesel from both the parent oils with the international standard showed that it meets most of the requirements.
The study paves the way for a green route for biodiesel production and would promote the use of non-edible vegetable oils over edible ones to produce biodiesel. Further, it is a right step to use lipases in biodiesel production as compared to chemical catalysts. Ethanol, which can also be produced from biomass fermentation, can be used as acyl acceptor to produce biodiesel and this makes the process eco-friendly. Moreover, Burkholderia cepacia lipase is a good choice among lipases to get high biodiesel yields successfully from SPSO and CCO at low oil-to-ethanol molar ratios.
A novel phosphatidyl nanoprodrug system can be selectively released parent drugs in cancer cells, triggered by the local overexpression of phospholipase D (PLD). This system significantly reduces the intrinsic disadvantages of conventional chemotherapeutic drugs. However, the separation and purification processes of phosphatidyl prodrug, the precursor of phosphatidyl nanoprodrug, have not been established, and the preparation of nanocrystals with good stability and tumor-targeting capability is still challenging.
In this study, we established a successive elution procedure for the phosphatidyl prodrug—phosphatidyl mitoxantrone (PMA), using an initial ten-bed volume of chloroform/methanol/glacial acetic acid/water (26/10/0.8/0.7) (v/v/v/v) followed by a five-bed volume (26/10/0.8/3), with which purity rates of 96.93% and overall yields of 50.35% of PMA were obtained. Moreover, to reduce the intrinsic disadvantages of conventional chemotherapeutic drugs, phosphatidyl nanoprodrug—PMA nanoprodrug (NP@PMA)—was prepared. To enhance their stability, nanoparticles were modified with polyethylene glycol (PEG). We found that nanoprodrugs modified by PEG (NP@PEG–PMA) were stably present in RPMI-1640 medium containing 10% FBS, compared with unmodified nanoprodrug (NP@PMA). To enhance active tumor-targeting efficiency, we modified nanoparticles with an arginine-glycine-aspartic acid (RGD) peptide (NP@RGD–PEG–PMA). In vitro cytotoxicity assays showed that, compared with the cytotoxicity of NP@PEG–PMA against tumor cells, that of NP@RGD–PEG–PMA was enhanced. Thus, RGD modification may serve to enhance the active tumor-targeting efficiency of a nanoprodrug, thereby increasing its cytotoxicity.
A process for the preparation and purification of novel phosphatidyl prodrugs was successfully established, and the nanoprodrug was modified using PEG for enhanced nanoparticle stability, and using RGD peptide for enhanced active tumor-targeting efficiency. These procedures offer considerable potential in the development of functional antitumor prodrugs.
Citrus limetta peels (CLP), a waste material generated by juice industries, has scarcely been reported for the production of yeast enzymes. The study was conducted to obtain a multienzyme preparation from a yeast consortium under solid-state fermentation of CLP. The substrate, CLP, was pretreated using either acid or alkali, and factors affecting production of multienzyme were studied by generating two separate Plackett–Burman designs. Since, alkali-pretreated CLP yielded higher titers; therefore, significant factors affecting multienzyme preparation using this substrate were optimized by employing Box–Behnken design. The analysis revealed that under optimized conditions, i.e., cultivation of yeast strains for 72 h to alkali-pretreated CLP moistened with mineral salt medium having pH 5 yielded more than 10 IU mL−1 of cellulase, xylanase, and amylase. The multienzyme was studied for its application to saccharify fruit and non-fruit wastes and for orange juice clarification. The data showed that the enzyme preparation could release 3.03 mg L−1 h−1 of reducing sugars from various crude substrates and was able to reduce turbidity of orange juice by 11% with substantial decrease in viscosity and acidity. Hence, CLP appeared as a promising substrate to produce multienzyme preparation from yeast consortium.
Conversion of spent coffee grounds through the Thermo-Catalytic Reforming system (TCR®) is evaluated in this study. While, the TCR® is a technology that has been developed by Fraunhofer UMSICHT, which combines an intermediate pyrolysis and a catalytic reforming. The temperature of the catalytic reformer is varied between 500 and 700 °C to achieve an optimum yield quantities and qualities of the products. The hydrogen concentration is maximized at a reforming temperature of 700 °C, and a gas yield up to 52 wt% is achieved. The thermal stable bio-oil produced at 700 °C has the highest calorific value of 36.8 MJ/kg with significantly low oxygen and water content, low viscosity and low TAN (total acid number). Furthermore, the maximum bio-oil and char yields are obtained at the lowest reforming temperature of 500 °C. Overall spent coffee grounds show a great potential as feedstock in the Thermo-Catalytic Reforming for energy and bio-chemicals production.
Calotropis procera fiber (CPF) is the fruit fiber of C. procera and belongs to a typical cellulosic fiber. In this study, Calotropis procera fiber (CPF) was first purified in the pretreatment process including delignification and bleaching before the isolation of cellulose nanocrystal. Chemical composition of Calotropis procera fiber was determined according to TAPPI standard method. It was composed of 64.0 wt% cellulose, 19.5 wt% hemicelluloses, and 9.7 wt% of lignin. The morphology of the Calotropis procera fiber and fiber after each pretreatment process was also investigated. Cellulose nanocrystal was extracted by classical sulfuric acid hydrolysis of the pretreated Calotropis procera fiber. TEM and SEM were used to analyze the morphologies of the obtained CNC. The crystallinity, thermal stability and suspension stability of the CNC were also investigated. The interesting results proved that this under-utilized biomass could be exploited as a new source of cellulose raw material for the production of cellulose nanocrystal.
Organofluorines are widely used in a variety of applications, ranging from pharmaceuticals to pesticides and advanced materials. The widespread use of organofluorines also leads to its accumulation in the environment, and two major questions arise: how to synthesize and how to degrade this type of compound effectively? In contrast to a considerable number of easy-access chemical methods, milder and more effective enzymatic methods remain to be developed. In this review, we present recent progress on enzyme-catalyzed C–F bond formation and cleavage, focused on describing C–F bond formation enabled by fluorinase and C–F bond cleavage catalyzed by oxidase, reductase, deaminase, and dehalogenase.
E-waste management is extremely difficult to exercise owing to its complexity and hazardous nature. Printed circuit boards (PCBs) are the core components of electrical and electronic equipment, which generally consist of polymers, ceramics, and heavy metals.
The present study has been attempted for removal of heavy metals from printed circuit board by metal-resistant actinobacterium Streptomyces albidoflavus TN10 isolated from the termite nest. This bacterium was found to recover different heavy metals (Al 66%, Ca 74%, Cu 68%, Cd 65%, Fe 42%, Ni 81%, Zn 82%, Ag 56%, Pb 46%) within 72 h under laboratory conditions. The metal content of PCB after bioleaching was analyzed by ICP-MS. The crude PCB and bioleaching residue were characterized by FT-IR, XRD, SEM for the determination of structural and functional group changes for confirmation of bioleaching.
The findings of the present study concluded that Streptomyces albidoflavus TN10 is a promising candidate for bioleaching of heavy metals from the printed circuit board as an eco-friendly and cost-effective process.
In this study, the waste oil of rendered pork (WO-RP) from a food processing industry was studied as a source of biodiesel. The WO-RP was characterized and was found to have a high acid value of 4.30 mg KOH/g. A pre-treatment using H2SO4 was done through the standard titration method that resulted in a reduction of acid value to 0.75 mg KOH/g. The transesterification process over the KOH catalyst was carried out and optimized using the central composite design (CCD) using the Design Expert 7.0 software. The optimum conditions were found at 3:1 methanol–oil molar ratio, 0.55% catalyst loading, and 45-min reaction time. At optimum conditions, the biodiesel yield was 95.28 ± 0.15%. Its chemical characteristics were tested in terms of acid value at 0.75 mg KOH/g, ash content at 0.01 wt%, density at 0.86 g/cm3, HHV at 39.98 MJ/kg, water content at 0.10%, and kinematic viscosity at 6.9 mm2/s. The FAME profile shows the presence of linoleic, palmitic, oleic and stearic acid as major fatty acid components and functional group shows carbonyl group with traces of carboxylic at 1719 cm−1 and the sharp peak of esters at 1749 cm−1 indicating that the derived product is biodiesel.
Herein, autotrophic metabolism of Cupriavidus necator H16 growing on CO2, H2 and O2 gas mixture was analyzed by metabolic pathway analysis tools, specifically elementary mode analysis (EMA) and flux balance analysis (FBA). As case studies, recombinant strains of C. necator H16 for the production of short-chain (isobutanol) and long-chain (hexadecanol) alcohols were constructed and examined by a combined tools of EMA and FBA to comprehensively identify the cell’s metabolic flux profiles and its phenotypic spaces for the autotrophic production of recombinant products. The effect of genetic perturbations via gene deletion and overexpression on phenotypic space of the organism was simulated to improve strain performance for efficient bioconversion of CO2 to products at high yield and high productivity. EMA identified multiple gene deletion together with controlling gas input composition to limit phenotypic space and push metabolic fluxes towards high product yield, while FBA identified target gene overexpression to debottleneck rate-limiting fluxes, hence pulling more fluxes to enhance production rate of the products. A combination of gene deletion and overexpression resulted in designed mutant strains with a predicted yield of 0.21–0.42 g/g for isobutanol and 0.20–0.34 g/g for hexadecanol from CO2. The in silico-designed mutants were also predicted to show high productivity of up to 38.4 mmol/cell-h for isobutanol and 9.1 mmol/cell-h for hexadecanol under autotrophic cultivation. The metabolic modeling and analysis presented in this study could potentially serve as a valuable guidance for future metabolic engineering of C. necator H16 for an efficient CO2-to-biofuels conversion.
A methanotrophic community was enriched in a semi-continuous reactor under non-aseptic conditions with methane and ammonia as carbon and nitrogen source. After a year of operation, Methylosinus sp., accounted for 80% relative abundance of the total sequences identified from potential polyhydroxyalkanoates (PHAs) producers, dominated the methane-fed enrichment. Prior to induction of PHA accumulation, cells harvested from the parent reactor contained low level of PHA at 4.0 ± 0.3 wt%. The cells were later incubated in the absence of ammonia with various combinations of methane, propionic acid, and valeric acid to induce biosynthesis of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Previous studies reported that methanotrophic utilization of odd-chain fatty acids for the production of PHAs requires reducing power from methane oxidation. However, our findings demonstrated that the PHB-containing methanotrophic enrichment does not require methane availability to generate 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV)—when odd-chain fatty acids are presented. The enrichment yielded up to 14 wt% PHA with various mole fractions of 3HV monomer depending on the availability of methane and odd-fatty acids. Overall, the addition of valeric acid resulted in a higher PHA content and a higher 3HV fraction. The highest 3HV fraction (up to 65 mol%) was obtained from the methane–valeric acid experiment, which is higher than those previously reported for PHA-producing methanotrophic mixed microbial cultures.
Carbohydrate-active enzymes (CAZymes) are industrially important enzymes, which are involved in synthesis and breakdown of carbohydrates. CAZymes secreted by microorganisms especially fungi are widely used in industries. However, identifying an ideal fungal candidate is costly and time-consuming process. In this regard, we have developed a web-database “CAZymes Based Ranking of Fungi (CBRF)”, for sorting and selecting an ideal fungal candidate based on their genome-wide distribution of CAZymes. We have retrieved the complete annotated proteomic data of 443 published fungal genomes from JGI-MycoCosm web-repository, for the CBRF web-database construction. CBRF web-database was developed using open source computing programing languages such as MySQL, HTML, CSS, bootstrap, jQuery, JavaScript and Ajax frameworks. CBRF web-database sorts complete annotated list of fungi based on three selection functionalities: (a) to sort either by ascending (or) descending orders; (b) to sort the fungi based on a selected CAZy group and class; (c) to sort fungi based on their individual lignocellulolytic abilities. We have also developed a simple and basic webpage “S-CAZymes” using HTML, CSS and Java script languages. The global search functionality of S-CAZymes enables the users to understand and retrieve information about a specific carbohydrate-active enzyme and its current classification in the corresponding CAZy family. The S-CAZymes is a supporting web page which can be used in complementary with the CBRF web-database (knowing the classification of specific CAZyme in S-CAZyme and use this information further to sort fungi using CBRF web-database). The CBRF web-database and S-CAZymes webpage are hosted through Amazon® Web Services (AWS) available at http://13.58.192.177/RankEnzymes/about. We strongly believe that CBRF web-database simplifies the process of identifying a suitable fungus both in academics and industries. In future, we intend to update the CBRF web-database with the public release of new annotated fungal genomes.
Natural astaxanthin is mainly derived from Haematococcus pluvialis. In the photoinduction phase, astaxanthin accumulation ability can be significantly affected by the characteristics of H. pluvialis cells in the proliferation phase. Based on sequential heterotrophy–dilution–photoinduction (SHDP) technology, the authors’ previous study showed that high astaxanthin accumulation ability is accompanied by high chlorophyll content of H. pluvialis heterotrophic cell; whereas the mechanism of this result remained largely obscure. Therefore, transcriptome analysis was conducted to explain this mechanism.
RNA-seq analysis showed that the transcription level of chlorophyll synthesis-related genes was negatively correlated with genes related to astaxanthin synthesis. A metabolic network between chlorophyll synthesis and astaxanthin accumulation was proposed.
The relationship between chlorophyll synthesis and astaxanthin accumulation was clarified. Chlorophyll degradation products might be used for astaxanthin synthesis through certain pathways. This study enlightens on the mechanism for the transformation of pigment and is conducive to optimize culture process of H. pluvialis by improving the chlorophyll content of heterotrophic cell.
Protein stability is not only fundamental for experimental, industrial, and therapeutic applications, but is also the baseline for evolving novel protein functions. For decades, stability engineering armed with directed evolution has continued its rapid development and inevitably poses challenges. Generally, in directed evolution, establishing a reliable link between a genotype and any interpretable phenotype is more challenging than diversifying genetic libraries. Consequently, we set forth in a small picture to emphasize the screening or selection techniques in protein stability-directed evolution to secure the link. For a more systematic review, two main branches of these techniques, namely cellular or cell-free display and stability biosensors, are expounded with informative examples.