Jinsong Shi

Nitrilases in nitrile biocatalysis: recent progress and forthcoming research

Over the past decades, nitrilases have drawn considerable attention because of their application in nitrile degradation as prominent biocatalysts. Nitrilases are derived from bacteria, filamentous fungi, yeasts, and plants. In-depth investigations on their natural sources function mechanisms, enzyme structure, screening pathways, and biocatalytic properties have been conducted. Moreover, the immobilization, purification, gene cloning and modifications of nitrilase have been dwelt upon. Some nitrilases are used commercially as biofactories for carboxylic acids production, waste treatment, and surface modification. This critical review summarizes the current status of nitrilase research, and discusses a number of challenges and significant attempts in its further development. Nitrilase is a significant and promising biocatalyst for catalytic applications.

Exploring flavour-producing core microbiota in multispecies solid-state fermentation of traditional Chinese vinegar.

Multispecies solid-state fermentation (MSSF), a natural fermentation process driven by reproducible microbiota, is an important technique to produce traditional fermented foods. Flavours, skeleton of fermented foods, was mostly produced by microbiota in food ecosystem. However, the association between microbiota and flavours and flavour-producing core microbiota are still poorly understood. Here, acetic acid fermentation (AAF) of Zhenjiang aromatic vinegar was taken as a typical case of MSSF. The structural and functional dynamics of microbiota during AAF process was determined by metagenomics and favour analyses. The dominant bacteria and fungi were identified as Acetobacter, Lactobacillus, Aspergillus, and Alternaria, respectively. Total 88 flavours including 2 sugars, 9 organic acids, 18 amino acids, and 59 volatile flavours were detected during AAF process. O2PLS-based correlation analysis between microbiota succession and flavours dynamics showed bacteria made more contribution to flavour formation than fungi. Seven genera including Acetobacter, Lactobacillus, Enhydrobacter, Lactococcus, Gluconacetobacer, Bacillus and Staphylococcus were determined as functional core microbiota for production of flavours in Zhenjiang aromatic vinegar, based on their dominance and functionality in microbial community. This study provides a perspective for bridging the gap between the phenotype and genotype of ecological system, and advances our understanding of MSSF mechanisms in Zhenjiang aromatic vinegar.

Purification and characterisation of a bifunctional alginate lyase from novel Isoptericola halotolerans CGMCC 5336

A novel halophilic alginate-degrading microorganism was isolated from rotten seaweed and identified as Isoptericola halotolerans CGMCC5336. The lyase from the strain was purified to homogeneity by combining of ammonium sulfate fractionation and anion-exchange chromatography with a specific activity of 8409.19 U/ml and a recovery of 25.07%. This enzyme was a monomer with a molecular mass of approximately 28 kDa. The optimal temperature and pH were 50 °C and pH 7.0, respectively. The lyase maintained stability at neutral pH (7.0-8.0) and temperatures below 50 °C. Metal ions including Na(+), Mg(2+), Mn(2+), and Ca(2+) notably increased the activity of the enzyme. With sodium alginate as the substrate, the Km and Vmax were 0.26 mg/ml and 1.31 mg/ml min, respectively. The alginate lyase had substrate specificity for polyguluronate and polymannuronate units in alginate molecules, indicating its bifunctionality. These excellent characteristics demonstrated the potential applications in alginate oligosaccharides production with low polymerisation degrees.

Bioassay-guided isolation of DPP-4 inhibitory fractions from extracts of submerged cultured of Inonotus obliquus.

Inonotus obliquus is a medicinal mushroom used in Russian and Eastern European folk medicine for the treatment of gastrointestinal cancer, cardiovascular disease and diabetes. Previous studies in our laboratory have demonstrated that the mycelium powders of I. obliquus possess significant antihyperglycemic effects in a mouse model of diabetic disease induced by alloxan. However, the active ingredients of mycelium powders responsible for the diabetes activity have not been identified. This study aims to identify the active ingredients of I. obliquus mycelium powders by a bioassay-guided fractionation approach and explore the mechanism of action of these active ingredients by using a well-established DPP-4 (an important enzyme as a new therapeutic target for diabetes) inhibitory assay model. The results showed the chloroform extract of mycelium was potential inhibitory against DPP-4. Bioactivity guided fractionation led to the identification of 19 compounds using UPLC-Q-TOF-MS. Molecular docking between the compounds and DPP-4 revealed that compounds 5, 8, 9, 14, 15 may be the active components responsible for the DPP-4 inhibitory activity.

Enhancement of steroid hydroxylation yield from dehydroepiandrosterone by cyclodextrin complexation technique.

The cyclodextrins (CDs) complexation technique was performed for the enhancement of hydroxylation yield from dehydroepiandrosterone (DHEA) by Colletotrichum lini ST-1. Using DHEA/methyl-β-cyclodextrin (M-β-CD) or DHEA/hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complexes as substrate (10g/L), the hydroxylation yields were increased by 14.98% and 20.54% respectively, and the biotransformation course was shortened by 12h. X-ray diffractometry, differential scanning calorimetry, and phase solubility analyses showed an inclusion complex was formed between CDs and DHEA at a molar ratio of 1:1, which remarkably increased the solubility of DHEA, and then improved substrate biotransformation efficiency and hydroxylation yield. Meanwhile, results of thermodynamic parameters (ΔG, ΔH, ΔS and Ks) analysis revealed the complexation process was spontaneous and DHEA/CDs inclusion complex was stable. Scanning electron microscopy and transmission electron microscopy showed that the enhancement of DHEA hydroxylation yield also depended on the improvement of cell permeability through interaction between cytomembrane and CDs. These results suggested that the CDs complexation technique was a promising method to enhance steroids hydroxylation yield by increasing steroids solubility and decreasing membrane resistance of substrate and product during biotransformation process.

Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises.

Abstract Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.

Elucidating and Regulating the Acetoin Production Role of Microbial Functional Groups in Multispecies Acetic Acid Fermentation.

ABSTRACT Acetoin (3-hydroxy-2-butanone) formation in vinegar microbiota is crucial for the flavor quality of Zhenjiang aromatic vinegar, a traditional vinegar produced from cereals. However, the specific microorganisms responsible for acetoin formation in this centuries-long repeated batch fermentation have not yet been clearly identified. Here, the microbial distribution discrepancy in the diacetyl/acetoin metabolic pathway of vinegar microbiota was revealed at the species level by a combination of metagenomic sequencing and clone library analysis. The results showed that Acetobacter pasteurianus and 4 Lactobacillus species (Lactobacillus buchneri, Lactobacillus reuteri, Lactobacillus fermentum, and Lactobacillus brevis) might be functional producers of acetoin from 2-acetolactate in vinegar microbiota. Furthermore, A. pasteurianus G3-2, L. brevis 4-22, L. fermentum M10-3, and L. buchneri F2-5 were isolated from vinegar microbiota by a culture-dependent method. The acetoin concentrations in two cocultures (L. brevis 4-22 plus A. pasteurianus G3-2 and L. fermentum M10-3 plus A. pasteurianus G3-2) were obviously higher than those in monocultures of lactic acid bacteria (LAB), while L. buchneri F2-5 did not produce more acetoin when coinoculated with A. pasteurianus G3-2. Last, the acetoin-producing function of vinegar microbiota was regulated in situ via augmentation with functional species in vinegar Pei. After 72 h of fermentation, augmentation with A. pasteurianus G3-2 plus L. brevis 4-22, L. fermentum M10-3, or L. buchneri F2-5 significantly increased the acetoin content in vinegar Pei compared with the control group. This study provides a perspective on elucidating and manipulating different metabolic roles of microbes during flavor formation in vinegar microbiota. IMPORTANCE Acetoin (3-hydroxy-2-butanone) formation in vinegar microbiota is crucial for the flavor quality of Zhenjiang aromatic vinegar, a traditional vinegar produced from cereals. Thus, it is of interest to understand which microbes are driving the formation of acetoin to elucidate the microbial distribution discrepancy in the acetoin metabolic pathway and to regulate the metabolic function of functional microbial groups in vinegar microbiota. Our study provides a perspective on elucidating and manipulating different metabolic roles of microbes during flavor formation in vinegar microbiota.

Biochemical characterization of an extreme alkaline and surfactant-stable keratinase derived from a newly isolated actinomycete Streptomyces aureofaciens K13

Keratinase has attracted increasing attention in the field of biocatalysis in recent years because of its critical role in keratin resource exploitation and keratin waste degradation. However, conventional studies focused on keratinases from bacterial and fungal strains, especially those of the Bacillus genus, keratinase resources from actinomycetes are far from being fully explored. In this study, a novel keratinase-producing strain was isolated with wool as the sole carbon and nitrogen source and identified as Streptomyces aureofaciens K13. The keratinase was purified to electrophoretic homogeneity with a molecular mass of 46 kDa. The purified enzyme exhibited optimum activity at 75 °C and pH of 12.0. It remained extremely stable at alkaline pH values between 6 and 12 and at a high reaction temperature of 65 °C. The keratinase displayed significant activity toward casein, keratin, BSA and wool. It could be activated in the presence of K+, Cu2+, Mn2+, Ca2+, Li+, and Sr2+. The keratinase was completely inhibited by PMSF and moderately inhibited by EDTA, indicating that this keratinase is a metallo-serine keratinase. This enzyme could remain stable and even be promoted in the presence of surfactants, including SDS, Tween, and Triton; especially, 1% of Tween 80 and Triton X-100 could substantially enhance the activity by 46% and 38%, respectively. These results indicated certain advantages over conventional keratinases. The keratinase can completely remove blood stains when combined with detergents. The improvement effect of S. aureofaciens K13 keratinase by various surfactants and the favourable washing performance might indicate its significant application potential in the detergent industry. There are rare reports on keratinase production from S. aureofaciens.

Alpha-terpineol promotes triterpenoid production of Antrodia cinnamomea in submerged culture

Antrodia cinnamomea is a medicinal mushroom producing potent bioactive triterpenoids. However, triterpenoids of A. cinnamomea in submerged culture are much less than those in fruiting bodies. Here we evaluated effects of different extracts from a host-related species, Cinnamomum camphora, on the mycelial growth and triterpenoid production of A. cinnamomea in submerged culture. The hot water extract of the stem showed the strongest promotion of the mycelial growth. The petroleum ether extract of the stem (PES) (0.05 g L(-1)) showed the greatest stimulatory effect on content and production of triterpenoids. A total of 39 compounds including terpenoids, phenolic and aromatic compounds were identified in the PES by GC-MS analysis. Furthermore, the effects of seven compounds contained in the PES on the mycelial growth and triterpenoid production of A. cinnamomea were evaluated. Among them, α-terpineol (0.5 mg L(-1)) showed the greatest stimulatory effect on the triterpenoid content (23.31 mg g(-1)) and triterpenoid production (91.33 mg L(-1)) of A. cinnamomea. Results of LC-MS analysis showed that α-terpineol (0.5 mg L(-1)) stimulated the syntheses of six triterpenoids in the mycelia of A. cinnamomea. This indicates that α-terpineol can act as an elicitor for triterpenoid biosynthesis in A. cinnamomea.

Bench-scale biosynthesis of isonicotinic acid from 4-cyanopyridine by Pseudomonas putida

Pseudomonas putida CGMCC3830 harboring nitrilase was used in isonicotinic acid production from 4-cyanopyridine. This nitrilase showed optimum activities towards 4-cyanopyridine at pH 7.5 and 45°C. The half-life of P. putida nitrilase was 93.3 h, 33.9 h, and 9.5 h at 30°C, 38°C, and 45°C, respectively. 4-Cyanopyridine (100 mM) was fully converted into isonicotinic acid within 20 min. The bench-scale production of isonicotinic acid was carried out using 3 mg of resting cells per mL in a 1 L system at 30°C and finally, 123 g L−1 of isonicotinic acid were obtained within 200 min without any by-products. The conversion reaction suffered from the product inhibition effect after the tenth feeding. The volumetric productivity was 36.9 g L−1 h−1. P. putida shows significant potential in nitrile hydrolysis for isonicotinic acid production. This paper is the first report on isonicotinic acid biosynthesis using Pseudomonas putida and it represents the highest isonicotinic acid production reported so far.