Glutamate dehydrogenases (GluDHs) are promising biocatalysts for the synthesis of chiral aamino acids by asymmetric reductive amination of a-keto acids. However, their strict substrate specificity limits their applications. To address this problem, we developed a molecular engineering method for GluDHs that enhances the asymmetric reductive amination of bulky a-keto acids. Based on rational design, a "cave" located in the active site pocket of PpGluDH (GluDH from Pseudomonas putida), which plays an essential role in substrate recognition, was tailored to facilitate the accepting of bulky substrates. Two mutants (A167G and V378A) were discovered to have significantly enhanced catalytic activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinyl]butyric acid (PPO) and several other bulky substrates. This molecular engineering method was then applied to ten other GluDHs from different sources and with different properties. All engineered GluDHs acquired substantial improvements in PPO-oriented catalytic activity. The most efficient mutant of NADP + (nicotinamide adenine dinucleotide phosphate)-specific GluDHs showed up to 1820-fold increased activity and the specific activity reached 111.02 U/mg-protein. The NAD + (nicotinamide adenine dinucleotide)-specific GluDHs, which have no detectable wild type activity toward PPO, acquired a considerable level of activity (1.90-29.48 U/mg-protein). In batch production of L-phosphinothricin, these "cave-tailored" GluDHs exhibited markedly improved catalytic efficiencies compared with their wild types and ee values of > 99%. The space-time yields (STY) varied from 818.16 to 1482.96 g · L À1 · d À1 , suggesting potential practical applications of these mutants.

The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, PpGluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.

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Sour rot, caused by Geotrichum citri-aurantii, is a major postharvest disease in citrus and results in significant economic losses. The genus Beauveria is recognized as a promising source of biocontrol agents for agricultural applications. Herein, we established a targeted strategy by integrating genomics and metabolomics to accelerate the discovery of new cyclopeptides from antagonistic metabolites produced by the marine-derived fungus Beauveria felina SYSU-MS7908. As a result, we isolated and characterized seven cyclopeptides, including six new molecules, isaridins I-N (1−6). Their chemical structures and conformational analysis were extensively elucidated using spectroscopic techniques (NMR, HRMS, and MS'MS data), modified Mosher's and Marfey's methods, and single-crystal X-ray diffraction. Notably, isaridin K (3) contains a peptide backbone with an N-methyl-2aminobutyric acid residue rarely found in natural cyclopeptides. Bioassays showed that compound 2 could significantly inhibit the mycelial growth of G. citri-aurantii by destroying the cell membrane. These findings provide an effective strategy for searching for new fungal peptides for potential agrochemical fungicides and also pave the way for further exploration of applications in agriculture, food, and medicine.

Hyperoside (quercetin 3-O-galactoside) exhibits many biological functions, along with higher bioactivities than quercetin. In this study, three UDP-dependent glycosyltransferases (UGTs) were screened for efficient hyperoside synthesis from quercetin. The highest hyperoside production of 58.5 mg·L−1 was obtained in a recombinant Escherichia coli co-expressing UGT from Petunia hybrida (PhUGT) and UDP-glucose epimerase (GalE, a key enzyme catalyzing the conversion of UDP-glucose to UDP-galactose) from E. coli. When additional enzymes (phosphoglucomutase (Pgm) and UDP-glucose pyrophosphorylase (GalU)) were introduced into the recombinant E. coli, the increased flux toward UDP-glucose synthesis led to enhanced UDP-galactose-derived hyperoside synthesis. The efficiency of the recombinant strain was further improved by increasing the copy number of the PhUGT, which is a limiting step in the bioconversion. Through the optimization of the fermentation conditions, the production of hyperoside increased from 245.6 to 411.2 mg·L−1. The production was also conducted using a substrate-fed batch fermentation, and the maximal hyperoside production was 831.6 mg·L−1, with a molar conversion ratio of 90.2% and a specific productivity of 27.7 mg·L−1·h−1 after 30 h of fermentation. The efficient hyperoside synthesis pathway described here can be used widely for the glycosylation of other flavonoids and bioactive substances.

Reaction coupling separation systems using calcium fumarate as a substrate can break the reaction equilibrium and promote the production of l-malate. However, the low reusability and stability of fumarase limit its further application. In this study, partially purified fumarase of Thermus thermophilus (87.0 U mg−1) was immobilized within konjac-κ-carrageenan beads. An amalgamation of konjac and carrageenan gum (2%) was used to form the beads, and polyethylene polyamine (0.2%) and glutaraldehyde (0.1%) were used as the cross-linking agents. The pH and temperature profiles of free and immobilized fumarases were remarkably similar. The diffusion limit of immobilized fumarase caused a decline in the maximal velocity (Vmax), whereas the kinetic constant (Km) value increased. Optimization of the parameters for biotransformation by immobilized fumarase showed that 88.3% conversion of 200 mM calcium fumarate could be achieved at 55 °C within 8 h. The beads were stored for 30 days at 4 °C with minimal loss in activity and were reusable for up to 20 cycles with 78.1% relative activity. By recycling the reaction supernatant, a total amount of 3.98 M calcium fumarate was obtained with a conversion of 99.5%, which is the highest value ever reported.

BACKGROUND The regioselective hydration of alicyclic α,ω‐dinitrile 1‐cyanocyclohexaneacetonitrile (1‐CCHAN), followed by hydrogenation of ω‐cyano group is a green and elegant route to gabapentin. As the selective chemical hydration of dinitrile is virtually impossible, a bioprocess for regioselective hydration of 1‐CCHAN was developed using microbial nitrile hydratase. RESULTS A newly isolated NHase producing strain, Rhodococcus aetherivorans ZJB1208, was successfully used for hydration of 1‐CCHAN. Some key parameters of the biocatalytic process, including reaction temperature, pH, catalyst loading and substrate loading, were optimized. The fed‐batch biotransformation was performed in non‐buffered water system with the continuous precipitation of 1‐cyanocyclohexaneacetamide. The substrate loading was increased up to 864 g L−1 (6.0 mol L−1), giving a product concentration of 966.7 g L−1 and biocatalyst yield (gproduct/gcat) of 204.2. CONCLUSION This study is the first example of microbial nitrile hydratase with both excellent regioselectivity and strong substrate tolerance for alicyclic dinitrile and affords a potentially industrial route to gabapentin. © 2015 Society of Chemical Industry