Hualei Wang

Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction

BackgroundA nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(−)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(−)-mandelic acid without the unwanted byproduct is needed.ResultsA novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(−)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters Vmax, Km, Kcat, and Kcat/Km toward mandelonitrile were 45.4 μmol/min/mg, 0.14 mM, 15.4 s-1, and 1.1×105 M-1s-1, respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h.ConclusionsPESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(−)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(−)-mandelic acid in the industry.

Characterization of a novel dextran produced by Gluconobacter oxydans DSM 2003.

A novel water-soluble dextran was synthesized from maltodextrin by cell-free extract of Gluconobacter oxydans DSM 2003. The dextran was purified by size exclusion chromatography, and the structure was determined by Fourier transform infrared spectroscopy, nuclear magnetic resonance, and gas chromatography–mass spectrometer. Based on the spectral data, we found that the dextran contained only d-glucose residues. The ratio of nonreducing end glucopyranosyl (Glcp) to 6-linked Glcp to 4,6-linked Glcp was estimated to be 8.62:78.79:12.59 by methylation analysis. This result indicated the existence of a small proportion of α(1,4) branches in α(1,6) glucosyl linear chains. Here, we reported the first time a novel dextran was synthesized by G. oxydans DSM 2003.

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of (R)-o-Chloromandelic Acid

ABSTRACT The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2% ee for the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system, o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7% ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.

Enhanced production of intracellular dextran dextrinase from Gluconobacter oxydans using statistical experimental methods

Optimization of the fermentation medium for DDase production by Gluconaobacter oxydans M5 was carried out in the shake flasks using two kinds of statistical methods. Four variables, namely glucose, tryptone, yeast extract and sodium chloride, were found to influence DDase production significantly by the Plackett-Burman screening. A four-factor five-level central composite design (CCD) was chosen to explain the combined effects of the four medium constituents. The optimum medium consisted of glucose (17.670 g/L), maltobiose (30 g/L), tryptone (12.198 g/L), yeast extract (13.528 g/L), ammonium nitrate (15 g/L), copper sulfate (0.01 g/L), zinc sulfate (0.01 g/L), and sodium chloride (0.009 g/L); the initial pH 6.0 was set prior to sterilization. The DDase yield obtained from optimized medium increased by 17-fold (0.238 U/mL) or so. Under these optimal conditions, the experimental values agreed with the predicted values, indicating that the chosen method of optimization of medium composition was efficient, relatively simple, time reducing and material saving.

Enhancement of ethyl (S)-4-chloro-3-hydroxybutanoate production at high substrate concentration by in situ resin adsorption

Asymmetric reduction of ethyl 4-chloro-3-oxobutyrate (COBE) by carbonyl reductases presents an efficient way to produce Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important chiral intermediate for the synthesis of hydroxymethylglutaryl-CoA reductase inhibitors such as Lipitor®. In this study, an NADPH-dependent carbonyl reductase (SrCR) from Synechocystis sp. was characterized to demonstrate a broad substrate spectrum, and the highest activity (53.1U/mg protein) with COBE. To regenerate the cofactor NADPH, Bacillus subtilis glucose dehydrogenase was successfully coexpressed with SrCR. Owing to the product inhibition, no more than 400mM of COBE could be completely reduced to (S)-CHBE using the recombinant Escherichia coli/pET-SrCR-GDH. The macroporous adsorption resin HZ 814 was applied to adsorb (S)-CHBE in situ to alleviate the product inhibitio. Consequently, 3000mM (494g/L) of COBE was bioconverted within 8h, resulting in a (S)-CHBE yield of 98.2%, with 99.4% ee and total turnover number of 15,000, revealed great industrial potential of (S)-CHBE production.

Switching a nitrilase from Syechocystis sp. PCC6803 to a nitrile hydratase by rationally regulating reaction pathways

The development of robust biocatalysts producing a large range of organic amides by hydration of nitriles is an important pursuit and challenge. A nitrilase with a broad range of nitrile substrates was switched to a nitrile hydratase by rationally regulating the reaction pathways. Five mutants improved the amide formation in the product, and four of them formed >50% amide. F193N, with the highest amide formation among the four mutants, improved its amide product up to 73%, which was 35-fold that of the wild type, while maintaining 50% activity relative to the wild type. This study would afford a new synthetic route to amides from nitriles and could be a valuable addition to the synthetic repertoire. Further protein engineering may expand the reaction range of an enzyme to afford more additional pathways to synthetic biology.