Hualei Wang

East China University of Science and Technology, Shanghai, Shanghai Shi, China

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Publications (15)34.07 Total impact

  • Huihui Sun · Wenyuan Gao · Hualei Wang · Dongzhi Wei ·
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    ABSTRACT: Objectives: To identify a novel nitrilase with S-selectivity toward mandelonitrile that can produce (S)-mandelic acid in one step. Results: A novel nitrilase PpL19 from Pseudomonas psychrotolerans L19 was discovered by genome mining. It showed S-selectivity with an enantiomeric excess of 52.7 % when used to hydrolyse (R, S)-mandelonitrile. No byproduct was observed. PpL19 was overexpressed in Escherichia coli BL21 (DE3) and formed inclusion bodies that were active toward mandelonitrile and stable across a broad range of temperature and pH. In addition, PpL19 hydrolysed nitriles with diverse structures; arylacetonitriles were the optimal substrates. Homology modelling and docking studies of both enantiomers of mandelonitrile in the active site of nitrilase PpL19 shed light on the enantioselectivity. Conclusions: A novel nitrilase PpL19 from P. psychrotolerans L19 was mined and distinguished from other nitrilases as it was expressed as an active inclusion body and showed S-selectivity toward mandelonitrile.
    Biotechnology Letters 11/2015; DOI:10.1007/s10529-015-1992-0 · 1.59 Impact Factor
  • Huihui Sun · Hualei Wang · Wenyuan Gao · Lifeng Chen · Kai Wu · Dongzhi Wei ·
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    ABSTRACT: Nitrilase PpL19 from Pseudomonas psychrotolerans L19 can hydrolyze racemic mandelonitrile to (S)-mandelic acid with an enantiomeric excess (ee) value of 52.7%. In this study, random mutagenesis combined with site-directed mutagenesis was performed to identify the key residues responsible for nitrilase enantioselectivity. Five enzyme mutants exhibiting distinct selectivity were generated and four "hot spots" (M113, R128, A136, and I168) responsible for enantioselectivity toward mandelonitrile were identified and characterized. Furthermore, through saturation mutagenesis, positions 113 and 128 were confirmed to substantially influence the enantioselectivity of PpL19, and certain replacements of the methionine at position 113, in particular, were found to reverse the enantioselectivity of PpL19 from S- to R-selectivity. Two other single mutants of the enzyme, PpL19-A136Y and -I168Y, also showed reversed selectivity and preferentially produced (R)-mandelic acid (ee values: 66.7% and 74.3%, respectively). By combining the beneficial mutations, two enantiocomplementary nitrilase mutants, PpL19-LH and PpL19-GYY, were created, which exhibited high S- and R-selectivity toward mandelonitrile, respectively: PpL19-LH showed the highest S-selectivity toward mandelonitrile ever reported (91.1% ee), and, notably, the PpL19-GYY mutant was identified to be highly R-selective (90.1% ee) and thus an unexpected enantiocomplementary mutant for mandelonitrile.
    Biochemical and Biophysical Research Communications 11/2015; DOI:10.1016/j.bbrc.2015.11.038 · 2.30 Impact Factor
  • Li Zhao · Jinping Lin · Hualei Wang · Jingli Xie · Dongzhi Wei ·
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    ABSTRACT: In this work, a two-step process was developed for the production of 3-hydroxypropionic acid from glycerol. In the first step, glycerol was converted to 1,3-propanediol by Klebsiella pneumonia. In the second step, the 1,3-propanediol was converted into 3-hydroxypropionic acid by Gluconobacter oxydans. In a 7.0 L bioreactor, the whole process took 54 h, consumed 480 g glycerol and produced 242 g 3-hydroxypropionic acid. The conversion rate of glycerol to 3-hydroxypropionic acid was 50.4 % (g g(-1)). The final concentration of 3-hydroxypropionic acid arrived 60.5 g L(-1). The process was effective for 3-HP production from glycerol and it might provide a new approach to the biosynthesis of 3-HP from a cheap starting material. Moreover, in this paper, it was first reported that the by-product of 3-hydroxypropionic acid production from 1,3-propandeiol was acrylic acid.
    Bioprocess and Biosystems Engineering 10/2015; DOI:10.1007/s00449-015-1486-4 · 2.00 Impact Factor
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    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 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), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess (ee : compared to 89.2% ee of 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 combined 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 low amount of biocatalyst (10 g/L 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.
    Applied and Environmental Microbiology 10/2015; DOI:10.1128/AEM.02688-15 · 3.67 Impact Factor
  • Kai Wu · Hualei Wang · Huihui Sun · Dongzhi Wei ·
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    ABSTRACT: Enantioselective hydrolysis of racemic epoxides mediated by epoxide hydrolases (EHs) is one of the most promising approaches to obtain enantiopure epoxides. In this study, we identified and characterized a novel EH (TpEH1) from Tsukamurella paurometabola by analyzing the conserved catalytic residues of EH. TpEH1 was overexpressed and purified, and its catalytic properties were studied using racemic phenyl glycidyl ether (PGE) and its derivatives as substrates. TpEH1 showed excellent enantioselectivity to the substrates PGE, 3-methylPGE, and 3-nitroPGE. The highest enantioselectivity (E > 100) was achieved when 3-nitroPGE was used as the substrate. The recombinant Escherichia coli TpEH1 demonstrated high substrate tolerance toward PGE and could hydrolyze PGE at concentrations of up to 400 mM (60 g/L) with high enantioselectivity (E = 65), giving (R)-PGE with enantiomeric excess of more than 99 % ee and 45 % yield within 1 h. This concentration of PGE is the highest reported concentration catalyzed by native EHs to date. Thus, the easily available and highly active E. coli TpEH1 showed great potential for the practical preparation of optically pure (R)-PGE.
    Applied Microbiology and Biotechnology 06/2015; 99(22). DOI:10.1007/s00253-015-6716-9 · 3.34 Impact Factor
  • Huihui Sun · Wenyuan Gao · Haiyang Fan · Hualei Wang · Dongzhi Wei ·
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    ABSTRACT: To examine nitrilase-mediated hydrolysis of nitriles to produce optically pure α-hydroxycarboxylic acids. A novel nitrilase, GPnor51, from Luminiphilus syltensis NOR5-1B was discovered by genomic data mining. It could hydrolyze racemic o-chloromandelonitrile to (R)-o-chloromandelic acid with high enantioselectivity (ee 98.2 %). GPnor51 was overexpressed in Escherichia coli BL21 (DE3), purified, and its catalytic properties studied. GPnor51 had a broad substrate acceptance toward various nitriles with structure diversity. It was an arylacetonitrilase that uses arylacetonitriles as optimal substrates. The V max and K m of GPnor51 towards o-chloromandelonitrile were 1.9 μmol min(-1) mg(-1) protein and 0.38 mM, respectively. GPnor51 also demonstrated high enantioselectivity toward mandelonitrile and other substituted mandelonitrile. This enzyme has a great potential for commercial production of optically pure (R)-mandelic acid and its derivatives.
    Biotechnology Letters 04/2015; 37(8). DOI:10.1007/s10529-015-1830-4 · 1.59 Impact Factor
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    ABSTRACT: Nitrilases are enzymes widely expressed in prokaryotes and eukaryotes that utilize a Cys–Glu–Lys catalytic triad to hydrolyze non-peptide carbon–nitrogen bonds. Nitrilase from Syechocystis sp. Strain PCC6803 (Nit6803) shows hydrolysis activity towards a broad substrate spectrum, ranging from mononitriles to dinitriles and from aromatic nitriles to aliphatic nitriles. Yet, the structural principle of the substrate specificity of this nitrilase is still unknown. We report the crystal structure of Nit6803 at 3.1 Å resolution and propose a structural mechanism of substrate selection. Our mutagenesis data exhibited that the aromaticity of the amino acid at position 146 of Nit6803 is absolutely required for its nitrilase activity towards any substrates tested. Moreover, molecular docking and dynamic simulation analysis indicated that the distance between the sulfhydryl group of the catalytic cysteine residue and the cyano carbon of the substrate plays a crucial role in determining the nitrilase catalytic activity of Nit6803 and its mutants towards different nitrile substrates.
    Journal of Structural Biology 10/2014; 188(2). DOI:10.1016/j.jsb.2014.10.003 · 3.23 Impact Factor
  • Hualei Wang · Huihui Sun · Wenyuan Gao · Dongzhi Wei ·
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    ABSTRACT: A solvent engineering approach and an extended fed-batch reaction mode were introduced to increase the activity and enantioselectivity and alleviate the substrate inhibition of nitrilase BCJ2315 from Burkholderia cenocepacia J2315 toward o-chloromandelonitrile. Among the seven water-miscible organic solvents tested, ethanol (30%, v/v) demonstrated the highest reaction conversion (55.7%) and enantioselectivity (enantiomeric excess, 98.2% ee) compared with those of the control [which did not contain any organic solvent (13% and 89.2%, respectively)] and was thus chosen as the suitable cosolvent. In the extended fed-batch reaction mode, o-chloromandelonitrile (solubilized in ethanol, 5 M) was continuously fed into the reaction mixture containing ethanol as cosolvent (20%, v/v) to ensure an optimal reaction rate by adjusting the feeding rate and simultaneously increasing the enantioselectivity due to the increased concentration of ethanol. Finally, a maximum of 415 mM of product was produced with an enantiomeric excess value of 97.6% ee. The hydrolysis process was easily scaled up to 2 L, demonstrating that the described biocatalytic process was rationally designed and could be applied further on an industrial scale.
    Organic Process Research & Development 09/2013; 18(6):767–773. DOI:10.1021/op400174a · 2.53 Impact Factor
  • Hualei Wang · Guinan Li · Mingyang Li · Dongzhi Wei · Xuedong Wang ·
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    ABSTRACT: In this study, a novel nitrilase gene from Rhodobacter sphaeroides was cloned and overexpressed in Escherichia coli. The open reading frame of the nitrilase gene includes 969 base pairs, which encodes a putative polypeptide of 322 amino acid residues. The molecular weight of the purified native nitrilase was about 560 kDa determined by size exclusion chromatography. This nitrilase showed one single band on SDS-PAGE with a molecular weight of 40 kDa. This suggested that the native nitrilase consisted of 14 subunits with identical size. The optimal pH and temperature of the purified enzyme were 7.0 and 40 °C, respectively. The kinetic parameters V max and K m toward 3-cyanopyridine were 77.5 μmol min(-1) mg(-1) and 73.1 mmol/l, respectively. The enzyme can easily convert aliphatic nitrile and aromatic nitriles to their corresponding acids. Furthermore, this enzyme demonstrated regioselectivity in hydrolysis of aliphatic dinitriles. This specific characteristic makes this nitrilase have a great potential for commercial production of various cyanocarboxylic acids by hydrolyzing readily available dinitriles.
    World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology) 07/2013; 30(1). DOI:10.1007/s11274-013-1445-7 · 1.78 Impact Factor
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    ABSTRACT: The recombinant Escherichia coli M15/BCJ2315 which harbored a mandelonitrilase from Burkholderia cenocepacia J2315 was immobilized via catecholic chitosan and functionalized with magnetism by iron oxide nanoparticles. The immobilized cells showed high activity recovery, enhanced stability and good operability in the enantioselective hydrolysis of mandelonitrile to (R)-(-)-mandelic acid. Furthermore, the immobilized cells were reused up to 15cycles without any activity loss in completely hydrolyzing mandelonitrile (100mM) within 1h in aqueous solution. The ethyl acetate-water biphasic system was built and optimized. Under the optimal conditions, as high as 1M mandelonitrile could be hydrolyzed within 4h with a final yield and ee value of 99% and 95%, respectively. Moreover, the successive hydrolysis of mandelonitrile was performed by repeated use of the immobilized cells for 6 batches, giving a final productivity (g•L(-1)•h(-1)) and relative production (g•g(-1)) of 40.9 and 38.9, respectively.
    Journal of Biotechnology 07/2013; 167(4). DOI:10.1016/j.jbiotec.2013.07.024 · 2.87 Impact Factor
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    Hualei Wang · Huihui Sun · Dongzhi Wei ·
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    ABSTRACT: Background A 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. Results A 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. Conclusions PESSP 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.
    BMC Biotechnology 02/2013; 13(1):14. DOI:10.1186/1472-6750-13-14 · 2.03 Impact Factor
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    Kefeng Ni · Xu Zhou · Li Zhao · Hualei Wang · Yuhong Ren · Dongzhi Wei ·
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    ABSTRACT: The magnetic chitosan nanocomposites have been studied intensively and been used practically in various biomedical and biological applications including enzyme immobilization. However, the loading capacity and the remained activity of immobilized enzyme based on existing approaches are not satisfied. Simpler and more effective immobilization strategies are needed. Here we report a simple catechol modified protocol for preparing a novel catechol-chitosan (CCS)-iron oxide nanoparticles (IONPs) composites carrying adhesive moieties with strong surface affinity. The ω-transaminase (ω-TA) was immobilized onto this magnetic composite via nucleophilic reactions between catechol and ω-TA. Under optimal conditions, 87.5% of the available ω-TA was immobilized on the composite, yielding an enzyme loading capacity as high as 681.7 mg/g. Furthermore, the valuation of enzyme activity showed that ω-TA immobilized on CCS-IONPs displayed enhanced pH and thermal stability compared to free enzyme. Importantly, the immobilized ω-TA retained more than 50% of its initial activity after 15 repeated reaction cycles using magnetic separation and 61.5% of its initial activity after storage at 4°C in phosphate buffered saline (PBS) for 15 days. The results suggested that such adhesive magnetic composites may provide an improved platform technology for bio-macromolecules immobilized.
    PLoS ONE 07/2012; 7(7):e41101. DOI:10.1371/journal.pone.0041101 · 3.23 Impact Factor
  • Shu Wang · Xiangzhao Mao · Hualei Wang · Jinping Lin · Fuli Li · Dongzhi Wei ·
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    ABSTRACT: 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.
    Applied Microbiology and Biotechnology 04/2011; 91(2):287-94. DOI:10.1007/s00253-011-3267-6 · 3.34 Impact Factor
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    ABSTRACT: 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.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 03/2010; 9(8):1180-1189. · 0.57 Impact Factor
  • Yanlong Xing · Xiangzhao Mao · Shu Wang · Hualei Wang · Dongzhi Wei ·
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    ABSTRACT: Enzymes play such a pivotal role in cellular metabolism that enzyme assays are important for bio-engineering, disease diagnoses and drug discovery. Among the reported methods, fluoremetry has attracted more and more attention due to its high sensitivity and possibility of continuous dynamic monitoring. The recent progresses and applications in enzyme assays using fluorescent probes were reviewed. Different methods were classified into direct fluorescence detection and indirect fluorescence detection according to their labeled substrates and detection mechanisms. Our writing purpose is to provide the readers with a flavor of the kinds of tools and strategies available in enzyme assays with fluorescent probes. Also, the research situation and prospects were disucssed
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 12/2009; 25(12):1765-9.