Hualei Wang

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

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Publications (8)17.23 Total impact

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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. · 2.74 Impact Factor
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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; · 1.35 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; · 3.18 Impact Factor
  • Source
    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 mumol/min/mg, 0.14 mM, 15.4 s-1, and 1.1x105 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. · 2.17 Impact Factor
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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 01/2012; 7(7):e41101. · 3.53 Impact Factor
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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. · 3.69 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; 9:1180-1189. · 0.57 Impact Factor
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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.

Publication Stats

13 Citations
17.23 Total Impact Points

Institutions

  • 2011–2013
    • East China University of Science and Technology
      Shanghai, Shanghai Shi, China
  • 2009–2011
    • Chinese Academy of Sciences
      Peping, Beijing, China
  • 2010
    • Qingdao Agricultural University
      Tsingtao, Shandong Sheng, China