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Pan H.,Zhejiang University | Xie Z.,Zhejiang University | Bao W.,Zhejiang University | Cheng Y.,Zhejiang University | And 3 more authors.
FEBS Letters | Year: 2011

Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16-23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Pan H.,Zhejiang University | Bao W.,Hangzhou Bioking Biochemical Engineering Co. | Xie Z.,Zhejiang University | Zhang J.,Zhejiang University | And 2 more authors.
Biotechnology Letters | Year: 2010

Immobilization of cis-epoxysuccinate hydrolase-containing E. coli for d(-)-tartaric acid production was screened by various methods. The highest recovery of activity was obtained by entrapment in κ-carrageenan gel. 23. 6 g biomass/l and 43. 4 g κ-carrageenan/l were the best immobilization conditions optimized by response surface methodology with 83% yield (114 U/g). Cell autolysis was observed after immobilization. Immobilized cells showed high pH (5-10) stability, thermal (up to 65°C) stability, conversion rate (>99. 5%), enantioselectivity (ee> 99. 6%), and were less affected by metal ions and surfactants compared with free cells. Conversion rate for immobilized cells preserved 93% after 10 repeated batches (5% for free cells). © 2009 Springer Science+Business Media B.V.

Pan H.-F.,Zhejiang University | Bao W.-N.,Hangzhou Bioking Biochemical Engineering Co. | Xie Z.-P.,Zhejiang University | Zhang J.-G.,Zhejiang University | Zhang J.-G.,Hangzhou Bioking Biochemical Engineering Co.
African Journal of Biotechnology | Year: 2010

Response surface methodology was applied to identify and optimize the medium composition for the cis-epoxysuccinate hydrolase production in recombinant Escherichia coli. Plackett-Burman design was used in the first step to evaluate the effects of 8 variables on the enzyme activity. CaCl2, corn steep liquor and lactose were screened as significant factors and their concentrations were further optimized using response surface methodology based on 23 full factorial rotatable central composite design. The optimum predicted medium for maximum expression of recombinant cis-epoxysuccinate hydrolase was found to comprise: 17.1 g/l Na2HPO4· 12H2O, 2.0 g/l KH2PO4, 0.5 g/l NaCl, 1.0 g/l NH4Cl, 0.0111 g/l CaCl2 and 0.5 g/l MgSO 4·7H2O, 17.18 ml/l corn steep liquor and 9.74 g/l lactose, with a predicted enzyme activity of 35490 U/g biomass, which was very close to the experimental activity of 36318 U/g biomass resulting in 1.7-fold increment after optimization. © 2010 Academic Journals.

Pan H.F.,Zhejiang University | Bao W.N.,Hangzhou Bioking Biochemical Engineering Co. | Xie Z.P.,Zhejiang University | Zhang J.G.,Zhejiang University | And 2 more authors.
Journal of Microbiology and Biotechnology | Year: 2010

A cis-epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52 was purified 51.4-fold with a yield of 27.1% using ammonium sulfate precipitation, ionic exchange, hydrophobic interaction, molecular sieve chromatography and an additional anion-exchange chromatography. The CESH was stable in a broad range of temperature (up to 50°C) and pH (4.0-10.0) with optima of 40°C and pH 6.5, respectively. It could be partially inhibited by EDTA-Na2, Ag+, SDS, and DTT, and slightly enhanced by Ba2+ and Ca2+. The enzyme exhibited high stereospecificity in d(-)-tartaric acid (enantiomeric excess value higher than 99%) with Km and Vmax values of 18.67 mM and 94.34 μM/min/mg for disodium cis-epoxysuccinate, respectively. The Bordetella sp. BK-52 CESH gene, which contained 885 nucleotides (open reading frame) encoding 294 amino acids with a molecular mass of about 32 kDa, was successfully overexpressed in Escherichia coli using a T7/lac promoter vector and the enzyme activity was increased 42-times compared with the original strain. It may be an industrial biocatalyst for the preparation of d(-)-tartaric acid. © The Korean Society for Microbiolgy and Biotechnology.

Li B.,Zhejiang University | Pan H.,Zhejiang University | Pan H.,Hangzhou Bioking Biochemical Engineering Co. | Sun W.,Hangzhou Bioking Biochemical Engineering Co. | And 4 more authors.
Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology | Year: 2014

Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 5-keto-D-gluconic acid (5-KGA), a precursor of industrially important L(+)-tartaric acid. To increase the yield of 5-KGA, fermentation conditions of 5-KGA production was optimized. Under the optimum medium and culture conditions in the shake flask, the highest 5-KGA production reached 19.7 g/L, increased by 43.8% after optimization. In a 5-L bioreactor, the pH was controlled at 5.5 and dissolved oxygen (DO) at 15%, 5-KGA production reached 46.0 g/L, raised at least 1.3 times than in the shake flask. Glucose feeding fermentation process was further developed, and the highest 5-KGA production of 75.5 g/L with 70% of yield was obtained, 32.0% higher than the highest reported value. Therefore, this newly developed fermentation process provided a practical and effective alternative for the commercial production of 5-KGA, and further of L(+)-tartaric acid. © 2014 by the Institute of Microbiology, the Chinese Academy of Sciences and the Chinese Society for Microbiology

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