Hangzhou Bioking Biochemical Engineering Co.

Hangzhou, China

Hangzhou Bioking Biochemical Engineering Co.

Hangzhou, China
SEARCH FILTERS
Time filter
Source Type

Liu J.,Jiangnan University | Xie Z.,Hangzhou Bioking Biochemical Engineering Co. | Shin H.-D.,Georgia Institute of Technology | Li J.,Jiangnan University | And 3 more authors.
Journal of Biotechnology | Year: 2017

Aspergillus oryzae finds wide application in the food, feed, and wine industries, and is an excellent cell factory platform for production of organic acids. In this work, we achieved the overproduction of L-malate by rewiring the reductive tricarboxylic acid (rTCA) pathway and L-malate transport pathway of A. oryzae NRRL 3488. First, overexpression of native pyruvate carboxylase and malate dehydrogenase in the rTCA pathway improved the L-malate titer from 26.1 g L−1 to 42.3 g L−1 in shake flask culture. Then, the oxaloacetate anaplerotic reaction was constructed by heterologous expression of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase from Escherichia coli, increasing the L-malate titer to 58.5 g L−1. Next, the export of L-malate from the cytoplasm to the external medium was strengthened by overexpression of a C4-dicarboxylate transporter gene from A. oryzae and an L-malate permease gene from Schizosaccharomyces pombe, improving the L-malate titer from 58.5 g L−1 to 89.5 g L−1. Lastly, guided by transcription analysis of the expression profile of key genes related to L-malate synthesis, the 6-phosphofructokinase encoded by the pfk gene was identified as a potential limiting step for L-malate synthesis. Overexpression of pfk with the strong sodM promoter increased the L-malate titer to 93.2 g L−1. The final engineered A. oryzae strain produced 165 g L−1 L-malate with a productivity of 1.38 g L−1 h−1 in 3-L fed-batch culture. Overall, we constructed an efficient L-malate producer by rewiring the rTCA pathway and L-malate transport pathway of A. oryzae NRRL 3488, and the engineering strategy adopted here may be useful for the construction of A. oryzae cell factories to produce other organic acids. © 2017 Elsevier B.V.


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.


Zhang C.,Zhejiang University | Pan H.,Zhejiang University | Pan H.,Hangzhou Bioking Biochemical Engineering Co. | Yao L.,Zhejiang University | And 6 more authors.
Biotechnology Letters | Year: 2016

Objectives: To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate. Results: By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg−1 (kcat/Km, 9.8 ± 0.1 mM−1 s−1), which was 230 % higher than that of wild-type enzyme 355 U mg−1 (kcat/Km, 5.3 ± 0.1 mM−1 s−1). However, the thermostability of the mutant F10Q slightly decreased. Conclusions: The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis. © 2016, Springer Science+Business Media Dordrecht.


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.,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 | 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.


Bao W.,Zhejiang University | Bao W.,Hangzhou Bioking Biochemical Engineering Co. | Pan H.,Zhejiang University | Pan H.,Hangzhou Bioking Biochemical Engineering Co. | And 6 more authors.
Journal of Bioscience and Bioengineering | Year: 2015

A novel cis-epoxysuccinate hydrolase (CESH) producing strain of Labrys sp. BK-8 for production of l(+)-tartaric acid was isolated and identified. After optimization, a maximum activity of 3597±151U/g was achieved in batch culture in a 10L fermentor. When Labrys sp. BK-8 was immobilized on κ-carrageenan, the immobilized cells showed a high conversion rate (99%), enantioselectivity (EE99.5%) and storage stability (90d). A conversion rate of 97% was still achieved after 10 repeat batches. The CESH was stable over a broad range of temperatures (up to 45°C) and pH values (4.0-10.0). The Labrys sp. BK-8 isolate provides a new alternative with good stability for the industrial biosynthesis of l(+)-tartaric acid. © 2014 The Society for Biotechnology, Japan.


Cheng Y.,Zhejiang University | Pan H.,Zhejiang University | Pan H.,Hangzhou Bioking Biochemical Engineering Co. | Bao W.,Hangzhou Bioking Biochemical Engineering Co. | And 6 more authors.
Biotechnology Letters | Year: 2014

The gene encoding a novel cis-epoxysuccinate hydrolase, which hydrolyzes cis-epoxysuccinate to l (+)-tartaric acid, was cloned from Klebsiella sp. BK-58 and expressed in Escherichia coli. The ORF was 825 bp encoding a mature protein of 274 amino acids with a molecular mass of 30.1 kDa. Multiple sequence alignment showed that the enzyme belonged to the haloacid dehalogenase-like super family. Homology modeling and site-directed mutagenesis were performed to investigate the structural characteristics of the enzyme. Its overall structure consisted of a core domain formed by six-stranded parallel β-sheets flanked by seven α-helices and a subdomain that had a four helix bundle structure. Residues D48, T52, R85, N165, K195, Y201, A219, H221, and D224 were catalytically important forming the active pocket between the two domains. An 18O-labeling study suggested that the catalytic reaction of the enzyme proceeded through a two-step mechanism. © 2014, Springer Science+Business Media Dordrecht.


Patent
Hangzhou Bioking Biochemical Engineering Co. | Date: 2015-02-25

The present invention relates to a cis-epoxysuccinate hydrolase-encoding gene, a polypeptide encoded by the gene and related application thereof. The cis-epoxysuccinate hydrolase-encoding gene has a nucleotide sequence as shown in SEQ ID NO.1, and cis-epoxysuccinate hydrolase encoded by the gene has an amino acid sequence as shown in SEQ ID NO.2. A prokaryotic expression vector constructed by the cis-epoxysuccinate hydrolase gene is transformed into Escherichia coli to construct genetically engineered bacteria which can be induced to efficiently convert cis-epoxysuccinic acid or its salts into L(+)-tartaric acid or its salts. The cis-epoxysuccinate hydrolase produced from E. coli has the ability to efficiently convert cis-epoxysuccinic acid or its salts into L(+)-tartaric acid or its salts.


PubMed | Hangzhou Bioking Biochemical Engineering Co. and Zhejiang University
Type: Journal Article | Journal: Biotechnology letters | Year: 2016

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 Umg(-1) (k cat /K m , 9.80.1mM(-1)s(-1)), which was 230% higher than that of wild-type enzyme 355Umg(-1) (k cat /K m , 5.30.1mM(-1)s(-1)). However, the thermostability of the mutant F10Q slightly decreased.The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

Loading Hangzhou Bioking Biochemical Engineering Co. collaborators
Loading Hangzhou Bioking Biochemical Engineering Co. collaborators