Studies On Cis-epoxysuccinic Acid Hydrolase From Bordetella Sp. Strain 1-3

As an important chiral pool,D(-)-tartaric acid is mainly used as chiral resolving agent in pharmacy industry,sour additive and preservative in food industry,whereas it is rarely exist as a natural resource.Producing of D(-)-tartaric acid with chemical resolution is restricted because of the complex techniques,high costs and environmental pollution.Having high effectiveness,high selectivity,mild reaction conditions and environmental friendliness,biotransformation becomes an ideal alterative method to produce D(-)-tartaric acid.Cis-epoxysuccinic acid hydrolase can hydrolyze cis-epoxysuccinic acid to produce D(-)-tartaric acid.At present,research report about this hydrolase is rare except for two patents from Japan.In this study,a bacterium named strain 1-3 was isolated from soil and the biotransformation of D(-)-tartaric acid was achieved by the immobilized cells of strain 1-3.Subsequently,the fermentation conditions of this bacterium were optimized by using response surface method.The cis-epoxysuccinic acid hydrolase was then purified and its characteristics were studied.Finally,the gene of this enzyme was cloned and expressed in E.coli.The results of this research were sumerized as follows:1.The isolation and identification of cis-epoxysuccinic acid hydrolase producing strain:A bacterium strain which can produce D(-)-tartaric acid was isolated after enrichment culture and streaking plate purification.Based on its morphological studies,physiochemical identification,16S rDNA sequencing and phylogenetic analysis,this isolate was identified to be Bordetella sp.2.Biotransformation by using immobilized cells:After immobilization with gel-embedding method,the cells were used to transform cis-epoxysuccinic acid.The production of biotransformation was purified and a transparent crystal was obtained. After the analysis of Fourier transform infrared spectroscopy,nuclear magnetic resonance and specific optical rotation,the production was identified to be D(-)-tartaric acid. 3.Optimization of fermentation conditions:The optimal fermentation conditions were studied using Response Rurface Methodology with biomass and enzyme activity as variants.The optimal culture medium included yeast extraction 7.8 g/L,cis-epoxysuccinic acid 9.8 g/L,KH₂PO₄ 1.12g/L,K₂HPO₄·3H₂O 3g/L, MgSO₄·7H₂O 0.625g/L,element solution 12.5 mL/L,pH 7.0.After fermentation at 30℃for 30h,the enzyme activity reached 9.27 U/mL.4.Purification and characterization of cis-epoxysuccinic acid hydrolase: Cells were harvested from 10 1 culture medium by centrifugation and the cells were disrupted with ultrasonic to obtain crude enzyme.The enzyme was purified 177 folds after gradient precipitation of ammonium sulfate,DEAE-Cellulose ion exchange chromatography,Toyopearl HW-65C hydrophobic chromatography and Sephadex G-75 molecular-exclusion chromatography.The purified enzyme was homogeneous as judged by PAGE,and the subunit molecular mass was estimated to be 33 KDa by SDS-PAGE.Studies on characteristics showed that the optimal reaction temperature was 45℃and the optimal pH was 7.0.The cis-epoxysuccinic acid hydrolase was stable between pH 5.0～10.0 and at the temperature below 55℃.The Km value for cis-epoxysuccinic acid was 10 mmol/L.The enzyme was markedly inhibited by Hg²⁺, EDTA and o-phenanthrolin.ICP-MS analysis indicated that this hydrolase had Zn²⁺as a cofactor.5.Cloning and expression of cis-epoxysuccinic acid hydrolase:The DNA fragment encoding cis-epoxysuccinic acid hydrolase was amplified with PCR and it was then inserted into pET-15b expression vector.Recombinant pET-15b was transformed into E.coli.BL21 to express the cis-epoxysuccinic acid hydrolase.The amount of enzyme production by E.coli.BL21 was increased more than 70 folds than that of produced by the strain 1-3.The studies on the characteristics of the recombinant enzyme showed that it was stable in the range of pH 5.0～9.0,which was appreciably narrower than that of the original's,whereas other characteristics are same as the original one.