Study On The Catalytic Mechanism Of A Novel Hydrolase CESH And Its Catalvtic Process With A High Efficiency

cis-Epoxysuccinate hydrolase (CESH), a kind of hydrolase, could transform cis-epoxysuccinic acid into tartaric acid. Tartaric acid is widely used in food, pharmaceutical, chemistry, textile, electroplating industry and et al., resulting in its quantity demand increasing progressively year by year. A novel approach for tartaric acid production using stereospecific CESH from microorganism has become an inevitable development trend. Characters of tartaric acid and CESH enzyme are reviewed here, and we study on the catalytic mechanism of a novel CESH and its catalytic process with a high effieciency.A novel wild-type strain with CESH activity was isolated from vegetable field for biotransforming cis-epoxysuccinate into D(-)-tartaric acid. It was assigned to genus Bordetella and named Bordetella sp. BK-52. The maximum CESH activity reached764U/g after optimization its fermentation condition. Then the CESH was purified from the wild-type strain, and enzyme properties assay showed that CESH had high temperature and pH stability, was weakly affected by most metal ions and exhibited high stereospecificity, which indicated that it was potential for use in industry as a biologic catalyst. In addition, Bordetella sp. CESH gene (GenBank Accession No. EU053208) was firstly obtained.According to the above CESH gene sequence, the gene engineering bacteria were constructed and its maximum activity was40129U/g, which was53-fold higher than wild-type strain, after optimizing expression conditions to enhance CESH production in Escherichia coli by response surface methodology. Then, κ-carrageenan gel entrapment was selected as the best matrix through12methods for immobilization cells.83%yield of activity was got after optimization immobilization conditions by response surface methodology. The immobilized cells were relatively stable in various environmental conditions, such as temperature, pH, metal ions and surfactants, and still remain93%residual activity after10repeated batches, indicating its potential and prospect in industry.Rhodococcus opacus CESH could transform cis-epoxysuccinic acid into L(+)-tartaric acid. It was belong to haloacid dehalogenase like superfamily according to the results of amino acid sequence alignments and second structure predictions.9residues played indispensable roles in the catalytic function, including Asp18, Asp193, Arg55, Lys164, His190, Thr22, Tyr170, Asn134and Alal88, which were determined by site-directed mutagenesis of29conserved residues, circular dichroism spectra analysis, enantioselectivity analysis and enzyme properties assay. Single and multiple turnover reaction in H218O showed that the O atom of water firstly transfer into the eznzyme and then to the product in the reaction. Then3D structure of Rhodococcus opacus CESH was predicted. Superpositions in the whole structure and in the active site structure indicated that CESH is structurely similar to those3D-structure-reported haloacid dehalogenase (Hdl, L-DEX and Dh1B). We proposed that its reaction proceeds through the two-step mechanism and characterized by Asp18-His190-Asp193catalytic triad. Specifically, Asp18of CESH firstly nucleophilic attack on the substrate cis-epoxysuccinic acid, resulting in the formation of an ester bond between the enzyme and substrate. The His190and Asp193, in the active site, activate the water molecule, and hydrolyse the ester bond allowing the release of product L(+)-tartaric acid.