【作者】 潘海峰；

【导师】 李永泉； 张建国；

【作者基本信息】 浙江大学 ， 生化与分子生物学， 2011， 博士

【摘要】 顺式环氧琥珀酸水解酶(cis-epoxysuccinate hydrolase, CESH),是一种水解酶,能催化顺式环氧琥珀酸水解为酒石酸。酒石酸广泛应用于食品、医药、化工、纺织、电镀等行业,其需求量正在逐年递增,目前利用具有立体选择特异性的微生物来源CESH生产酒石酸已逐步成为酒石酸生产技术的新发展方向。本文概述了酒石酸和CESH的研究进展,并研究了新颖水解酶CESH的催化机制及其高效催化工艺。从田园土中筛选到一株产新颖CESH的野生菌,它能将顺式环氧琥珀酸立体特异性的转化为D(-)-酒石酸,经鉴定为博德特氏菌属(Bordetella),并命名为Bordetella sp. BK-52。对该野生菌产酶条件进行优化,最高酶活达764U/g。随后我们从该野生菌中分离纯化得到CESH,酶学性质研究显示,该酶具有温度稳定性好、pH稳定性范围广、金属离子耐受性好,立体特异性高等特点,表明该酶很适合作为工业生物催化剂。此外,我们还首次获得Bordetella sp. CESH的基因序列(GenBank登录号为EU053208)。根据上述CESH基因序列,构建基因工程菌,通过响应面法优化了CESH在大肠杆菌中的表达条件,使工程菌的酶活达到40129U/g,为野生菌的53倍。随后从12种细胞固定化方法中筛选到K-卡拉胶包埋法,通过响应面法优化其固定化条件,使固定化工程菌细胞的酶活回收率达83%。该固定化细胞对温度、pH、金属离子、表面活性剂等多种外界条件具有较强的稳定性,连续使用10批后,残余活力仍为93%,显示出良好的工业应用潜能与前景。红球菌(Rhodococcus opacus) CESH可催化顺式环氧琥珀酸生成L(+)-酒石酸。序列比对和二级结构预测分析显示,该CESH属于卤烷基脱卤酶超家族成员,29个保守位点的定点突变实验、圆二色谱分析、立体特异性分析和酶学性质分析证实,下列9个氨基酸在该酶催化过程中起重要作用：Aspl8、Aspl93、Arg55、Lysl64、Hisl90、Thr22、Tyrl70、Asnl34和Alal88。同位素H218O标记的单转化和多转化实验表明,在水解过程中,水分子中的O原子首先转移到酶中,然后再转移到产物中。接着预测了Rhodococcus opacus CESH的三维结构,整体三维结构和重要活性位点的重叠结果显示,该酶与一些三维结构已知的卤烷基脱卤酶(Hdl、L-DEX和Dh1B)的结构非常相似,采用两步法的催化机制,并推测该催化机制通过催化三联体Asp18-His190-Asp193进行,即CESH中的Asp18首先亲核进攻底物顺式环氧琥珀酸,形成“底物-酶酯中间体”,接着位于活性位点的His190和Asp193激活水分子,进一步水解酯键,并释放产物L(+)-酒石酸。更多还原

【Abstract】 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.更多还原