巧克力微杆菌SIT101酯酶基因的克

发布时间：2018-04-02 15:07

  本文选题：巧克力微杆菌　切入点：酯酶　出处：《上海应用技术大学》2016年硕士论文

【摘要】：酯酶(Esterases,EC 3.1.1.1)是一类可以催化酯键水解和形成的酶,现在已经成为一种重要的生物催化剂应用于制药、食品和生物降解等领域。我们课题组在前期的工作中,筛选出一株巧克力微杆菌SIT101,其所产酯酶能够高立体选择性的催化内消旋的生物素中间体二甲酯不对称水解为对应的(4S,5R)构型的半酯,它是合成D-生物素的重要手性中间体。由于野生菌产酶水平较低,将酯酶克隆表达于重组菌将有可能显著提高其表达水平,提升其工业应用潜力。本文首先在测定巧克力微杆菌SIT101基因组草图的基础上,采用基因组数据挖掘结合计算机辅助虚拟筛选的策略,预测了 5种可能具有催化活性的酯酶序列,通过酶基因的克隆表达以及活性和选择性的测试确定了酯酶EstSIT101能够催化生物素中间体二甲酯不对称水解为相应的(4S,5R)-半甲酯,对映体过量值高达99%。其次,从温度、pH值、底物特异性、动力学等方面对酯酶EstSIT101的酶学性质进行了表征,为进一步应用奠定了基础。主要实验结论如下:(1)巧克力微杆菌SIT01酯酶的筛选、克隆表达与纯化。首先应用基因组挖掘的方法从Microbacterium chocolatum SIT 101基因组中预测了31个酯水解酶基因,采用Swiss-Model软件对它们进行了同源建模,并将其与底物分子进行了分子对接,以二酯酯键021与活性位点组氨酸的咪唑的H原子之间的距离作为计算机辅助筛选的重要判据,从中预测了五种酯酶可能会立体选择性催化水解生物素中间体二甲酸,对结合能最低的三种酯酶进行了克隆表达和活性测试。发现只有酯酶EstSIT01具有催化生物素中间体二甲酯水解生成(4S,5R)生物素半甲酯。经过镍柱纯化和超滤离心后的纯化酶浓度为3.56mg/mL,目标酶占细胞总的可溶性蛋白的28.2%。SDS-PAGE结果显示酯酶EstSIT01分子量约为39kDa,与理论分子量为39.35kDa相符。酯酶EstSIT01包含370个氨基酸,同源建模的结果表明,EstSIT01拥有典型的α/β水解酶折叠结构和保守的催化三联体Ser110-His330-Asp268。(2)酯酶EstSIT01酶学性质的表征。酯酶EstSIT01倾向于水解短链酯类,而且该酶立体选择性水解生物素二甲酯生成生物素半甲酯的产率和e.e值均高达99%。酯酶EstSIT01对对硝基苯酚乙酸酯(p-NPA)和生物素中间体二甲酯的最适反应温度分别为65℃和45℃,最适pH分别为9.5和10.0。在30℃下,EstSIT01比较稳定,而过碱(pH10)不利于酶的稳定性。并且以生物素二甲酯为底物,温度和pH对e.e值没有影响。当温度为30℃,pH 8.0时,Km和Vmax分别为0.1468mM和8.856μM/min。酯酶EstSIT01高选择性和高活力表明它是一种在制药工业上有前景的生物催化剂。
[Abstract]:Esterasesus (EC 3.1.1.1) is a kind of enzyme which can catalyze the hydrolysis and formation of ester bonds. It has been used as an important biocatalyst in pharmaceutical, food and biodegradation fields.In our earlier work, a strain of microbacillus chocolate SIT101 was screened. The esterase produced by SIT101 can catalyze asymmetric hydrolysis of dimethyl ester, a biotinylated intermediate with high stereoselectivity, into a corresponding semi-ester with a 4S5R configuration, and the esterase produced by SIT101 can catalyze asymmetric hydrolysis of dimethyl ester, a biotin intermediate, with high stereoselectivity.It is an important chiral intermediate for the synthesis of D-biotin.Due to the low level of enzyme production by wild bacteria, it is possible to improve the expression level of esterase by cloning esterase in recombinant bacteria and enhance its potential for industrial application.In this paper, based on the determination of SIT101 genome sketches of microbacillus chocolate, five possible esterase sequences with catalytic activity were predicted by using genomic data mining combined with computer-aided virtual screening strategy.It was confirmed that esterase EstSIT101 could catalyze asymmetric hydrolysis of dimethyl ester, an intermediate of biotin, into corresponding semi-methyl ester by cloning, expression and activity of enzyme gene. The enantiomeric overdose of esterase EstSIT101 was as high as 99g.Secondly, the enzymatic properties of esterase EstSIT101 were characterized from temperature and pH value, substrate specificity and kinetics, which laid a foundation for further application.The main results are as follows: 1) screening, cloning, expression and purification of SIT01 esterase from microbacillus chocolate.First, 31 esterase genes were predicted from the genome of Microbacterium chocolatum SIT 101 by the method of genome mining, and the homologous modeling of them was carried out by Swiss-Model software, and they were docked with the substrate molecule.Using the distance between diester ester bond 021 and H atom of active site histidine imidazole as an important criterion of computer-aided screening, it is predicted that five esterases may catalyze stereoselective hydrolysis of biotin intermediate dicarboxylic acid.Three esterases with the lowest binding energy were cloned, expressed and tested for their activity.It was found that only esterase EstSIT01 could catalyze the hydrolysis of dimethyl ester, an intermediate of biotin, to form biotinyl hemimethyl ester.After purification by nickel column and ultrafiltration centrifugation, the concentration of purified enzyme was 3.56 mg / mL. The result of 28.2%.SDS-PAGE showed that the molecular weight of esterase EstSIT01 was about 39 kDa, which was consistent with the theoretical molecular weight of 39.35kDa.Esterase EstSIT01 tends to hydrolyze short chain esters, and the yield and e.e value of stereoselective hydrolysis of biotinyldimethyl ester to biotin hemimethyl ester are as high as 99e.The optimum reaction temperature of esterase EstSIT01 for p-nitrophenol acetate (p-NPA) and biotin intermediate dimethyl ester was 65 鈩，

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