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首页> 外文期刊>Journal of Biotechnology >Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553
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Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

机译：马克斯克鲁维酵母CBS 1553的对映选择性酯酶的纯化和鉴定

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摘要

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.
机译：纯化并表征了来自酵母克鲁维酵母CBS 1553的细胞内酯酶，该酶对1,2-O-异亚丙基甘油（IPG）的外消旋酯具有有趣的对映选择性水解活性。研究了以5升发酵规模获得对映选择性酯酶的最佳培养条件。粗细胞提取物中的两种酯酶活性（EST1和EST2）通过具有特异性活性染色的天然PAGE进行鉴定，并通过阴离子交换色谱法彼此分离。 EST1在外消旋IPG乙酸酯的拆分中比EST2具有更高的活性和对映选择性，并通过疏水相互作用色谱和制备性电泳进一步纯化（最终比活度约为300 U mg（-1），显示出一条主要的蛋白带，分子量为29 kDa。EST1在pH 8.0和10.0之间显示最佳活性，并在7.0-10.0的pH范围内保持稳定。此外，它具有较高的热稳定性，并在高达80摄氏度的温度下具有活性，并且在15％（ v / v），该酶在IPG的乙酸酯水解中显示出相似的Vmax，而对（S）-IPG乙酸酯的Km值显着低于对（R）-对映体的Km值（5.3和分别为70 microM和70 microM）。最后，在存在不同甘油酯和具有不同链长的合成底物的情况下，EST1活性的比较表明，该生物催化剂非常适合短链底物特斯

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