基于分子结构评价的Bacillus subtilis β-1,4-内切木聚糖酶胰蛋白酶抗性的理性设计  

Trypsin-resistant Improvement of Bacillus subtilis β-1,4-endoxylanase by Rational Design Based on Molecular Structure Evaluation

作　　者：虞晓丹[1,2] 吴秀秀[1,2] 姚冬生[1,2] 刘大岭[1,3] 谢春芳[1,3] 

机构地区：[1]暨南大学生物医药研究院,广州510632 [2]暨南大学微生物技术研究所,广州510632 [3]暨南大学生物工程系,广州510632

出　　处：《中国生物工程杂志》2016年第8期80-88,共9页China Biotechnology

基　　金：国家高技术研究发展计划(863计划)(2013AA102801);广东省省级科技计划项目(2013B090600141)资助项目

摘　　要：饲料用酶在饲喂过程中,不可避免地会受到消化道中蛋白酶的破坏而令其使用效率受到影响,因此,饲料酶的蛋白酶抗性是其十分重要的性质之一。基于酶反应原理与计算化学理论对Bacillus subtilis 168β-1,4-内切木聚糖酶(Xyn A)的胰蛋白酶抗性进行理性设计,最终得到三个突变体——Xyn A^(R121C)、Xyn A^(R121C/K98Q)和Xyn A^(K39I)。酶学性质分析显示Xyn A^(R121C)和Xyn A^(R121C/K98Q)与野生型(Xyn A)的最适反应温度均为60℃,Xyn A^(K39I)为40℃;突变体酶与野生型酶的最适p H都是6.0;人工肠液(p H 6.8,10 mg/ml胰蛋白酶溶液)处理野生型及其突变体后,Xyn A^(R121C/K98Q)和Xyn A^(R121C)的残留酶活力均明显比野生型高,它们的半衰期分别是野生型的2.02倍和1.52倍,Xyn A^(K39I)在胰蛋白酶溶液中的半衰期为90min,比野生型缩短了37 min。In the feeding process, the decomposition of the digestive tract proteases is one of the important reasons affecting the efficiency of feed enzymes. Therefore, the feed enzymes having protease resistance is a very important property. The rational design of protein modification with resistance to protease based on the method of the biocatalysis and computational chemistry was used, 168 1,4-13-endoxylanase with resistance to protease and which has been established to improve Bacillus subtilis to get the excellent properties. Single site mutation to R121 and K39 and double sites mutations to R121/K98 were utilized to get the fine properties of mutant enzymes XynATM, XynAR121c/K98Q and XynAK391. Fortunately, the excellent properties were gotten finally. The optimal treatment with simulated intestinal fluid （pH 6.8, 10mg/ml trypsin solution） , the remained enzyme activity of mutants was much higher than the wild type. As for XynAR121C, its half-life period was 193 min, 1.52 times higher than XynA. As for XynAR121c/K98Q, its half-life period was 257 min, 2.02 times higher than XynA. As for XynAK391, after incubation with simulated intestinal fluid at 40℃ for different time, its half-life period was 90 min, only 37min shorter than XynA.

关 键 词：理性设计 木聚糖酶 胰蛋白酶抗性 酶稳定性 

分 类 号：Q819[生物学—生物工程]