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首页> 外文期刊>Biochemistry >Circular Permutation of Bacillus circulans Xylanase: A Kinetic and Structural Study
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Circular Permutation of Bacillus circulans Xylanase: A Kinetic and Structural Study

机译：环状芽孢杆菌木聚糖酶的环状排列：动力学和结构研究。

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ABSTRACT: The 20 kDa Bacillus circulans Bcx is a well-studied endoxylanase with a /3-jellyroll fold that places its N- and C-termini in salt bridge contact. Initial experiments verified that Bcx could be circularly permuted by PCR methods to introduce new termini in loop regions while linking its native termini directly or via one or two glycines. Subsequently, a library of circular permutants, generated by random DNase cleavage of the circularized Bcx gene, was screened for xylanase activity on xylan in Congo Red-stained agar. Analysis of 35 unique active circular permutants revealed that, while many of the new termini were introduced in external loops as anticipated, a surprising number were also located within /3-strands. Furthermore, several permutations placed key catalytic residues at or near the new termini with minimal deleterious effects on activity and, in one case, a 4-fold increase. The structure of one permutant was determined by X-ray crystallography, whereas three others were probed by NMR spectroscopy. These studies revealed that the overall conformation of Bcx changed very little in response to circular permutation, with effects largely being limited to increased local mobility near the new and the linked old termini and to a decrease in global stability against thermal denaturation. This library of circularly permuted xylanases provides an excellent set of new start points for directed evolution of this commercially important enzyme, as well as valuable constructs for intein-mediated replacement of key catalytic residues with unnatural analogues. Such approaches should permit new insights into the mechanism of enzymatic glycoside hydrolysis.
机译：摘要：20 kDa环状芽孢杆菌Bcx是一种经过充分研究的内切木聚糖酶，具有/ 3-果冻折叠，使其N-和C-末端与盐桥接触。最初的实验证实，Bcx可以通过PCR方法循环置换，在环区域引入新的末端，同时直接或通过一个或两个甘氨酸连接其天然末端。随后，通过随机DNase切割环化的Bcx基因生成的环状置换物文库，筛选了刚果红染色琼脂中木聚糖酶对木聚糖的活性。对35个独特的活动圆形置换的分析表明，尽管许多新的末端已按预期引入外部循环中，但令人惊奇的数目也位于/ 3股内。此外，几个排列将关键的催化残基放置在新末端处或附近，对活性的有害影响最小，在一种情况下，增加了4倍。一个置换体的结构通过X射线晶体学确定，而其他三个通过NMR光谱进行探测。这些研究表明，Bcx的整体构型响应圆排列变化很小，其作用很大程度上限于新末端和相连末端附近的局部迁移性增加以及抗热变性的整体稳定性降低。该环状排列的木聚糖酶文库为该商业上重要的酶的定向进化提供了极好的新起点集，以及用非天然类似物进行内含肽介导的关键催化残基替代的有价值的构建体。此类方法应允许对酶苷水解的机理有新的认识。

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