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首页> 外文期刊>Applied Microbiology >Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues
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Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

机译：通过引入脯氨酸和谷氨酸残基提高链霉菌木聚糖酶的热稳定性

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Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris . Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.
机译：蛋白质工程通常用于提高酶在高温下的活性和稳定性的鲁棒性。在这项研究中，我们确定了预期会影响链霉菌sp。的热稳定性的四个残基。菌株S9木聚糖酶XynAS9通过多序列分析（MSA）和分子动力学模拟（MDS）。采用定点诱变通过用脯氨酸或谷氨酸（V81P，G82E，V81P / G82E，D185P / S186E和V81P / G82E / D185P / S186E）取代这些残基来构建五个突变体，以及突变体和野生型酶在毕赤酵母中表达。与野生型XynAS9相比，所有五个突变酶均显示出改善的热性质。活性和稳定性分析（包括圆二色性和差示扫描量热法）显示，位置81和82处的突变比185和186位处的突变提高了热性能。具有组合取代的突变体（V81P / G82E和V81P / G82E （/ D185P / S186E）表现出最佳温度变化最明显，向上约17°C，并且它们在70°C时热失活的半衰期和熔化温度分别增加了9倍和约7.0°C。 V81P和G82E在相邻位置的突变组合使单突变的作用增加了一倍以上。两个突变区均位于长二级结构元件的末端，并可能使局部结构硬化。 MDS表明位于内部β-桶末端的位置81和82之后的长环区域易于展开。刚性位点的主链和由活动位点峡谷底部的突变填充的凹槽可以稳定突变体，从而提高其热稳定性。

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