在肺炎克雷伯氏菌嗜盐 α-淀粉酶的 329 位氨基酸进行定点饱和突变会影响酶的性质。

Site-saturation mutagenesis at amino acid 329 of Klebsiella pneumoniae halophilic α-amylase affects enzymatic properties.

机构信息

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, China.

出版信息

J Biosci Bioeng. 2020 Feb;129(2):155-159. doi: 10.1016/j.jbiosc.2019.09.002. Epub 2019 Sep 28.

DOI:10.1016/j.jbiosc.2019.09.002

PMID:31575478

Abstract

Halophilic α-amylases possess optimal activity in high salt concentrations. Therefore, they can be used in many extreme conditions in industrialised production. In the present work, a halophilic α-amylase (KP) from Klebsiella pneumoniae was characterised, and it exhibited a high specific activity of 3512 U/mg under optimal conditions of 2 M NaCl at 50°C and pH 6.5, but only 97 U/mg in the absence of salt. Furthermore, threonine at position 329 (Thr-329) was found to be related to the non-halophilic properties of KP according to PCR-based site-saturation mutagenesis. The activity of a mutant KP in which this threonine was replaced by aspartic acid was improved 14.6-fold compared with the native enzyme under salt-free conditions, and was increased by 14.8% in the absence of salt. Additionally, the optimal enzymatic properties of KP, including pH and temperature, were altered very little by the amino acid replacement. A further three halophilic α-amylases displayed similar mutational results. The findings provide a reference for bidirectional transformation of KP and similar halophilic enzymes.

摘要

嗜盐 α-淀粉酶在高盐浓度下具有最佳活性。因此，它们可以在工业化生产的许多极端条件下使用。本研究对肺炎克雷伯氏菌来源的嗜盐 α-淀粉酶（KP）进行了表征，其在最适条件 2 M NaCl、50°C 和 pH6.5 下的比活高达 3512 U/mg，但在无盐条件下仅为 97 U/mg。此外，根据基于 PCR 的定点饱和突变，发现位置 329 的苏氨酸（Thr-329）与 KP 的非嗜盐特性有关。与野生型酶相比，将该苏氨酸突变为天冬氨酸的突变体 KP 的活性在无盐条件下提高了 14.6 倍，在无盐条件下提高了 14.8%。此外，KP 的最佳酶学特性，包括 pH 和温度，在氨基酸替换后变化很小。另外三种嗜盐 α-淀粉酶也显示出类似的突变结果。这些发现为 KP 和类似嗜盐酶的双向转化提供了参考。