Rational design of thermostability in bacterial 1,3-1,4-β-glucanases through spatial compartmentalization of mutational hotspots
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- 作者：Chengtuo Niu ; Linjiang Zhu ; Xin Xu ; Qi Li
- 关键词：1 ; 3 ; 1 ; 4 ; β ; Glucanase ; Iterative saturation mutagenesis ; Molecular dynamics simulation ; Spatial compartmentalization ; Thermostability
- 刊名：Applied Microbiology and Biotechnology
- 出版年：2017
- 出版时间：February 2017
- 年：2017
- 卷：101
- 期：3
- 页码：1085-1097
- 全文大小：
- 刊物类别：Chemistry and Materials Science
- 刊物主题：Microbiology; Microbial Genetics and Genomics; Biotechnology;
- 出版者：Springer Berlin Heidelberg
- ISSN：1432-0614
- 卷排序：101
文摘
Higher thermostability is required for 1,3-1,4-β-glucanase to maintain high activity under harsh conditions in the brewing and animal feed industries. In this study, a comprehensive and comparative analysis of thermostability in bacterial β-glucanases was conducted through a method named spatial compartmentalization of mutational hotspots (SCMH), which combined alignment of homologous protein sequences, spatial compartmentalization, and molecular dynamic (MD) simulation. The overall/local flexibility of six homologous β-glucanases was calculated by MD simulation and linearly fitted with enzyme optimal enzymatic temperatures. The calcium region was predicted to be the crucial region for thermostability of bacterial 1,3-1,4-β-glucanases, and optimization of four residue sites in this region by iterative saturation mutagenesis greatly increased the thermostability of a mesophilic β-glucanase (BglT) from Bacillus terquilensis. The E46P/S43E/H205P/S40E mutant showed a 20 °C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature (Tm) compared to wild-type BglT. Its half-life values at 60 and 70 °C were 3.86-fold and 7.13-fold higher than those of wild-type BglT. The specific activity of E46P/S43E/H205P/S40E mutant was increased by 64.4 %, while its stability under acidic environment was improved. The rational design strategy used in this study might be applied to improve the thermostability of other industrial enzymes.