Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
Department of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata University, Yamagata, Japan.
J Basic Microbiol. 2022 Feb;62(2):174-184. doi: 10.1002/jobm.202100335. Epub 2021 Nov 22.
A moderately halotolerant serine protease was previously isolated from Bacillus subtilis from salted, fermented food. Eight mutation sites on the protein surface were selected for protein engineering based on sequence and structural comparisons with moderately halotolerant proteases and homologous non-halotolerant proteases. The newly constructed multiple mutants with substituted Asp and Arg residues were compared with the recombinant wild type (rApr) and the previously constructed mAla-8 substituted with Ala to analyze the contribution of protein surface charge to the salt adaptation of the protease. The three mutants showed >1.2-fold greater halotolerance than rApr. In addition, the mutants showed a broader range of pH stability than rApr, retaining >80% of their maximum activity in the pH range 5.0-11. The mutants also retained >75% of their activity after incubation for 1 h at pH 8.0 and 55°C or at pH 11.5 and 25°C. The Asp and Arg residues exchanged by multiple substitution probably played a role in increasing protein surface hydration and solubility in high salt conditions. This study illustrated that increasing a high proportion of the negative or positive charge on the surface of the Bacillus serine protease stably improved the protein's salt adaptation.
先前从腌制发酵食品中的枯草芽孢杆菌中分离到一种耐盐性中等的丝氨酸蛋白酶。根据序列和结构比较，以及与耐盐性中等蛋白酶和同源非耐盐性蛋白酶的比较，在蛋白质表面选择了 8 个突变位点进行蛋白质工程改造。与重组野生型（rApr）和先前构建的用丙氨酸取代的 mAla-8 相比，比较了新构建的带有取代的天冬氨酸和精氨酸残基的多个突变体，以分析蛋白质表面电荷对蛋白酶耐盐性的贡献。与 rApr 相比，这三种突变体的耐盐性提高了>1.2 倍。此外，与 rApr 相比，突变体具有更宽的 pH 稳定性范围，在 pH 5.0-11.0 范围内保留了>80%的最大活性。在 pH 8.0 和 55°C 或 pH 11.5 和 25°C 下孵育 1 小时后，突变体的活性也保留了>75%。多重取代交换的天冬氨酸和精氨酸残基可能在增加蛋白质表面在高盐条件下的水合和溶解度方面发挥了作用。本研究表明，增加枯草芽孢杆菌丝氨酸蛋白酶表面的高比例负电荷或正电荷可稳定提高蛋白质的耐盐性。
