Improvement of the halotolerance of a Bacillus serine protease by protein surface engineering
Improvement of the halotolerance of a Bacillus serine protease by protein surface engineering
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DOI：
10.1002/jobm.202100335
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发表时间：
2021-11
影响因子：
3.1
通讯作者：
S. Takenaka;Airi Takada;Y. Kimura;M. Watanabe;Ampin Kuntiya
中科院分区：
文献类型：
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作者：
S. Takenaka;Airi Takada;Y. Kimura;M. Watanabe;Ampin Kuntiya
先前从盐渍发酵食品中分离的枯草芽孢杆菌中分离出一种中等耐盐性的丝氨酸蛋白酶。通过与中度耐盐蛋白水解酶和同源非耐盐蛋白水解酶的序列和结构比较，选择了蛋白质表面的8个突变位点进行蛋白质工程。将新构建的含有Asp和Arg残基的多重突变体与重组野生型(RapR)和先前构建的Ala替代的Mala-8进行比较，以分析蛋白质表面电荷对该酶盐适应性的贡献。这三个突变体表现出比RapR高1.2倍的耐盐性。此外，突变体具有比RapR更宽的pH稳定性，在pH 5.0-11的范围内保持了80%的最大活性。在pH 8.0和55℃或在pH 11.5和25℃孵育1 h后，突变体仍保持了75%的活性。通过多次取代交换的天冬氨酸和精氨酸残基可能在高盐条件下增加了蛋白质的表面水化和溶解性。这项研究表明，增加芽孢杆菌丝氨酸蛋白酶表面高比例的负电荷或正电荷可以稳定地提高蛋白质的盐适应性。
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.