基于理性设计提高枯草芽孢杆菌角蛋白酶的热稳定性

Improving the thermostability of Bacillus subtilis keratinase by rational design

角蛋白酶KerZ1在其最适反应温度60℃下能够有效地催化水解羽毛角蛋白废物,但热稳定性较差。以KerZ1为研究对象,利用蛋白的理性设计来提高其热稳定性。首先,通过分子动力学模拟软件AMBER16以及HotSpot Wizard服务器的B-factor分析预测出KerZ1中对热敏感的柔性环区域;然后,结合β-转角残基的统计分析将引入位置的原有残基替换为保守性较高的氨基酸。结果表明,突变体酶A128D的失活半衰期(t_(1/2))和半失活温度(T_(50))较KerZ1提高了12.32 min和5.8℃。同时,突变体的动力学参数(k_(cat)/K_(m))表征结果显示,其催化效率未受到显著影响。另外,分析突变位点以及周围氨基酸残基的分布,结果表明突变体酶A128D的表面亲水性增加可能是提高其热稳定性的主要原因。该研究通过理性设计柔性环区域提高了角蛋白酶的热稳定性,为推动角蛋白酶的工业化应用奠定了基础。

Bacillus subtilis keratinase(KerZ1)could effectively degrade keratin waste at 60℃,however,its thermostability is not preferable.The rational design method was applied to increase its thermostability.First,the B-factor analysis and molecular dynamics simulation software AMBER16 were used to predict the flexible loop regions.Subsequently,combined with theβ-turn residue statistic,the residues at the introduced position were replaced with conserved amino acids.The results showed that the half-life(t_(1/2))and half inactivation temperature(T_(50))of the A128D mutant increased by 12.32 min and 5.8℃,respectively.And the catalytic efficiency was not significantly affected.In addition,the analysis of the distribution of mutant residues and surrounding amino acid residues indicated that the increased surface hydrophilicity of the A128D mutant might be the main reason for thermostability.In conclusion,this strategy improves the thermostability of keratinase by modifying the flexible loop region,and lays the foundation for the industrial application of keratinase.