蛋白质分子表面氨基酸突变提高植酸酶Yi APPA的活性和热稳定性

Mutation of Amino Acid Residues on the Protein Surface Improves the Activity and Thermostability of Phytase YiAPPA

为了提高植酸酶的活性和热稳定性,增加其在食品加工领域的应用潜力,对植酸酶Yi APPA进行同源模建,结合蛋白质分子表面氨基酸突变策略,选择位于分子表面的赖氨酸和甘氨酸进行定点突变,构建单位点突变体。通过活性和热稳定性筛选,获得活性和热稳定性显著提高的突变体K216R以及热稳定性提高的突变体K189R。通过有益突变位点叠加策略,进一步构建并表征组合突变体K189R/K216R的酶活力及热稳定性。结果表明:与Yi APPA相比,K189R/K216R于80℃半衰期由14.81 min延长至23.35 min,半失活温度由55.12℃提升至62.44℃,热解折叠温度由48.36℃提升至53.18℃。并且K189R/K216R于37℃、pH 4.5的酶活力由3959.98 U/mg提高至4469.13 U/mg。分子结构建模和分子动力学模拟的结果显示:K189R/K216R中引入了新的氢键,能够提高酶部分结构单元的稳定性,使其热稳定性得到提高;同时K189R/K216R的催化口袋体积增大是其活性提高的主要原因。本研究通过蛋白质分子表面氨基酸突变策略可有效提高植酸酶Yi APPA的活性和热稳定性,为植酸酶及其他类型酶的分子改造提供参考依据。

In order to improve the activity and thermal stability of phytase and to enhance its application potential in the food processing industry,single-site mutants were constructed by homology modeling of phytase YiAPPA and directed mutagenesis of lysine(Lys)and glycine(Gly)residues located on the molecular surface.The mutant K216R with significantly improved activity and thermostability and the mutant K189R with improved thermostability were obtained.Subsequently,the combined mutant K189R/K216R was constructed and its enzyme activity and thermal stability were characterized.Compared with YiAPPA,the half-life of K189R/K216R at 80℃was increased from 14.81 to 23.35 min,the half-inactivation temperature(T5030)from 55.12 to 62.44℃,the melting temperature(Tm)from 48.36 to 53.18℃,and the specific enzymatic activity of K189R/K216R at 37℃and pH 4.5 from 3959.98 to 4469.13 U/mg.Molecular structure modeling analysis and molecular dynamics simulation revealed that the introduction of new hydrogen bonds into K189R/K216R could improve the stability of some structural units of the enzyme,thereby enhancing its thermostability.The improvement in its activity was mainly attributed to the increase in the volume of the catalytic pocket of K189R/K216R.In conclusion,the present study demonstrated that the strategy of amino acid residue mutation on the protein surface effectively improves the activity and thermostability of phytase YiAPPA.Our findings can serve as a reference for molecular modification of phytase and other enzymes.