理性设计改造牛肠激酶的热稳定性

Engineering Thermostability of Bovine Enterokinase by Rational Design Method

以牛肠激酶作为研究对象,利用理性设计的方法提高其热稳定性。首先通过分子动力学模拟软件Gromacs v 4.5.5,Flex Service以及B-FITTER软件预测出了肠激酶的柔性区Fragment 64~69,Fragment 85~90;然后结合β-转角序列统计学信息以及引入位置原有的残基不参与形成氢键的原则,确立了3个突变位点S67P,R87P以及Y136P;通过Quik Change^(TM)定点突变的方法引入突变位点,并进行了酶热稳定性分析。结果表明,R87P突变体酶的失活半衰(t_(1/2))和T_(50)^(10)较野生型分别提高了3.1 min和11.8℃,同时,动力学常数(K_m/k_(cat))测定结果显示酶活未受到显著影响。该策略有潜力应用于其他工业酶分子的热稳定性改造,为推动生物酶的工业化应用奠定基础。

The rational design method was applied to increase the thermostability of bovine enterokinase. Molecular dynamics software Gromacs v4.5.5, FlexService and B-FITFER were used to predict the flexibility profile of the bovine enterokinase structures. Fragment 64 - 69 and Fragment 85 ~ 90 were confirmed to represent the flexible region. Subsequently, β-turn sequence statistics and stereoscopic criteria of introducing proline were combined to pinpoint appropriate substitution sites by and Y136P） were introduced within the fexible region prolines. Finally, site-directed mutations （ S67P, R87P using Quik ChangeTM method and the thermostability of wild-type and the enterokinase mutants were investigated. The result demonstrated that the half-life （t1/2 ） and half inactivation （T50） temperature of the R87P mutant increased by 3.1 min and 11.8℃ when compared to that of the wild-type enzyme. Meanwhile, the catalytic efficiency （ Km/kcat ） of the R87P mutant enzyme remained nearly unchanged. This strategy has potential to be applied to engineer thermostability of other enzyme, which is beneficial for the wider aoolication of biocatalvsts in industry.