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为提高烷基卤脱卤酶Dha A对芥子气的活性和热稳定性,采用Autodock软件计算Dha A突变前后与芥子气的结合情况,利用重叠延伸PCR和DNA无缝拼接结合的方法,改变Dha A活性空腔进出口通道的大小,构建包含5个位点的Dha A突变体(Ile135Phe+Cys176Tyr+Val245Phe+Leu246Ile+Tyr273Phe);将Dha A及其突变体构建在p ET-28a载体上后,在Escherichia coli BL21(DE3)中进行表达,比较纯化后的野生型与突变体在酶学性质方面的变化情况.Autodock分子对接结果显示,突变后的Dha A与芥子气的结合能、结合效率和抑制常数均小于野生酶,突变体对芥子气的比活性提高了1.4倍,对10 mg/m L芥子气的降解率提高了近20%.热稳定性实验发现,Dha A突变体在50℃水浴1 h后残余酶活为76%,比野生型Dha A提高了19%.Dha A突变体的Tm值为56℃,比野生型Dha A提高了6℃.综上表明改变Dha A活性空腔内的进出口通道可以提高Dha A的热稳定性和对芥子气的催化活性.
In order to improve the activity and thermostability of the alkyl halide dehalogenase Dha A to mustard gas, Autodock software was used to calculate the binding of mustard gas before and after Dha A mutation. The overlap extension PCR and DNA splicing were used to change the Dha A activity Dha A mutant containing 5 sites (Ile135Phe + Cys176Tyr + Val245Phe + Leu246Ile + Tyr273Phe) was constructed. After Dha A and its mutants were constructed on p ET-28a vector, Escherichia The expression of wild-type and mutant in purified protein was compared with that of wild-type and mutant.Atodock molecular docking showed that the binding energy, binding efficiency and inhibitory constant Less than the wild-type enzyme, the mutant had a 1.4-fold higher specific activity for mustard gas and a nearly 20% more degradation rate for the mustard gas of 10 mg / m L. The thermal stability test revealed that the Dha A mutant remained after 1 h of water bath at 50 ℃ The enzyme activity was 76%, which was 19% higher than that of the wild type Dha A. The Tm value of the Dha A mutant was 56 ° C, which was 6 ° C higher than that of the wild type Dha A. In conclusion, Channels can improve the thermal stability of Dha A and mustard The catalytic activity.