多点突变提高DhaA对芥子气的活性和热稳定性  

Improvement in the thermostability and activity of DhaA against sulfur mustard by multipoint mutagenesis

作　　者：赵渊中 钟近艺[1,2] 郭楠[1,2] 董志扬[3] 林洁[3] 

机构地区：[1]中国人民解放军防化研究院,北京102205 [2]国民核生化灾害防化国家重点实验室,北京102205 [3]中国科学院微生物研究所微生物资源前期开发国家重点实验室,北京100101

出　　处：《应用与环境生物学报》2017年第4期714-718,共5页Chinese Journal of Applied and Environmental Biology

摘　　要：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 thermostability and activity of DhaAs against sulfur mustard, we engineered DhaA thermostability and activity against sulfur mustard by modification of residues in the access tunnel. A recombinant plasmid pET28a-DhaA-Ile135Phe ＋ Cys176Tyr ＋ Val245Phe ＋ Leu246Ile ＋ Tyr273Phe containing five mutant genes was constructed by overlap extension PCR and seamless cloning. The recombinant vector was transformed into Escherichia coli BL21 （DE3）. The native and mutated DhaAs were separately expressed in E. coli BL21 （DE3）. Both DhaAs were purified and characterized. The mutated DhaA showed 1.4-fold higher catalytic efficiency than the native DhaA. Moreover, the degradation rate of 10 mg/mL sulfur mustard increased by 20%. Thermostability analysis revealed that the relative activity after storing at 50 ℃ for 1 h was 76%, and was 19% higher than that of the native DhaA. Furthermore, melting temperature （Tm） of the mutated DhaA was 56 ℃, which was 6 ℃ higher than that of the native DhaA. Analysis data of the molecular docking was matched with the experimental result of the mutants. Therefore, engineering the access tunnels of DhaA is a feasible strategy to improve the thermostability and activity against sulfur mustard.

关 键 词：多点突变 进出口通道 DHA A 芥子气 催化活性 热稳定性 

分 类 号：O623.82[理学—有机化学]