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[1]赵渊中,钟近艺,郭楠,等.多点突变提高DhaA对芥子气的活性和热稳定性[J].应用与环境生物学报,2017,23(04):714-718.[doi:10.3724/SP.J.1145.2016.08012]
　ZHAO Yuanzhong,ZHONG Jinyi,*,et al.Screening, biodiversity, and growth characteristics of cold-adapted nitrobacteria isolated from Arctic Ocean sediments[J].Chinese Journal of Applied & Environmental Biology,2017,23(04):714-718.[doi:10.3724/SP.J.1145.2016.08012]
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多点突变提高DhaA对芥子气的活性和热稳定性()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
23卷
期数:
2017年04期
页码:
714-718
栏目:
研究论文
出版日期:
2017-08-25

文章信息/Info

Title:
Screening, biodiversity, and growth characteristics of cold-adapted nitrobacteria isolated from Arctic Ocean sediments
作者:
赵渊中; 钟近艺; 郭楠; 董志扬; 林洁
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
Author(s):
ZHAO Yuanzhong1;  ZHONG Jinyi1;  2*;  GUO Nan1;  2;  DONG Zhiyang3 & LIN Jie3
1Research Institute of Chemical Defence, Beijing 102205, China 2State Key Laboratory of NBC Protection for Civilian, Beijing 102205, China 3State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
关键词:
多点突变; 进出口通道; DhaA; 芥子气; 催化活性; 热稳定性
Keywords:
multipoint mutagenesis;  access tunnel;  DhaA;  sulfur mustard;  catalytic activity;  thermostability
分类号:
X172 : Q55
DOI:
10.3724/SP.J.1145.2016.08012
摘要:
为提高烷基卤脱卤酶DhaA对芥子气的活性和热稳定性，采用Autodock软件计算DhaA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DhaA活性空腔进出口通道的大小，构建包含5个位点的DhaA突变体（Ile135Phe + Cys176Tyr + Val245Phe + Leu246Ile + Tyr273Phe）；将DhaA及其突变体构建在pET-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DhaA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/mL芥子气的降解率提高了近20%. 热稳定性实验发现，DhaA突变体在50 ℃水浴1 h后残余酶活为76%，比野生型DhaA提高了19%. DhaA突变体的Tm值为56 ℃，比野生型DhaA提高了6 ℃. 综上表明改变DhaA活性空腔内的进出口通道可以提高DhaA的热稳定性和对芥子气的催化活性. （图11 表2 参10）
Abstract:
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.

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更新日期/Last Update: 2017-08-25