食品科学2017，Vol.38Issue(10)：18-23,6.DOI:10.7506/spkx1002-6630-201710004
Pseudoalteromonas carrageenovora芳香基硫酸酯酶突变文库热稳定性提高突变体的筛选及鉴定
Screening and Characterization of Mutant with Improved Thermostability from a Random Mutant Library of Pseudoalteromonas carrageenovora Arylsulfatase
摘要
Abstract
A library of Pseudoalteromonas carrageenovora arylsulfatase mutants was constructed by random mutagenesis using error-prone PCR.After screening,one mutant strain named 4-153 was obtained whose arylsulfatase had improved thermal stability.It was found that there were two amino acid substitutions in the mutant,including D84A and H260L.When p-nitrophenyl sulfate was used as a substrate,the optimal reaction temperature for the mutant enzyme was 55 ℃.Mutant arylsulfatase 4-153 (M4-153) retained 85％,83％,48％,and 13％ of its initial activity after incubation at 45,50,55 and 60 ℃ for 30 min,respectively.Meanwhile,wild-type arylsulfatase (WT) retained 79％,68％,21％,and 1％ of its initial activity after incubation at 45,50,55 and 60 ℃ for 30 min,respectively.These results showed that M4-153 had a better thermal stability than WT.M4-15 3 had an optimum pH of 8.0,and it was stable over the pH range of 5.0-9.0.Inhibition assay with EDTA indicated that metal ions played an important role in the catalytic process of the mutant enzyme.The recombinant arylsulfatase 4-153 showed a relatively strong tolerance to some detected detergents including Triton X-100,Tween 20,Tween 80,and Chaps.The desulfuration ratio of the crude polysaccharides from Gracilaria lemaneiformis by M4-153 was 79.5％.关键词
芳香基硫酸酯酶/易错聚合酶链式反应/热稳定性提高/突变体性质Key words
arylesterase/error-prone PCR/improved thermostability/mutant property分类
生物科学引用本文复制引用
乔超超,王新侠,李鹤宾,倪辉,肖安风,朱艳冰..Pseudoalteromonas carrageenovora芳香基硫酸酯酶突变文库热稳定性提高突变体的筛选及鉴定[J].食品科学,2017,38(10):18-23,6.基金项目
国家自然科学基金青年科学基金项目(31401632) （31401632）
福建省高校新世纪优秀人才支持计划项目(B15139) （B15139）
