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Zhu Yanbing,Yin Xiaoqian,Liu Han,Li Hebin,Chen Yanhong,Li Lijun,Xiao Anfeng,Ni Hui. 2019. Substitution of His260 residue alters the thermostability of Pseudoalteromonas carrageenovora arylsulfatase. Acta Oceanologica Sinica, 38(6):75－82

Substitution of His260 residue alters the thermostability of Pseudoalteromonas carrageenovora arylsulfatase

His260残基的替代改变食鹿角菜假交替单胞菌芳香基硫酸酯酶的热稳定性

Received:May 23, 2018

DOI：10.1007/s13131-019-1356-z

Key words:arylsulfatase Pseudoalteromonas carrageenovora directed evolution error-prone PCR thermostability

中文关键词: 芳香基硫酸酯酶食鹿角菜假交替单胞菌定向进化易错PCR 热稳定性

基金项目:The National Natural Science Foundation of China under contract No. 31401632; the Program for New Century Excellent Talents in Fujian Province University, China under contract No. B15139.

Author Name	Affiliation	E-mail
Zhu Yanbing	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China
Yin Xiaoqian	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Liu Han	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China
Li Hebin	Department of Pharmacy, Xiamen Medical College, Xiamen 361023, China
Chen Yanhong	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China
Li Lijun	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China
Xiao Anfeng	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China
Ni Hui	College of Food and Biological Engineering, Jimei University, Xiamen 361021, China Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China	nihui@jmu.edu.cn

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Abstract:

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A library of P. carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, two mutants of H260L and D84A/H260L showed enhanced thermal stability than the wild-type predecessor (WT). Site-directed mutagenesis demonstrated that only amino acid residue at Position 260 plays an important role in the thermostability of P. carrageenovora arylsulfatase. Thermal inactivation analysis showed that the half-life (t_1/2) values at 55℃ for H260L, H260I, H260Q, H260F and H260R were 40.6, 48.4, 30.9, 29.1 and 34.5 min, respectively, while that of WT was 9.1 min. Structure modeling demonstrated that the additional hydrogen bonds and/or optimization of surface charge-charge interactions could be responsible for the increased thermostability imparted by H260L, H260I, H260Q, H260F and H260R.

中文摘要:

本研究旨在提高食鹿角菜假交替单胞菌芳香基硫酸酯酶的热稳定性。通过使用易错PCR引入随机突变来构建食鹿角菜假交替单胞菌芳香基硫酸酯酶的突变体文库。通过筛选，获得的两个突变体酶H260L和D84A/H260L比野生型酶（WT）具有更高的热稳定性。定点突变分析表明，只有260位的氨基酸残基在该芳香基硫酸酯酶的热稳定性中起重要作用。热失活分析表明，H260L、H260I、H260Q、H260F和H260R在55℃的半衰期（t_1/2）值分别为40.6、48.4、30.9、29.1和34.5 min，而WT在55℃的半衰期为9.1 min。结构模拟分析表明，氢键的增加和/或表面电荷-电荷相互作用的优化可能是H260L、H260I、H260Q、H260F和H260R热稳定性提高的原因。

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