Arid
DOI10.18388/abp.2015_1074
Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp S9 isolated from Spitsbergen island soil
Wicka, Monika; Wanarska, Marta; Krajewska, Ewelina; Pawlak-Szukalska, Anna; Kur, Jozef; Cieslinski, Hubert
通讯作者Cieslinski, Hubert
来源期刊ACTA BIOCHIMICA POLONICA
ISSN0001-527X
EISSN1734-154X
出版年2016
卷号63期号:1页码:117-125
英文摘要

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9 Delta (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9 Delta protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35 degrees C and pH 9.0. Additionally, the EstS9N esterase retained similar to 90% of its activity from 25-40 degrees C and similar to 40% of its activity at 10 degrees C. Moreover, analysis of its kinetic parameters (K-m, k(cat), k(cat)/K-m) toward p-nitrophenyl butyrate determined at 15 degrees C and 25 degrees C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.


英文关键词GDSL-family cold-active esterase autotransporter Pseudomonas sp S9
类型Article
语种英语
国家Poland
收录类别SCI-E
WOS记录号WOS:000372547900015
WOS关键词PSEUDOALTEROMONAS SP 643A ; ANTARCTIC DESERT SOIL ; ADAPTED ESTERASE ; METAGENOMIC LIBRARY ; GENOME SEQUENCE ; STRAIN ; FAMILY ; ENZYMES ; PSYCHROTROPH ; SECRETION
WOS类目Biochemistry & Molecular Biology
WOS研究方向Biochemistry & Molecular Biology
资源类型期刊论文
条目标识符http://119.78.100.177/qdio/handle/2XILL650/190917
作者单位Gdansk Univ Technol, Dept Mol Biotechnol & Microbiol, Gdansk, Poland
推荐引用方式
GB/T 7714
Wicka, Monika,Wanarska, Marta,Krajewska, Ewelina,et al. Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp S9 isolated from Spitsbergen island soil[J],2016,63(1):117-125.
APA Wicka, Monika,Wanarska, Marta,Krajewska, Ewelina,Pawlak-Szukalska, Anna,Kur, Jozef,&Cieslinski, Hubert.(2016).Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp S9 isolated from Spitsbergen island soil.ACTA BIOCHIMICA POLONICA,63(1),117-125.
MLA Wicka, Monika,et al."Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp S9 isolated from Spitsbergen island soil".ACTA BIOCHIMICA POLONICA 63.1(2016):117-125.
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