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DOI | 10.18388/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
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ISSN | 0001-527X |
EISSN | 1734-154X |
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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