Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil

• Authors: Monika Wicka , Marta Wanarska , Ewelina Krajewska , Anna Pawlak-Szukalska , Józef Kur , Hubert Cieśliński
• Languages of publication: EN

Abstracts

EN

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 EstS9Δ (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 EstS9Δ 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°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°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.

Keywords

EN

GDSL-family; cold-active; esterase; autotransporter; Pseudomonas sp. S9

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2016
• Volume: 63
• Issue: 1
• Pages: 117-125

Dates

published

2016

received

2015-06-01

revised

2015-06-28

accepted

2015-11-22

(unknown)

2016-01-28

Contributors

author

Monika Wicka

• Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland

author

Marta Wanarska

• Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland

author

Ewelina Krajewska

• Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland

author

Anna Pawlak-Szukalska

• Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland

author

Józef Kur

• Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland

author

Hubert Cieśliński

• Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland

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Identifiers

DOI

10.18388/abp.2015_1074