title: Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.
abstract: 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.
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Wicka
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Wanarska
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Krajewska
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Pawlak-Szukalska
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Józef
authors.initials: J
authors.lastname: Kur
authors.affiliation: Department of Molecular Biotechnology and Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
authors.forename: Hubert
authors.initials: H
authors.lastname: Cieśliński
mesh_terms: D003001:Cloning, Molecular; D003080:Cold Temperature; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D007700:Kinetics; D011549:Pseudomonas / Q000201:enzymology*; D012988:Soil Microbiology; D000071436:Thermotolerance / Q000235:genetics
chemical_list: D004950:Esterases