title: Cloning, expression, and characterization of a recombinant esterase from cold-adapted Pseudomonas mandelii.
abstract: A gene coding for the extracellular esterase (EstK) was cloned from the psychrotrophic bacterium Pseudomonas mandelii based on its partial amino acid sequence as determined by mass spectrometry. The entire open reading frame consisting of 1,011 bp was expressed in Escherichia coli as a soluble protein and purified by nickel-chelated affinity chromatography and Capto Q column chromatography. Here, we show that the 33-kDa recombinant EstK protein (rEstKsp) had a substrate preference for esters of short-chain fatty acids, especially, p-nitrophenyl acetate. Optimum activity of rEstKsp was at pH 8.5 and 40 °C. The esterase activity remained similar from a range of 4∼20 °C, but the maximum activity varied depending upon pH. With p-nitrophenyl acetate as the substrate, K (M) was 210 μM and k (cat) was 3.4 s(-1). Circular dichroism and fluorescence spectroscopy results revealed that rEstKsp had a predominantly α-helical structure and maintained its folded state at 4∼40 °C. Interestingly, the tertiary structure of rEstKsp was predicted based on the structures of other hyperthermophilic esterases. Our results demonstrated that both native and rEstKsp are active at low temperatures and have a unique substrate preference for p-nitrophenyl acetate.
authors.affiliation: Department of Biomedical Science, Daegu University, Gyeongsan 712-714, South Korea.
authors.forename: Changwoo
authors.initials: C
authors.lastname: Lee
authors.forename: Junyoung
authors.initials: J
authors.lastname: Kim
authors.forename: Seunghee
authors.initials: S
authors.lastname: Hong
authors.forename: Bonlon
authors.initials: B
authors.lastname: Goo
authors.forename: Seungyeon
authors.initials: S
authors.lastname: Lee
authors.forename: Sei-Heon
authors.initials: SH
authors.lastname: Jang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D004950:Esterases