Low-temperature lipase from psychrotrophic Pseudomonas sp. strain KB700A.
We have previously reported that a psychrotrophic bacterium, Pseudomonas sp. strain KB700A, which displays sigmoidal growth even at -5 degrees C, produced a lipase. A genomic DNA library of strain KB700A was introduced into Escherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open reading frame (KB-lip) consisting of 1,422 nucleotides that encoded a protein (KB-Lip) of 474 amino acids with a molecular mass of 49,924 Da. KB-Lip showed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily I.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activity in the presence of Ca2+. Addition of EDTA completely abolished lipase activity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn2+ and Sr2+ also led to enhancement of lipase activity but to a much lower extent than that produced by Ca2+. The optimal pH of KB-Lip was 8 to 8.5. The addition of detergents enhanced the enzyme activity. When p-nitrophenyl esters and triglyceride substrates of various chain-lengths were examined, the lipase displayed highest activity towards C10 acyl groups. We also determined the positional specificity and found that the activity was 20-fold higher toward the 1(3) position than toward the 2 position. The optimal temperature for KB-Lip was 35 degrees C, lower than that for any previously reported Subfamily I.3 lipase. The enzyme was also thermolabile compared to these lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily I.3, indicating that KB-Lip has evolved to function in cold environments, in accordance with the temperature range for growth of its psychrotrophic host, strain KB700A.
Rashid
N
N
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.
Shimada
Y
Y
Ezaki
S
S
Atomi
H
H
Imanaka
T
T
D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003080:Cold Temperature; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D003902:Detergents; D008049:Lipase