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 degreesC, produ ced a lipase. A genomic DNA library of strain KB700A was introduced into Es cherichia coli TG1, and screening on tributyrin-containing agar plates led to the isolation of the lipase gene. Sequence analysis revealed an open rea ding 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 show ed 90% identity with the lipase from Pseudomonas fluorescens and was found to be a member of Subfamily 1.3 lipase. Gene expression and purification of the recombinant protein were performed. KB-Lip displayed high lipase activ ity in the presence of Ca2+. Addition of EDTA completely abolished lipase a ctivity, indicating that KB-Lip was a Ca2+-dependent lipase. Addition of Mn 2+ 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. T he 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 CIO acyl groups. We also dete rmined the positional specificity and found that the activity was 20-fold h igher toward the 1(3) position than toward the 2 position. The optimal temp erature for KB-Lip was 35 degreesC, lower than that for any previously repo rted Subfamily 1.3 lipase. The enzyme was also thermolabile compared to the se lipases. Furthermore, KB-Lip displayed higher levels of activity at low temperatures than did other enzymes from Subfamily 1.3, indicating that KB- Lip has evolved to function in cold environments, in accordance with the te mperature range for growth of its psychrotrophic host, strain KB700A.