Gene cloning, sequence analysis and heterologous expression of a novel cold-active lipase from Pseudomonas sp. PF16.

Abstract

A pychrotrophic bacterium secreing an extracellular alkaline cold-active lipase was isolated from refrigeratory. Based on morphology and 16S rRNA gene sequence analysis, the isolate which was named as PF16 was identified as a member of Pseudomonas. A novel lip-PF16 gene was obtained from the genomic DNA of strain PF16. Nucleotide sequence analysis showed that open reading frame (ORF) was consisting of 1,431 nucleotides encoding 476 amino acids long protein. Lip-PF16 showed 89.5% and 88% identity with the cold-adapted lipases from Pseudomonas fluorescens and Pseudomonas sp. KB700A, respectively. It was found to be a member of conserved subfamily I.3 bacterial lipase, which contained a lipase consensus sequence GXSXG. The lip-PF16 gene was expressed in Escherichia coli BL21 (DE3) and functional lipase was obtained successfully. The molecular weight was estimated to be 50 KDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The cloned lipase was active over broad range of temperature range with optimum activity at 4^°C, and found to be alkaline preferring with optimum activity at pH 9.0. This recombination lipase show high catalytic activity at low temperature.