Affiliations: Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan, China
Correspondence:
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Corresponding author: Yunlin Wei, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, China.[email protected]
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.
