Molecular cloning and characterization of a protease gene from antarctic psychrotrophic bacterium Pseudomonas sp. 3-37
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Date
2009
Authors
Tham, Hui Mian
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Abstract
Cold-active enzymes are usually characterized by a higher specific activity at low
temperature when compared to their mesophilic counterparts and offer a high potential
in numerous biotechnological applications. In this study, the gene encoding
metalloprotease of the psychrotrophic bacterium Pseudomonas sp. 3-37 isolated from
water samples collected at Odbert Island, Antarctica, was cloned and characterized. The
bacterium could survive a wide range of temperature from 4 ºC to 30 ºC and produces a
protease on Skim Milk Agar when cultured at 4 ºC. The metalloprotease structural gene
was isolated from the Pseudomonas sp. 3-37 genomic library by using a homologous
metalloprotease PCR fragment probe. Nucleotide sequence analysis revealed an open
reading frame (ORF) of 1431 bp encoding a 477 amino acid residues with an estimated
molecular mass of 53 kDa that exhibited 100% identity to extracellular alkaline
metalloprotease of Pseudomonas fluorescens A506 (accession no AAP4489). Two other
genes were identified flanking the metalloprotease gene. The 2 genes were putative Daminopeptidase
gene and zinc protease transporter gene. The metalloprotease gene was
expressed in a protease deficient strain of E. coli Tuner(DE3) and the functional activity
of the recombinant metalloprotease was confirmed by casein zymography. This
metalloprotease contains a zinc-binding motif (HExxH), repetitive glycine-rich
nonapeptide sequence motif (GGxGxDxux) and contains no cysteine residue, these
features are specifically found in serralysin subfamily.
(accession no CAA07699.1) and zinc-protease transporter of Pseudomonas fluorescens
CY091 , respectively. (Appendix H.3).
Description
Master
Keywords
Biological Science , Molecular cloning , Protease gene