Molecular cloning and characterization of a protease gene from antarctic psychrotrophic bacterium Pseudomonas sp. 3-37

Loading...
Thumbnail Image
Date
2009
Authors
Tham, Hui Mian
Journal Title
Journal ISSN
Volume Title
Publisher
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
Citation