Prydz Bay, Deep-sea Sediment Microbial Screening Of Low Temperature Enzyme, Gene Cloning Expression And The Nature Of Analysis

Microorganisms living in permanent cold environment, such as deep sea and Antarctic, presumably have developed particular characteristics that allow them to thrive at such environment. For example, the cold-active or cold-adaptive enzymes have attracted more and more interests as they not only are essential in some mechanism studies, but also provide potential for commercial development. Recent microbial studies of the Antarctic have led to significantly new discoveries of unusual microbial diversity, new species, and cold-adaptive enzymes. However, partially due to the great difficulties in collecting samples, it remains as one of the most unknown fields. Compared to organic synthesis, biocatalysts often have far better chemical precision, which can lead to more efficient production of single stereoisomers, fewer side reactions, and a lower environmental burden. The bulk uses of amylases, lipases, proteases and celluases in industrial sectors are including food and feed industry, peptide synthesis, leather industry, management of industrial and household waste, photographic industry.A total of 42 strains, isolated from deep sea sediment of Prydz Bay, Antarctic (74°25'E, 66°55'S, depth of 900 m), which grow only below 30℃were identified based on the morphological differences and SDS-PAGE of culture supernatant. 17 of them were found to have cold-adaptive amylase, lipase or protease activity. Six strains showing strong amylolytic activity, eight strains showing lipolytic activity, one showing proteolytic activity under 10℃were picked out for further study. They were affiliated withγ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequences. The amylaseproducing strains belonged to Pseudomonas, Rhodococcus and Nocardiopsis. The lipaseproducing strains belonged to Pseudomonas, Psychrobacter. The protease-producing strains belonged to Nocardiopsis. The optimal culture conditions for the enzyme production of strain 7197 were investigated; Cold-adaptive alpha-amylase produced by Nocardiopsis sp. 7326 was purified and characterization; Lipase encoding gene (lipA1) from Psychrobacter sp. 7195 was cloned and sequenced, and expressed in E. coll.; Lipase encoding gene (lipA) from Pseudomonas sp. 7323 was cloned and sequenced, and expressed in E. coli.; Amylase encoding gene (amyP) from Pseudomonas sp. 7197 was cloned and sequenced, and expressed in E. coli. We also obtained the lipase gene (lip3) through PCR cloning with metagenome DNA, which was extracted from the deep sea sediment of Prydz Bay. Then the gene was expressed in E. coli and characterization. The results and techniques will contribute to the further research in the abundant cold-adaptive enzymes from the Antarctic.