Characterization And Gene Clone Of Cold-active Chitinase From The Pseudoalteromonas Sp. AC444 And Chitinase Diversity In Deep Sea Sediment Of Prydz Bay, Antarctic

One of the heat spots for global ecosystem research is the study of ecology in Antarctica continent, which takes up an important part among global system. Prydz Bay,the largest bay embedded in the Antarctic continent, exerts important effects on the Antarctic marine ecosystem due to its special"bottle-neck"shape and the sea-bottom landform. By virtue of this, Prydz Bay thus is able to retain the sea water with high salinity, low temperature as well as high density. Chitin is the most abundant and important source of nutrients and energy in marine environment, especially in Antarctic sea area. The degradation of chitin by chitinase from microorganisms becomes a key step in the cycling of nutrients and energy. It's also proposed that chitinase can be served as one of the bio-monitors for the environment evaluation. The coldest environment of Antarctica throughout the world providing unique samples for the screening of cold active enzymes is of great significance in scientific exploitation. In this study, a cold active chitinase was screened and characterized from the deep sea sediment of Prydz Bay, and the chitinase gene diversity in this sediment sample was also surveyed. These results supplied the Antarctic ecosystem research with new materials.The bacteria, AC444, producing cold active chitinase, was isolated and studied. The morphological identification and 16S ribosome DNA (rDNA) analysis indicated that this strain belonged to the genus, Pseudoalteromonas. The optimal temperature and the highest temperature for the growth of Pseudoalteromonas sp. AC444 were 15°C and 30°C respectively, indicating that it was a psychrotrophic bacterium. Furthermore, the chitinase produced by Pseudoalteromonas sp. AC444 was secreted best at 20oC in the medium containing colloidal chitin and tryptone, and reach the highest activity at 13.47 U. The optimum temperature and pH value for the Pseudoalteromonas sp. strain AC444 chitinase were 35?C and 7 respectively, implying that it belonged to neutral cold-active enzyme. This chitinase could retain 25% residual activity under 10oC, and was sensitive to high temperature. The chiA gene of AC444 chitinase was cloned by multiple PCR. The nucleotide sequence contained an open reading frame of 3108bp, coding for 1035 amino acid residues. The gene sequence had high similarity to the chiA genes in database, but the protein showed quite different enzyme characteristics.The first molecular assessment of chitinase gene diversity of different layers of deep sea sediment collected from Prydz Bay was performed by primers...