Isolation And Characterization Of Marine Actinomycete C203 And Cloning Of Chitinase Gene

Researching and developing on the chitin and its ramifications such as chitin oligosaccharide are increasing rapidly, they show a good future in many fields such as medicine, foodstuff and environmental protection. Producing chitin oligosaccharide by bio-decomposing is a good way, which has moderation reaction conditions and does not pollute environment compared with chemical methods. Many microbes can produce chitinase, but only a few are used in practice because of their low chitinase activity. Screening microbes with high, stable and promising chitinase activity is an important way to solve the using problem.In this study, research was carried out on following aspects: isolation of marine microbes which produce chitinase, screening and identification of strains, cloning chitinase gene, separation of the chitinase and studying its characters, analyzing the chitinase protein.Thirty eight strains of microbes produce chitinase, which included thirty two bacteria, five actinomycetes and one fungus, were isolated from various samples of South Sea. An actinomycete was selected for further researching which shows strong activity to chitin even when glucose is present. The characters of morphological, physiological-biochemical, and 16S rDNA sequence suggested C203 belongs to Streptomyces, it was presumed that C203 may be a new marine genus by analyzing the phylogenetic tree.The liquid fermentation conditions and medium components were optimized. The effects of carbon source, nitrogen source, salinity, inorganic salt, pH value and incubation volume on the production of chitinase were studied and the ratio of nutrition component was optimized by orthogonal experiment. The optimum culture medium components include: colloid chitin (w/v 1%) 20% (v/v), glucose 0.5%, yeast extract 0.3%, K2HPO4 0.04%, old sea water. The optimum inoculation conditions are: initial pH7.0⁷.2, seed cultural time 48h, 5% inoculation amount, 28℃, 200r/min for 6d.An extracellular chitinase from the culture supernatant of C203 was purified to an apparent homogeneity by concentration, dialysis and PAGE. The chitinase had a molecular mass of 44KD. Physical and chemical characters of the chitinase were studied. The results showed that the optimal reaction temperature is 60℃and the optimal pH is 7.2, Mg²⁺ and Fe²⁺ can promote the activity of the chitinase, while Cu²⁺ and Zn²⁺ can inhibit the activity sharply, it can show the strongest activity when the chitin concentration is 1%.A conservative fragment of 609bp was obtained by designing a pair of primer based on the conservative region of chitinase gene, and then the both sequences of 3'-end and 5'-end were obtained by designing a series of random primers and special primers through TAIL-PCR method. The gene was 1563bp in all, which had 84% sequence similarity with the chitinase gene (Chi 40) of S. thermoviolaceus, and included an ORF of 1251bp. It was found that what had sequence similarity over 80% were all chitinase produced by microbes when the gene was translated into protein, this chitinase gene had the highest sequence similarity (83%) with the chitinase produced by S. aureofaciens.The chitinase gene was translated into protein through DNAMAN software, then its hydrophobicity profile, hydrophilicity profile, pI and MW were analyzed, its secondary structure was anticipated also. The results showed its pI is 5.86, MW is 44208.8.