Isolation Of A Chitinase-producing Strain And Charaeteration Of Its Chitinase

Bacillus thuringiensis(Bacillus thuringiensis, Bt) is an insect pathogenic microorganisms, it has been developed as a most successful bacterial insecticide because it can produce insecticidal crystal proteins.Chitinase genes are widely distributed in Bt, many kinds of chitinase genes were cloned from different subspecies of Bt strains so far, and a preliminary study of insect activity and disease prevention were conducted. In recent years, the distribution of chitinase in Bacillus thuringiensis, the conditions of enzyme production, the nature and structure of chitinase, the cloning chitinase genes, and the biocontrol research of chitinase were gradually increasing.The research of chitinase for the conversion of chitin resources has become a global hot research topic.Through transparent circle method, we got a strain of Bt DLD171 producing chitinase from the soil. There is no significant change in color by DNS colorimetric method to determinate its enzyme activity. According to Bt chitinase sequence to design full-length primers, chitinase gene of Bt DLD171 was cloned and expressed in E. coli Rosetta(DE3). Chi A73 genes encodes a protein of 67 k Da molecular weight, this protein has a modular structure which combines a catalytic domain and the region of chitin consisting. The results of using different fluorescent substrate material to detect chitinase activity showed that the original strain broth can hydrolyze both 4-MU-(Glc NAc)3 and 4-MU-Glc NAc, while expressing Chi A73 can only hydrolyze 4-MU-(Glc NAc)3. So Chi A73 is an endo-chitinase. Between p H4 to p H 10, this enzyme have hydrolase activity. The highest activity was at p H 8. The optimum temperature of chitinase Chi A73 is 40 ℃.In this paper, we selected 4 strains of non-Bt strains producing chitinase from 40 parts of soil by using transparent circle method. We used DNS method for determining their chitinase activity, the highest chitinase activity was 132 U / ml. This strain was inoculated on PDA plate medium, 30℃ cultured 2-3d, small colonies, white, smooth, neat edges, slightly protruding, like bacterial colonies, and opaque.NAfter 16 S rRNA sequence analysis indicated that: the strain is an Amycolatopsis bacteria which belongs to rare Actinomyces, we named it Amycolatopsis orientalis YM9-2. According to chitinase gene sequence of Amycolatopsis orientalis HCCB10007 in its whole genome sequence, designing two pairs of PCR primers, the strains were cloned chitinase genes. A size of about 1kb DNA sequence and a size of about 1.6kb DNA sequence were cloned, the results showed that the two sequences are chitinase genes. Chitinase gene(Gen Bank accession number KP338820) full-length sequence was 1056 bp, encoding 351 amino acids, predicted molecular mass of 36.9 k Da, isoelectric point of 8.26. Chitinase gene(Gen Bank accession number KP338819) full-length sequence was 1638 bp, encoding 545 amino acids, predicted molecular mass of 57.1 k Da, isoelectric point of 7.98. Amycolatopsis orientalis YM9-2 inhibition activity of fungal strains, we found that it can inhibite the strains of Fusarium culmorum and Fusarium avenaceum.