The Features Of Chitinase And Gene Cloning Of Chitinase Gene In Streptomyces Sampsonii KJ42

A strain of bacterial named KJ42, which can produce chitinase and be antistatic to Rhizoctonia violacea, was isolated from the soil of poplar. The strain KJ42, with straight and soft aerial hyphae and oval spore, grows well in seven different media and produces golden brown soluble pigments in PDA media. Its survival temperature and pH range from15℃to42℃and from pH3to pH12. The strain KJ42cannot grow under the environmen-tal temperature of45℃. The strain KJ42can fluidify the glutin, peptonize the milk, hy-drolyze the amylum and deoxidize nitrate, but do not hydrolyze cellulose and do not pro-duce H2S and melanin. It can utilize included glucose, maltose, glycerol, mannitol, sorbie-rite, but not sucrose, xylose, fructose, sodium tartrate and Inositol. It was found that most of its morphological, cultural, physiological and biochemical characteristics were similar to those of Streptomyces. In addition, BLAST analysis of its16S rDNA sequence suggested the identity of99.31%with Streptomyces sampsonii. In light of the polyphasic taxonomi-cal principle, the strain KJ42was accordingly named as Streptomyces sampsonii KJ42.The components were optimized in basic media by the addition of carbon source, ni-trogen source and phosphonium source. The optimum prescription was obtained by using the4*3orthogonal experimental design, effects of carbon source, salinity, inorganic salt, pH value and incubation. The optimum culture medium components include:colloid chitin (W/V2%)200ml/L, peptone0.4%, K2HP040.4g/L, KH2PO40.3g/L, MgSO4·7H2O0.5g, FeSO4·7H2O0.01g, ZnSO40.001g. Under the optimum inoculation conditions:tempera-ture26℃, rotate speed140r/min and initial pH7.0, the enzyme activity reach its peak after144hours.According to the chitinase gene, two pairs of conservative primers were designed. Uti-lizing the two pairs of primers, two sequences, respectively named chiLA and chiLB, were cloned based on the strain KJ42genomic DNA. BLAST analysis of chiLA sequence sug-gested it was99%identity to Streptomyces sp.VC-YC6651chitinase19gene. A ORF with453bp and encoding151amino acids was found by utilizing DNASTAR Program. And chiLB, with a402bp length ORF encoding134amino acids, was88%identity to Strepto- myces peucetius chitinaseC precursor gene. It was found that what had sequence similarity over80%were all chitinase produced by microbes when the gene was translated into pro-tein, this chitinase gene had the highest sequence similarity (83%) with the chitinase pro-duced by Streptomyces.The chitinase gene was translated into protein through DNAMAN software, then its, hydrophilicity profile, pI and MW were analyzed, and its secondary structure was antic-ipated also. The results showed that the pI and MW of chiLA and chiLB were6.07,4.31,17161.92Da and15521.24Da, respectively.