Nucleotide sequencine and expression of chitinase gene from Bacillus sp. PP8

Abstract

Bacillus sp. PP8 was isolated from PP Island, Krabi, Thailand. When Bacillus sp. PP8 was grown in liquid medium containing colloidal chitin, flake chitin, or powdered chitin, chitinase and chitosanase activities were detected However, when it was grown in mediun containing colloidal chitosan or 100%deacetylated flake chitosan, only chitosanase activity was detected. The best inducer for chitinase and chitosanase activity was colloidal chitin and colloidal chitosan, respectively. Chitinase and chitosanase activity was determined by modified Schale's method. Chitinase and chitosanase activities reached their maximum level at 84 and 60 hours of cultivation, respectively. Both activities gradually decreased after further cultivation. GlcNAc can induce low level of chitinase production from PP8, however GlcN cannot induce either chitinase or chitosanase production. The optimum pH and temperature of the chitinase and chitosanase was pH 8.0 / 50 ?C and pH 7.0 / 50 ?C, respectively. GlcNAc and (GlcNAc)2 are major products when colloidal chitin was used as substrate for hydrolysis by PP8 chitinase. When crude enzyme from PP8 was analyzed by SDS-PAGE followed by activity staining, we observed five bands of 145, 66, 55, 45 and less than 15 kDa in size containing chitinase activity. On the other hand, only a single band of 47.5 kDa with chitosanase activity was observed. A chitinase encoding gene was previously cloned using shot gun cloning. Three colonies producing different clear zones on the selective agar designated, pST24, pST847 and pST1691 were selected. The highest chitinase activity was found in clone pST847 containing the 6.3 kb inserted fragment. pST847 was chosen for further analysis. There are four derivatives; pSNXX-3.0, pSNPX-6.3, pSNBB-1.8 and pSNBP-4.5 of pST847 but only pSNXX-3.0 had chitinase activity. Nucleotide sequence of pSNXX-3.0 revealed an open reading frame of 1797 bp encoding for 599 amino acids with signal peptide corresponding to 66.20kDa. The chitinase gene expression was performed using pET19b and pTrcHis-2C. The chitinase activity was found in the culture medium and confirmed by SDS-PAGE followed by activity staining. The optimum pH, temperature, and products of colloidal chitin hydrolysis of the recombinant chitinase corresponded to the 66 kDa native chitinase from Bacillus sp. PP8. The enzyme did not degrade when kept in pH 5-8, at 4 ?C for 2 months. Partial purified of the enzyme was performed by chitin adsorption and about 90% yield were recovery.