Studies On Chitinase Activities From Bacillus Thuringiensis

Chitin is the second most abundant polysaccharides in nature. Chitinases are important and major enzymes which can degrade chitin. Bacillus thuringiensis is the most successfully used biological pesticide. Chitinase can increase the insecticidal activity of Bt. Herein, we present the cloning of two chitinase genes(chi74 and chi36) from a Bt strain(named BRC-Bt1) with high chitinase activity, as well as the expression in vitro, the domains of chitinases, the catalytic mechanisms, the regulation of chitinase genes expression and the bioactivity of chitinase hydrolysis products.The nucleotide sequence of chi74 gene from BRC-Bt1 consists of 2025 bases and encodes a putative protein of 674 amino acids residus with a predicted molecular weight of 74kDa and pI 5.77. Homology comparison revealed that the chi74 gene had high homology in nucleotide sequence with the genes encoded 70kDa chitinases of Bt reported. The predicted protein of Chi74 was a modular enzyme consisting of a signal peptide, a FN3 domain, a ChBD domain and a catalytic domain belonging to chitinase family 18. The chi74 gene was expressed with GST fusion in E. coli and the characteristic of Chi74 was investigated. When using the colloid chitin as substrate, the optimum pH, optimum temperature and thermal stability of Chi74 were pH 5, 60℃and 40℃, respectively. The enzyme exhibited activity in broad temperature range, from 20 to 80℃, and pH between 4.0 and 8.0. The chitinase activity of Chi74 was not influenced by Na⁺, K⁺, NO₃^- and Cl-; however, it was improved by Mg²⁺and SO₄^2-, and inhibited by Cu²⁺ and Ag⁺. HPLC analysis of the hydrolysis products indicated that it contained GlcNAc, (GlcNAc)2 and oligomers with higher degree of polymerization, and GlcNAc was predominant product increased gradually with the reaction time extension. The Chi74 maybe an endo-chitinase.Three different C-terminal truncation chitinase genes of chiA, chiAF and chiAC from chi74 were constructed, which encoded the putative proteins with the predicted molecular weights and the pI values of 54kDa and 6.04, 63kDa and 6.07, 66kDa and 5.74, respectively. The 3 truncation genes were expressed with GST fusion in E.coli. With colloidal chitin as substrate, the relative activities of ChiA, ChiAF and ChiAC were 81%, 72% and 39% compared with the native chitinase Chi74. The optimum temperature of three truncation enzymes were all 60℃and the curves of enzyme activity on different temperature were similar to Chi74. The activities of three truncation enzymes were activated by Mg²⁺ and the activation efficiency was higher than that of native enzyme, and were inhibited by Ag⁺ and the inhibition efficiency was higher than that of Chi74. HPLC analysis of the hydrolysates of three truncation enzymes showed that the contents of GlcNAc and chito-oligomers were at lower and higher level respectively, compared to Chi74. FN3 domain and ChBD domain may play important roles in insoluble chitin hydrolysis.The nucleotide sequence of chi36 gene from BRC-Bt1 consists of 1083 bases and encodes a putative protein of 360 amino acids residus with a predicted molecular weight of 40kDa and a pI value of 6.52. The chi36 gene showed high homology with the genes encoded 36kDa chitinases from Bt, Bacillus cereus and Bacillus anthracis. Analysis of the predicted protein structure of Chi36 revealed that it also belongs to chitinase family 18, and contains a signal peptide and a single catalytic domain which has little homology with Chi74. The chi36 gene was expressed with GST fusion in E. coli and the characteristic of Chi36 was also investigated. When using the colloid chitin as substrate, the optimum pH, optimum temperature and thermal stability of Chi36 were pH 5, 55℃and 40℃, respectively. The enzyme exhibited activity in broad temperature range, from 30 to 80℃, and pH between 4.0 and 8.0. The chitinase activity of Chi36 was not influenced by Na⁺, K⁺, Mg²⁺, NO₃^- and Cl-; however, it was improved by SO₄^2-, and inhibited by Fe³⁺, Cu²⁺ and Ag⁺. HPLC analysis of hydrolysates showed that it contained GlcNAc, (GlcNAc)2 and oligomers with higher degree of polymerization, and the content of GlcNAc was lower than chito-oligomers. The Chi36 maybe an endo-chitinase and its catalytic mechanism different from that of Chi74. The enzyme activity of the mixture of Chi36 and Chi74 with equal volume was the highest.Three different C-terminal prolonged chitinase genes of chi36F, chi36C and chi36FC were constructed by adding the different C-terminal fragments of Chi74 into the C-terminal of Chi36, which encoded the putative proteins with the predicted molecular weights of 49kDa, 52kDa and 60kDa, respectively. The three prolonged genes were expressed with GST in E. coli. With colloidal chitin as substrate, the relative activities of Chi36F, Chi36C and Chi36FC were 7%, 21% and 33% higher than native Chi36, respectively. The optimum temperature of three prolonged enzymes were all 55℃. The enzyme activities of three prolonged enzymes were inhibited by Ag+ and the inhibition efficiency was lower than Chi36. The components of colloid chitin hydrolysis products of three prolonged enzymes were similar with Chi36, but the concentrations of each component were higher than native enzyme. It supported that FN3 domain and ChBD domain may play important roles in insoluble chitin hydrolysis.The upstream sequences of chi74 and chi36 from 6 Bt strains were cloned and sequenced. Analysis of the sequence revealed that the chi74 and chi36 were expressed independently and spaced long distance. The promoters of chi36 were not similar to the promoter of chi74. The result of promoter prediction showed that the amounts of promoters from chi74 were more than from chi36. The transcriptional factor of CAP/CRP and NIT-2 were found in both upstream sequences of chi74 and chi36.The supernatants of BRC-Bt1 cultured in chitinase-produced medium and LB medium both inhibited spore germination of Colletotrichum musae, and the inhibition efficiency of the former was higher than the later. The supernatant of BRC-Bt1 cultured in chitinase-produced medium also inhibited spore germination and hyphal extension of Magnaporthe grisea. The mixture of Chi74 and Chi36 with the final concentration of 10mg/mL and 5mg/mL exhibited 87.5% and 54.5% mortality against Pinus massoniana, respectively. The mortality of hydrolysis products produced by mixture enzyme at 1h, 3h, 8h and 24h were 23.8%, 69.3%, 100% and 8.4%, respectively. Furthermore, the hydrolysis products exhibited in vitro anti-liver cancer cell (SMMC-7721) with IC₅₀69μg/ml, but had no influence on normal liver cell.