Design of DNA primers for chitinase gene cloning from Bacillus licheniformis PR-1

Abstract

Chitinase (EC3.2.1.14) is an enzyme that catalyzes the degradation of chitin. Bacillus licheniformis PR-1, isolated from soil in Thailand, is capable of producing chitinase. Chitinase from B. licheniformis PR-1 produced maximum chitinase activity in 8 and 5 day when cultured on colloidal chitin minimum medium with 0.05% and 0.25 % yeast extract, respectively. The optimum pH and temperature of crude chitinase was pH 5.0 in citrate buffer and 70 degree celcius, respectively. Crude chitinase hydrolyzed colloidal chitin the best followed by powder chitin, 80 % DD chitosan, flake chitin and regenerated chitin respectively. Products from this enzyme, analyzed by HPLC, were a mixture of chitobiose (GlcNAc)[subscript2] and N-acetylglucosamine (GlcNAc); chitobiose was the major products. SDS-PAGE and activity staining of crude enzymes showed three bands with chitinase activity. The estimated molecular weights of the major chitinase species were 70, 65 and 58 kDa. Designed primers (BP-I, II, V, VI, VII, VIII, IX, BP-F and BP-R) that were specific for familly 18 chitinases among Bacillus sp., were able to amplified full length chitinase genes. Shotgun cloning of the PstI partially cut genomic DNA of B. licheniformis PR-1 was performed. Two transformants from 8,000 colonies showed clear zones with inserted fragment of 5 and 3 kb, respectively. Only one plasmid, pPRChi65, had chitinase activity and produced highest chitinase activity in E.coli JM109. The promotor of Chi65 could be suppressed when grown in LB medium with glucose. Crude chitinase from pPRChi65 was characterized. The optimum pH and temperature were pH 5.0 in citrate buffer and 60 oC, respectively. Two open reading frame were found in the 5 kb inserted. One is 1,779 bp long encoding for a protein 593 amino acids, which correspond to 65,100 Da with isoelectric point of 5.84 and the other is not a complete sequence for chitodextrinase. The amino acid comparison indicated Chi65 is 89 % similar to chitinase from B. licheniformis TP-1 followed by 79 % chitinase from B. subtilis. Crude chitinase hydrolyzed colloidal chitin the best followed by 80 % DD, powder chitin, flake chitin and regenerated chitin. SDS-PAGE and activity staining of crude enzyme of recombinant clone showed three bands with were chitinase activity. The molecular weights were approximately 70, 65 and 58 kDa, which is the same as crude chitinase from B. licheniformis PR-1. Determination of hydrolytic products by HPLC, found a mixture of chitobiose and GlcNAc from pPRChi65.