Cloning of DNA fragment containing chitinase gene from Burkholderia cepacia

Abstract

Chitinase (EC 3.2.1.14) is an enzyme that catalyzes the degradation of chitin, an insoluble Beta-1,4-linked polymer of N-acetylglucosamine. Chitinases are present in a wide range of organisms, including bacteria, insects, plants and animals. Burkholderia cepacia, isolated in Thailand, is capable of chitinase production. After SDS-PAGE and activity staining of culture medium, at least one band of chitinase activity, molecular weight 47.5 kDa, was detected. The optimum pH and temperature of crude enzyme were 7.0 and 40ํC, respectively. Chitinase hydrolyzed colloidal chitin the best, followed by glycol chitin, flake chitin, crab shells, 78% deacetylated chitosan and 90% deacetylated chitosan. Chromosomal DNA of Burkholderia cepacia was completely digested with EcoR I, BamH I, Pst i and Hind III and subjected to Southern blot analysis using a degenerate probe specific for family 18 chitinase gene, CBI. Southern blot analysis showed 7.0 kb band, 7.8 kb and 10 kb bands, and 1.2 kb and 1.8 kb bands when the chromosomal DNA was digested with EcoR I, BamH I and Pst I, respectively. EcoR I digested DNA fragments between 6 to 9 kb and BamH I digested DNA fragments between 6 to 12 kb were cloned into E.coli DH5alpha using pBluescrip/KS+ and screen for chitinase gene. pKK243B gave a positive signal on dot blot analysis when hybridize with CB1. Southern blot analysis of pKK243B showed 7.8 kb band and 1.8 kb band when pKK243B was digested with BamH I and Pst I, respectively. Colonies containing pKK243B gave a clear zone around the colony when grown on LB-glycol chitin agar and stained with congo red. In cultured LB-colloidal chitin medium, transformant carrying pKK243B showed 33% of chitinase activity compared with activity compared with activity from Burkholderia cepacia (100%). The 1.8 kb DNA fragment from Pst I digestion of pKK243B was sequenced. A partial open reading frame of 206 amino acid translated from nucleotide 1-618 and an open reading frame of 244 amino acid translated from nucleotide 618-1349 were found. The deduced amino acid sequences were compared with protein sequences in the Genbank Database. The partial open reading frame of 206 amino acid is similar to amino acid sequence of putative sensor proteins. The reading frame of 244 amino acid sequence shares homology with amino acid sequence of putative 2-component transcriptional regulator. No open reading frame which have similarity with chitinase gene was found in 1.8 kb Pst I fragment.