Cloning and nucleotide sequencing of chitinase gene from Burkholderia cepacia TU09

Abstract

Chitinase (EC3.2.1.14) is an enzyme that catalyzes the degradation of chitin. Burkholderia cepacia TU09, isolated from soil in Thailand, is capable of producing chitinase. Chitinase from B. cepacia TU09 produced mostly 2-acetamido-2-deoxy-D-glucose from beta-chitin and alfa-chitin, over 85% yield was produced within 1 day and 7 days, respectively. Crude chitinase was able to hydrolyze 45% deacetylated chitiosan colloidal chitin well, followed by flake chitin and crab shells. After SDS-PAGE and activity staining of crude enzyme, at least 4 bands of chitinase activity, molecular weight 40, 50, 60 and 90 kDa were detected. The pH and temperature optimum of the crude enzyme was 5.0/8.0 and 37ํC/55ํC, respectively. Chromosomal DNA of B.cepacia was partially cut and shotgun cloned into E.coli, JM109. Clones harboring chitinase gene were detected by the formation of clear-zone around the colony on agar plate containing colloidal chitin. One out of seven thousand colonies was found to produce clear-zone. The positive clone contained a plasmid with 2.8 kb insert fragment, pKK Chi60. DNA sequencing analysis revealed that pKKChi60 contained chitinase gene with an open reading frame of 1,689 bp, Chi60. Chi60 encodes for a protein with 563 amino acid residues, and a deduced molecular weight of 60,942 Da. The amino acid sequence of Chi60 exhibited over 90% homology to chitinase A of Serratia marcescens. After SDS-PAGE and activity staining of culture medium from the E.coli tranformant, a single band of chitinase activity, molecular weight 60 kDa was observed. Subsequently, partial characterization of Chi60 demonstrated that the optimum pH and temperature was between 4.0-6.0 and 50-60ํC, respectively. The relative hydrolytic activity of Chi60 for crystalline chitin (alfa-chitin crab shells) were 50% and 70% of the activity observed on soluble chitin and amorphous chitin substrate, respectively. Analyses of the degradation product by HPLC showed that the cloned enzyme, Chi60, produces N, N'diacetylchitobiose from chitin.