Cloning, expression, purification and characterisation of a thermostable chitinase from Bacillus licheniformis A1

Abstract

The chitinase B gene (ch/B65) of Bacillus licheniformis A1 (BlicA1) isolated from Diyadin hotspring in Turkey was cloned and sequenced. The gene is 1779 bp long and encodes a protein 592 amino acids with a 35-amino acid signal peptide at N-terminal. The gene has 99% percent similarity to chiB gene of Bacillus licheniformis under the GenBank number AY205293. The gene without signal peptide was overexpressed in Escherichia coli and the recombinant protein purified by nickel affinity chromatography. The activity of enzyme was shown on SDS-PAGE with the flourogenic substrate 4-methylumbelliferyl ß-D-N,N′-diacetylchitobioside. Kinetic characterisation of the enzyme was performed at 65 °C by using chromogenic substrate p-nitrophenyl N,N′-diacetyl-ß-D-chitobioside, and Km and V max were found to be 0.02 μM and 1017 U/mg protein, respectively. Enzyme has maximal activity at pH 6.0 and was stable over a broad pH range of 5.0-9.0 for 24 h at room temperature and 4 h at 65 °C. Enzyme was 60% stable at 65 °C for 1.5 h. The inhibition or activation of some substances on the activity of enzyme was determined. High kinetic properties of enzyme open the possibility of an extensive structural and enzyme-substrate interaction studies.