Purification and characterization of a novel extracellular chitinase from thermophilic <i>Bacillus</i> sp. Hu1

Abstract

The purification and characterization of a new thermophilic chitinase from thermophilic Bacillus sp. HU1, originally isolated from a soil sample collected from hot spring of XinJiang Province, China, is presented in this paper. The purification steps included ammonium sulfate precipitation, with columns of DEAE-Sepharose anion exchange chromatography and Sephacryl S-400 high resolution gel chromatography on AKTA purifier 100 protein liquid chromatography. The method gave a 5.6 fold increase of the specific activity and had a yield of 15%. The molecular weight of the chitinase was found to be around 80.8 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. The optimal pH and optimal temperature of the protease were at pH 6.5 and 60°C, respectively. The thermostable chitinase still retained the activity after incubation for 100 min at 60°C and it was increased about 1.31 times than that of the control (before heating) when the enzyme solution heated at 60°C for 60 min. The chitinase activity was inhibited by Cu²⁺, dithiothreitol (DTT), Na₂.EDTA and activated by Mg²⁺, Ca²⁺ and Zn²⁺ whereas, Na⁺, K⁺ and Fe³⁺ did not show significant inhibitory effects against the chitinase. Substrates specificity tests indicated that, colloid chitin was the best substrate among the seven substrates tested (colloid chitin, particle chitinase, chitosan (80% degree of deacetylation, chitosan (95% degree of deacetylation), carboxymethyl cellulose (CMC), cellulose and soluble starch. It was concluded that the chitinase have high specificity in hydrolyzing glycosidic bond between GlcNAc-GlcNAc.

Key words: Thermostable chitinase, purification, characterization, thermophilic bacillus sp.