Purification, characterization, and molecular cloning of an extracellular chitinase from Bacillus licheniformis stain LHH100 isolated from wastewater samples in Algeria.

Abstract An extracellular chitinase (ChiA-65) was produced and purified from a newly isolated Bacillus licheniformis LHH100. Pure protein was obtained after heat treatment and ammonium sulphate precipitation followed by Sephacryl S-200 chromatography. Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 65,195.13 Da. The sequence of the 27 N-terminal residues of the mature ChiA-65 showed high homology with family-18 chitinases. Optimal activity was achieved at pH 4 and 75 °C. Among the inhibitors and metals tested, p -chloromercuribenzoic acid, N -ethylmaleimide, Hg 2+ , and Hg + completely inhibited enzyme activity. Chitinase activity was high on colloidal chitin, glycol chitin, glycol chitosane, chitotriose, and chitooligosaccharide. Chitinase activity towards synthetic substrates in the order of p -NP-(GlcNAc) n ( n  = 2–4) was p -NP-(GlcNAc) 2  >  p -NP-(GlcNAc) 4  >  p -NP-(GlcNAc) 3 . Our results suggest that ChiA-65 preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GlcNAc) n . ChiA-65 obeyed Michaelis-Menten kinetics, the K m and k cat values being 0.385 mg, colloidal chitin/ml and 5000 s −1 , respectively. The chiA-65 gene encoding ChiA-65 was cloned in Escherichia coli and its sequence was determined. Above all, ChiA-65 exhibited remarkable biochemical properties suggesting that this enzyme is suitable for bioconversion of chitin waste.