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

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 65.195.13Da. 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 degrees C. Among the inhibitors and metals tested, p-chloromercuribenzoic acid, N-ethylmaleimide, Hg2(+), 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-(GIcNAc)(n), (n=2-4) was p-NP-(GIcNAc)(2)>p-NP-(GIcNAc)(4)>p-NP-(GIcNAc)(3). Our results suggest that ChiA-65 preferentially hydrolyzed the second glycosidic link from the non-reducing end of (GIcNAc)(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 coil and its sequence was determined. Above all, ChiA-65 exhibited remarkable biochemical properties suggesting that this enzyme is suitable for bioconversion of chitin waste. (C) 2014 Elsevier B.V. All rights reserved.