Purification and characterization of thermostable chitinases from bacillus lichemiformis MB-2

Abstract

Chitinases are of great biotechnological interest and have received remarkable attention. A themophilic bacterium, Bacillus lichenifomis MB-2, from Tompaso hot spring, North Sulawesi Indonesia, secreted thermostable chitinases into culture media. Preliminary study showed that the enzymes occur in multiple forms and were stable at a high temperature and a broad range of pH. The objectives of this research were to purify and characterize thermostable chitinases from B. lichenrformis MB-2. The extracellular chitinases were isolated by successive column chromatographies on Phenyl Sepharose CL4B, DEAE Sephacel, and Superdex G-75. The purified enzymes had molecular weight of 67 (Chi-67) and 102 kDa (Chi-102), estimated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Chi-67 and Chi-102 had an endo- and exo- chitinolytic activities, respectively. The optimal temperature of Chi-67 was 70°C, whereas Chi-102 was 60 "C. The optimal pH for both enzymes was 6.0. Chi-67 wets stable below 60°C and over a broad pH range of 4-1 1 for 1 h. In contrast, Chi- 102 was not stable at temperature above 50 "C and only stable at its optimum pH for 1 h. Chi-47 was resistant to denaturation by urea, Tween-20 and Triton-X, but unstable toward organic solvents such as propanol, ethanol, DMSO (dimethyl sdfoxide), and PEG (polyethylene glycol), indicating that hydrophobic interaction of proteins plays an important role in maintaining enzyme activity. Ionic interactions are also important for Chi-67 fold as the activity was reduced by guanidine hydrochloride and NaCl (1 M). Chi- 1 02 was relatively more stable toward various organic solvents than Chi-67. Both enzymes hydrolyzed colloidal chitin, glycol chitin, or glycol chitosan, but were less active to regenerated chitin, fine powder of chitin, or methyl cellulose. The Michaelis constants &) for colloidal chitin, glycol chitin, 4- methylumbelliferyl N', W-diacetylchitobioside [MUF(GICNAC)~], 4- methyiumbelliferyl N', N', M-triacetylchitotrioside [MCTF(G~CNAC)f~o]r Chi-67 were 3.08 mg ml-', 0.3 15 mg ml-', 0.02 pmol d-' and 0.02 pmol dl, respectively. Meanwhile, the K, for colloidal chitin, glycol chitin and MUF(GlcNAc) for Chi-102 were 2.00 mg ml-', 1.32 rng ml-', and 0.03 mM ml-', respectively. The first 13 N-tertninal amino acids of Chi-67 were determined to be SGKNYKIIGYYPS, which is identical to chitinase from B. licImenIfomris PR- 1 (accession no. AA022 144) and B. circuImrs No. 4.1 (accession no. AM23 3 68).