Biochemical and molecular characterization of an acido-thermostable endo-chitinase from Bacillus altitudinis KA15 for industrial degradation of chitinous waste

This paper reports the isolation and identification of an acido-thermostable chitinase (ChiA-Ba43) which was purified, from the culture liquid of Bacillus altitudinis strain KA15, and characterized. Purification of ChiA-Ba43 produced a 69.6-fold increase in the specific activity (120,000 U/mg) of the chitinase, with a yield of 51% using colloidal chitin as substrate. ChiA-Ba43 was found to be a monomeric protein with a molecular mass of 43,190.05 Da as determined by MALDI-TOF/MS. N -terminal sequence of the first 27 amino-acids (aa) of ChiABa43 displayed homology to chitinases from other Bacillus species. Interestingly, ChiA-Ba43 exhibited optimum pH and temperature of 4-5.5 and 85 degrees C, respectively. Thin-layer chromatography (TLC) showed that the final hydrolyzed products of the enzyme from chitin-oligosaccharides and colloidal chitin are a mixture of (GlcNAc)(2), (GlcNAc)(3), (GlcNAc)(4), and (GlcNAc)(5), which indicates that ChiA-Ba43 possesses an endo-acting function. More interestingly, compared to ChiA-Mt45, ChiA-Hh59, Chitodextrinase (R), N-acetyl-beta-glucosaminidase (R), and ChiA65, ChiA-Ba43 demonstrated a high level of catalytic efficiency and outstanding tolerance towards various organic solvents. The chiA-Ba43 gene (1332 bp) encoding ChiA-Ba43 (409 aa) was cloned, sequenced, and expressed in Escherichia coli strain HB101. The biochemical properties of the recombinant chitinase (rChiA-Ba43) were equivalent to those of the natively expressed enzyme. These properties make ChiA-Ba43 an ideal candidate for industrial bioconversion of chitinous waste.