Cloning and characterization of a chitinase from Thermobifida fusca reveals Tfu_0580 as a thermostable and acidic endochitinase

摘要

Being capable of hydrolyzing chitin, chitinases have various applications such as production of N -acetylchitooligosaccharides (COSs) and N -acetylglucosamine (GlcNAc), degrading chitin as a consolidated bioprocessing, and bio-control of fungal phytopathogens. Here, a putative chitinase in Thermobifida fusca , Tfu_0580, is characterized. Tfu_0580 was purified by homogeneity with a molecular weight of 44.9?kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Tfu_0580 displayed a clear activity against colloidal chitin, which is comparable to a commercial Streptomyces griseus chitinase. Enzyme activities against p -nitrophenyl β-D- N , N ′, N ′′-triacetylchitotriose ( p -NP-(GlcNAc)₃), N , N ′-diacetyl-β-D-chitobioside ( p -NP-(GlcNAc)₂) and p -nitrophenyl N -acetyl-β-D-glucosaminide ( p -NP-(GlcNAc)) showed that Tfu_0580 exhibited highest activity against p -NP-(GlcNAc)₃. Further optimization of the enzyme activity conditions showed: 1) an optimum catalytic activity at pH 6.0 and 30?°C; 2) activity over broad pH (4.8–7.5) and temperature (20–55?°C); 3) stimulation of activity by the metallic ions Ca2+ and Mn2+.