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

Abstract

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+. Keywords: Tfu_0580, Chitin, Chitinase, N-acetylchitooligosaccharides, Thermobifida fuscacloning