Purification and characterization of a bifunctional enzyme with chitosanase and cellulase activity from commercial cellulase

Abstract

A bifunctional enzyme with chitosanase and carboxymethyl cellulase (CMCase) activity was purified from commercial cellulase, which was produced by trichoderma viride, through sequential steps of DEAE-Sepharose CL-6B ion-exchange chromatography, Phenyl Sepharose CL-4B hydrophobic interaction chromatography and Sephadxe G-75 gel filtration. The purified hydrolase was homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass was 66 kDa. The hydrolase exhibited chitosanase activity for chitosan hydrolysis and cellulase activity for carboxymethyl cellulose (CMC) hydrolysis. For chitosan hydrolysis, the enzyme had an optimum pH of 5.2, temperature of 60 °C and exhibited typical Michaelis-Menten kinetics with K m value and V max of 10 mg/ml and 0.164 U/ml, respectively. For CMC hydrolysis, the pH and temperature optima enzyme were 4.0 and 50 °C. Heavy metal ions such as Hg 2+, Ag + significantly or completely inhibited the enzyme activity. Identification of glucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) oligomers as depolymerized products indicated that the enzyme cleaved both GlcN-GlcN and GlcNAc-GlcN linkages. The chitosan hydrolysates were oligomers with one to four glucosamine residues and some oligomers with longer chain length.