Purification and characterization of exo-β-d-glucosaminidase from commercial lipase

A chitosanolytic enzyme without lipolytic activity was purified to apparent homogeneity from a commercial lipase preparation by using a combination of DEAE-Sepharose CL-6B exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, and Sephacryl S-200 gel filtration chromatography, and the purified enzyme was characterized. The molecular mass of the homodimeric protein was about 130 kDa. The optimum action temperature and pH of the enzyme on chitosan were 60 °C and 4.6–4.8, respectively, and the enzyme was stable at temperatures lower than 60 °C and at pH 4–9. The purified enzyme exhibited the highest activity toward chitosan which was 73–82% deacetylated. TLC analysis shows that the purified enzyme released glucosamine residues successively from the substrates and final hydrolysis products of both chitosan tetramer and pentamer were glucosamine, indicating that the enzyme exhibited exo-β-d-glucosaminidase activity.