Purification and characterization of a novel lichenase from Bacillus licheniformis GZ-2

A novel lichenase from Bacillus licheniformis GZ-2 was purified to homogeneity by two steps ion-exchange chromatography with a specific activity of 8231.3 U/mg. The purified enzyme showed as a single protein band with a molecular mass of 25 kDa. The optimum pH and temperature for the enzyme activity were 6.5 and 60 °C, respectively. The enzyme exhibited strict specificity for β-1,3-1,4-d-glucans. The kinetic parameters K_m and V_max were 5.11 mg/mL and 2097 μmol/Min/mg for lichenan and 7.42 mg/mL and 1440 μmol/Min/mg for barley β-glucan. Compared to most of the reported β-1,3-1,4-glucanases (lichenase), the activity of the purified enzyme for lichenan was much higher than that for barley β-glucan. The main products of β-glucan hydrolyzed by the lichenase were cellubiosyltriose (DP3) and cellutriosyltraose (DP4). The lichenase gene from B. licheniformis GZ-2 was cloned and sequenced. The open reading frame of gene gz-2 contained 642 bp coding for a 214 amino acid mature protein. The gene was cloned into an expression vector pET 28a and expressed in Escherichia coli BL21. The activity in cell lysate supernatant was 137.9 U/mg.