Cloning, Expression and Characterization of an Alginate Lyase in Bacillus subtilis WB600

The aim of this study was to further broaden the heterologous expression of alginate lyase from Vibrio alginolyticus in a Bacillus subtilis expression vector. A B. subtilis WB600/pP43NMK-alg62 strain was constructed. (NH4)(2)SO4 precipitation and Ni-affinity chromatography were performed to purify the enzyme. We then characterized the enzyme. Its molecular weight was 57.64 kDa, and it worked optimally at 30 degrees C with a pH of 8.0. Ca2+ markedly enhanced the enzymatic activity of Alg62 while Cu2+ and Ni2+ inhibited its activity. Alg62 had a wide range of substrate specificity, showing high activity toward sodium alginate and polyG. Following optimization of the fermentation process, the optimal conditions for the recombinant expression of Alg62 were as follows: temperature of 37 degrees C, pH of 7.0, medium consisting of glycerol 15 g/L, yeast powder 25 g/L and K+ 1.5 mmol/L. At these optimal conditions, enzyme activity reached 318.21 U/mL, which was 1.54 times higher than the initial enzyme activity.