Optimized Recombinant Expression and Characterization of Collagenase in Bacillus subtilis WB600
Yaqing Zhu, Linlin Wang, Kaixuan Zheng, Ping Liu, Wenkang Li, Jian Lin, Wenjing Liu, Shoushui Shan, Liqin Sun, Hailing Zhang
Abstract
Background: The collagenase encoding gene col was cloned into a pP43NMK vector and amplified in Escherichia coli JM109 cells. The shuttle vector pP43NMK was used to sub-clone the col gene to obtain the vector pP43NMK-col for the expression of collagenase in Bacillus subtilis WB600. The enzyme was characterized and the composition of the expression medium and culture conditions were optimized. Methods: The expressed recombinant enzyme was purified by ammonium sulfate, ultrafiltration, and through a nickel column. The purified collagenase had an activity of 9405.54 U/mg. Results: The recombinant enzyme exhibited optimal activity at pH 9.0 and 50 °C. Catalytic efficiency of the recombinant collagenase was inhibited by Fe3+ and Cu2+, but stimulated by Co2+, Ca2+, Zn2+, and Mg2+. The optimal conditions for its growth were at pH 7.0 and 35 °C, using 15 g/L of fructose and 36 g/L of yeast powder and peptone mixture (2:1) at 260 rpm with 11% inoculation. The maximal extracellular activity of the recombinant collagenase reached 2746.7 U/mL after optimization of culture conditions, which was 2.4-fold higher than that before optimization. Conclusions: This study is a first attempt to recombinantly express collagenase in B. subtilis WB600 and optimize its expression conditions, its production conditions, and possible scale-up.