| Summary: | Background: The collagenase encoding gene <i>col</i> was cloned into a pP43NMK vector and amplified in <i>Escherichia coli</i> JM109 cells. The shuttle vector pP43NMK was used to sub-clone the <i>col</i> gene to obtain the vector pP43NMK-<i>col</i> for the expression of collagenase in <i>Bacillus subtilis</i> 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 Fe<sup>3+</sup> and Cu<sup>2+</sup>, but stimulated by Co<sup>2+</sup>, Ca<sup>2+</sup>, Zn<sup>2+</sup>, and Mg<sup>2+</sup>. 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 <i>B. subtilis</i> WB600 and optimize its expression conditions, its production conditions, and possible scale-up.
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