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)₂SO₄ 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 °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 °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.

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.

In this study, the fermentation broth of the recombinant Pichia pastoris strain ncy-2 was studied. After pretreatment, separation, and purification, lysozyme was optimized using biofilm and ion exchange separation. Finally, lysozyme dry enzyme powder was prepared by concentrating and vacuum drying. The removal rate of bacterial cells was 99.99% when the fermentation broth was centrifuged at low temperature. The optimum conditions were: transmembrane pressure of 0.20 MPa, pH 6.5, 96.6% yield of lysozyme, enzyme activity of 2612.1 u/mg, which was 1.78 times higher than that of the original enzyme; D152 resin was used for adsorption and elution. Process conditions were optimized: the volume ratio of resin to liquid was 15%; the adsorption time was 4 h; the concentration of NaCl was 1.0 mol/L; the recovery rate of lysozyme activity was 95.67%; the enzyme activity was 3879.6 u/mL; and the purification multiple was 0.5, 3.1 times of the original enzyme activity. The enzyme activity of lysozyme dry enzyme powder was 12,573.6 u/mg, which had an inhibitory effect on microsphere lysozyme. Its enzymatic properties were almost the same as those of natural lysozyme, which demonstrated good application prospects and production potential.