Heterologous Expression and Characterization of Collagenases from Pseudomonas chlororaphis GP72

Collagenases can specifically degrade collagen, showing a wide application prospect in food, leather, waste utilization, biotechnology, and other industries. Currently, <i>Hathewaya histolytica</i> is commonly used in industry to produce collagenases, but its application is greatly limited by its pathogenicity. This study first identified five potential <i>Pseudomonas</i>-derived collagenases by sequence alignment. Bioinformatics tools were used to analyze their structures and functions. Heterologous expression of two <i>P. chlororaphis</i>-derived collagenases was achieved in <i>E. coli</i>, and their enzymatic properties were characterized. Bioinformatics analysis shows that the <i>Pseudomonas</i>-derived collagenases had low molecular weights (22.1~50.5 kDa) and good thermal stability (aliphatic index 73.73~88.81). Deletion of <i>P. chlororaphis</i> GP72ANO strain <i>colP1</i> and <i>colP2</i> genes had no significant effect on cell growth. The yields of collagenase ColP1 and ColP2 obtained from <i>E. coli</i> BL21(DE3) cultivation broth were 148 mg/L and 322 mg/L, respectively. The optimum temperature of each collagenase was 28 °C, and the soluble collagen activities of ColP1 and ColP2 were up to 42.64 U/mg and 21.21 U/mg, respectively. Collagenase ColP1 had the highest enzyme activity at pH 8, while collagenase ColP2 had the highest enzyme activity at pH 4. Metal ions such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, and Mn²⁺ inhibited the activity of collagenases to different degrees. This study successfully achieved recombinant expression and preliminary purification of <i>Pseudomonas</i>-derived collagenases in <i>E. coli</i> and explored their function and physicochemical properties.