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, Hathewaya histolytica is commonly used in industry to produce collagenases, but its application is greatly limited by its pathogenicity. This study first identified five potential Pseudomonas-derived collagenases by sequence alignment. Bioinformatics tools were used to analyze their structures and functions. Heterologous expression of two P. chlororaphis-derived collagenases was achieved in E. coli, and their enzymatic properties were characterized. Bioinformatics analysis shows that the Pseudomonas-derived collagenases had low molecular weights (22.1~50.5 kDa) and good thermal stability (aliphatic index 73.73~88.81). Deletion of P. chlororaphis GP72ANO strain colP1 and colP2 genes had no significant effect on cell growth. The yields of collagenase ColP1 and ColP2 obtained from E. coli 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 Pseudomonas-derived collagenases in E. coli and explored their function and physicochemical properties.