Heterologous Expression, Enzymatic Characterization, and Biofilm Eradication Activity of Cellulase CelL7 Derived from Marine Sources

In this study, a novel cellulase gene, CelL7, was cloned from the marine bacterium Zobellia sp. B2. Furthermore, a fusion gene, CelL7-CBM3, was constructed by fusing a carbohydrate-binding module family 3 (CBM3) to CelL7 and heterologously expressed in Escherichia coli BL21. The expressed fusion protein was purified by affinity column chromatography. The full length of the CelL7 gene was 1077 bp, encoding 358 amino acid residues, and the theoretical molecular mass of the encoded protein was 40.39 kDa. The specific enzyme activities of CelL7 and CelL7-CBM3 were 2249.81 and 2915.75 U/mg, respectively. The optimal reaction temperatures for both enzymes were 50 ℃, and the optimal pHs were 5.0 and 5.5, respectively. Mn²⁺ and Fe²⁺ activated the activity of CelL7, while Cu²⁺ inhibited it. CelL7 was capable of degrading carboxymethyl cellulose sodium, cellobiose, and xylan. When sodium carboxymethyl cellulose was used as a substrate, the Michaelis-Menten constant (K_m) of CelL7-CBM3 was 11.70 mg/mL, which was lower than that of CelL7 (K_m = 13.23 mg/mL), indicating that the fusion enzyme, with an added binding domain, exhibited enhanced affinity for carboxymethyl cellulose sodium. The maximum reaction rate (V_max) was 175.44 mg/(mL·min), the catalytic constant (K_cat) was 2.78 s^-1, and the K_cat/K_m was 0.24 mL/(mg·s), which were comparable to those of CelL7. Biofilm clearance experiments showed that concentrations of CelL7 ranging from 10.0 to 60.0 μg/mL and those of CelL7-CBM3 ranging from 30.0 to 60.0 μg/mL were effective in dispersing biofilm and reducing the amount of biofilm.