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 pro...
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| Format: | Article |
| Language: | English |
| Published: |
China Food Publishing Company
2025-03-01
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| Series: | Shipin Kexue |
| Subjects: | |
| Online Access: | https://www.spkx.net.cn/fileup/1002-6630/PDF/2025-46-6-015.pdf |
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| Summary: | 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 1 077 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 2 249.81 and 2 915.75 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 (Km) of CelL7-CBM3 was 11.70 mg/mL, which was lower than that of CelL7 (Km = 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 (Vmax) was 175.44 mg/(mL·min), the catalytic constant (Kcat) was 2.78 s-1, and the Kcat/Km 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. |
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| ISSN: | 1002-6630 |