Research
| Title： | Structural and functional analyses of microcystinase A: optimized heterologous expression, stability, and degradability |
|---|---|
| First author： | Yang, Siyu; Zuo, Jun; Huang, Licheng; Liu, Jin; Gan, Nanqin |
| Journal： | JOURNAL OF OCEANOLOGY AND LIMNOLOGY |
| Years： | 2024 |
| DOI： | 10.1007/s00343-024-4047-3 |
| Abstract： | Microcystinase (MlrA) is a key endopeptidase that catalyzes microcystin degradation without generating harmful byproduct. However, the application of MlrA in the field is primarily impeded by its limited productivity and short lifespan. Therefore, the MlrA's function was studied by modelling its structure, which subsequently increased its heterologous expression and high-temperature stability. Results demonstrate that after the irregular sequence at the C-terminus of MlrA was removed, enzyme solubility was significantly decreased. In addition, three fusion tags, namely maltose-binding protein, glutathione S-transferase (GST), and N-utilization substance A (NusA) were used to enhance the overexpression of soluble recombinant MlrA, among which NusA-MlrA exhibited the highest solubility. Moreover, NusA-MlrA was active in pH 4-10 at 20-80 degrees C; even at 80 degrees C, approximately 35.8% of fusion protein remained active. NusA-MlrA retained 89% of MlrA's activity even after 7 d of storage at 50 degrees C; and on day 7, the protein retained >90% of its activity at pH 7. Finally, a stable, soluble, and long-lasting heterologous MlrA was successfully constructed that could eliminate microcystins in Escherichia coli C43 (DE3). This study enriched the comprehension of MlrA's structure and enzymatic properties, by particularly addressing the endopeptidase's low expression and short lifespan, which improved its suitability for future applications. |
