Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense
Abstract
:1. Introduction
2. Results
2.1. Target Selection, Aspergillus Niger Transformation, and Screening
2.2. Purification of the Recombinant Laccase and Study of Physico-Chemical Properties
2.3. Kinetic Parameters
2.4. Enzyme Activity and Stability at Different pH and Temperature Values
2.5. Effect of Metal Ions, Inhibitors and Solvents on Laccase Activity
2.6. Effect of Sea Salt on Laccase Activity and Surface Charge of SlLac2
3. Discussion
4. Materials and Methods
4.1. Strains and Culture Conditions
4.2. Cloning and Expression of SlLac2-Encoding Gene
4.3. Screening of Transformants and Laccase Activity Assay
4.4. Production and Purification of Recombinant SlLac2
4.5. Bioinformatic Analysis
4.6. Determination of N-Terminal Amino Acid Sequence and Glycosylation Level
4.7. Substrate Specificity and Kinetics
4.8. Effect of pH and Temperature on the Activity and Stability of the Laccase
4.9. Effect of Metal Ions, Inhibitors, Solvents, and Sea Salt on Laccase Activity.
4.10. Nucleotide Sequence Accession Number
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Accession Number | Signal Peptide (Amino Acids) | Mature Protein (Amino Acids) | Molecular Mass (Da) | PI | Coordination Sites |
---|---|---|---|---|---|
Q70KY3 | 22 | 601 | 68958 | 5.18 | IHWHG WYHSH HPMHLH HCHIAWH |
KY554800 | 21 | 555 | 60904 | 6.09 | IHWHG WYHSH HPMHLH HCHIAWH |
KY554801 | 18 | 543 | 59813 | 4.10 | IHWHG WYHSH HPMHLH HCHIAWH |
APMZ2_prot14771 * | 21 | 592 | 64581 | 8.07 | IHWHG WYHSH HPIHLH HCHIAFH |
APMZ2_prot8345 | 23 | 578 | 63456 | 5.64 | IHWHG WYHSH HPIHLH HCHIAWH |
APMZ2_prot7177 | 21 | 615 | 68221 | 6.03 | IHFHG WYHSH HPIHKH HCHINNH |
APMZ2_prot10330 | No | 754 | 83453 | 5.40 | IHFHG WYHAH HPFHLH HCHNMWH |
APMZ2_prot15523 | No | 729 | 80156 | 5.37 | LHAHG WYHSH HPMHLH HCHLAWH |
APMZ2_prot2393 | No | 554 | 60438 | 4.66 | LHFHG WYHSH HPFHLH HCHIEWH |
APMZ2_prot9198 | 19 | 576 | 64532 | 5.08 | MHWHG FYHSH HPFHLH HCHVLQH |
Purification Steps | Volume (mL) | Total Activity (nKat) | Protein (mg) | Specific Activity (nkat mg−1) | Activity Yield (%) | Purification Factor (fold) |
---|---|---|---|---|---|---|
Culture medium | 930 | 78,027 | 176 | 443 | 100 | 1 |
Ultrafiltration | 500 | 99,000 | 156 | 635 | 126.9 | 1.4 |
CM-Sepharose | 325 | 20,775 | 15.4 | 1349 | 26.6 | 3.0 |
Sephacryl S-200HR | 40 | 18,782 | 7.2 | 2609 | 24.1 | 5.9 |
Substrate | KM (mM) | Kcat (s−1) | Kcat/KM (s−1 mM−1) | |
---|---|---|---|---|
ABTS | 0.0206 +/− 0.0039 | 0.153 +/− 0.0026 | 7.42 | |
DMP | 0.0240 +/− 0.0039 | 0.0059 +/− 0.0003 | 0.24 | |
Syringaldazine | 0.0035 +/− 0.00057 | 0.0074 +/− 0.0002 | 2.11 |
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Ali, W.B.; Ayed, A.B.; Turbé-Doan, A.; Bertrand, E.; Mathieu, Y.; Faulds, C.B.; Lomascolo, A.; Sciara, G.; Record, E.; Mechichi, T. Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense. Int. J. Mol. Sci. 2020, 21, 8402. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218402
Ali WB, Ayed AB, Turbé-Doan A, Bertrand E, Mathieu Y, Faulds CB, Lomascolo A, Sciara G, Record E, Mechichi T. Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense. International Journal of Molecular Sciences. 2020; 21(21):8402. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218402
Chicago/Turabian StyleAli, Wissal Ben, Amal Ben Ayed, Annick Turbé-Doan, Emmanuel Bertrand, Yann Mathieu, Craig B. Faulds, Anne Lomascolo, Giuliano Sciara, Eric Record, and Tahar Mechichi. 2020. "Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense" International Journal of Molecular Sciences 21, no. 21: 8402. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21218402