A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver
The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochro...
- Autores:
-
Bahramian Nasab, Soudeh
Homaei, Ahmad
FERNANDEZ LAFUENTE, ROBERTO
Del Arco, Jon
Fernández-Lucas, Jesús
- Tipo de recurso:
- Article of investigation
- Fecha de publicación:
- 2023
- Institución:
- Corporación Universidad de la Costa
- Repositorio:
- REDICUC - Repositorio CUC
- Idioma:
- OAI Identifier:
- oai:repositorio.cuc.edu.co:11323/10385
- Acceso en línea:
- https://hdl.handle.net/11323/10385
https://repositorio.cuc.edu.co/
- Palabra clave:
- Marine enzymes
Cytochrome P450 reductase
Purification
Biotechnology
- Rights
- openAccess
- License
- Atribución 4.0 Internacional (CC BY 4.0)
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dc.title.eng.fl_str_mv |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
title |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
spellingShingle |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver Marine enzymes Cytochrome P450 reductase Purification Biotechnology |
title_short |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
title_full |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
title_fullStr |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
title_full_unstemmed |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
title_sort |
A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liver |
dc.creator.fl_str_mv |
Bahramian Nasab, Soudeh Homaei, Ahmad FERNANDEZ LAFUENTE, ROBERTO Del Arco, Jon Fernández-Lucas, Jesús |
dc.contributor.author.none.fl_str_mv |
Bahramian Nasab, Soudeh Homaei, Ahmad FERNANDEZ LAFUENTE, ROBERTO Del Arco, Jon Fernández-Lucas, Jesús |
dc.subject.proposal.eng.fl_str_mv |
Marine enzymes Cytochrome P450 reductase Purification Biotechnology |
topic |
Marine enzymes Cytochrome P450 reductase Purification Biotechnology |
description |
The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6–9) and temperatures (40–60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol−1 K−1. The KM values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the kcat values were 206.79 s−1 and 202.93 s−1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min−1 mg1, the highest activity value described for a CPR up to date (3.2–4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst. |
publishDate |
2023 |
dc.date.accessioned.none.fl_str_mv |
2023-08-14T20:26:09Z |
dc.date.available.none.fl_str_mv |
2023-08-14T20:26:09Z |
dc.date.issued.none.fl_str_mv |
2023-01-29 |
dc.type.spa.fl_str_mv |
Artículo de revista |
dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_2df8fbb1 |
dc.type.content.spa.fl_str_mv |
Text |
dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/ART |
dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.coarversion.spa.fl_str_mv |
http://purl.org/coar/version/c_970fb48d4fbd8a85 |
format |
http://purl.org/coar/resource_type/c_2df8fbb1 |
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dc.identifier.citation.spa.fl_str_mv |
Bahramian Nasab, S.; Homaei, A.; Fernandez-Lafuente, R.; Del Arco, J.; Fernández-Lucas, J. A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver. Mar. Drugs 2023, 21, 99. https://doi.org/10.3390/ md21020099 |
dc.identifier.uri.none.fl_str_mv |
https://hdl.handle.net/11323/10385 |
dc.identifier.doi.none.fl_str_mv |
10.3390/ md21020099 |
dc.identifier.eissn.spa.fl_str_mv |
1660-3397 |
dc.identifier.instname.spa.fl_str_mv |
Corporación Universidad de la Costa |
dc.identifier.reponame.spa.fl_str_mv |
REDICUC - Repositorio CUC |
dc.identifier.repourl.spa.fl_str_mv |
https://repositorio.cuc.edu.co/ |
identifier_str_mv |
Bahramian Nasab, S.; Homaei, A.; Fernandez-Lafuente, R.; Del Arco, J.; Fernández-Lucas, J. A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver. Mar. Drugs 2023, 21, 99. https://doi.org/10.3390/ md21020099 10.3390/ md21020099 1660-3397 Corporación Universidad de la Costa REDICUC - Repositorio CUC |
url |
https://hdl.handle.net/11323/10385 https://repositorio.cuc.edu.co/ |
dc.relation.ispartofjournal.spa.fl_str_mv |
Marine Drugs |
dc.relation.references.spa.fl_str_mv |
1. Hale, S.S.; Buffum, H.W.; Hughes, M.M. Six decades of change in pollution and benthic invertebrate biodiversity in a southern New England estuary. Mar. Pollut. Bull. 2018, 133, 77–87. [CrossRef] [PubMed] 2. Gaur, N.; Narasimhulu, K.; PydiSetty, Y. Recent advances in the bio-remediation of persistent organic pollutants and its effect on environment. J. Clean. Prod. 2018, 198, 1602–1631. [CrossRef] 3. Ismail, M.; Akhtar, K.; Khan, M.; Kamal, T.; Khan, M.A.; Asiri, A.M.; Seo, J.; Khan, S.B. Pollution, toxicity and carcinogenicity of organic dyes and their catalytic bio-remediation. Curr. Pharm. Des. 2019, 25, 3645–3663. [CrossRef] 4. Razzaghi, M.; Homaei, A.; Vianello, F.; Azad, T.; Sharma, T.; Nadda, A.K.; Stevanato, R.; Bilal, M.; Iqbal, H. Industrial applications of immobilized nano-biocatalysts. Bioprocess Biosyst. Eng. 2022, 45, 237–256. [CrossRef] [PubMed] 5. Ranjbari, N.; Razzaghi, M.; Fernandez-Lafuente, R.; Shojaei, F.; Satari, M.; Homaei, A. Improved features of a highly stable protease from Penaeus vannamei by immobilization on glutaraldehyde activated graphene oxide nanosheets. Int. J. Biol. Macromol. 2019, 130, 564–572. [CrossRef] 6. Yan, H.; Wu, L.; Yu, J. The environmental impact analysis of hazardous materials and the development of green technology in the shipbreaking process. Ocean Eng. 2018, 161, 187–194. [CrossRef] 7. Homaei, A. Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain. J. Mol. Catal. B Enzym. 2015, 118, 16–22. [CrossRef] 8. Ufarte, L.; Laville, E.; Duquesne, S.; Potocki-Veronese, G. Metagenomics for the discovery of pollutant degrading enzymes. Biotechnol. Adv. 2015, 33, 1845–1854. [CrossRef] 9. Kadri, T.; Rouissi, T.; Brar, S.K.; Cledon, M.; Sarma, S.; Verma, M. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci. 2017, 51, 52–74. [CrossRef] 10. Homaei, A. Immobilization of Penaeus merguiensis alkaline phosphatase on gold nanorods for heavy metal detection. Ecotoxicol. Environ. Saf. 2017, 136, 1–7. [CrossRef] 11. Ahuja, S.K.; Ferreira, G.M.; Moreira, A.R. Utilization of enzymes for environmental applications. Crit. Rev. Biotechnol. 2004, 24, 125–154. [CrossRef] 12. Nikolaivits, E.; Dimarogona, M.; Fokialakis, N.; Topakas, E. Marine-derived biocatalysts: Importance, accessing, and application in aromatic pollutant bioremediation. Front. Microbiol. 2017, 8, 265. [CrossRef] [PubMed] 13. Menzorova, N.I.; Seitkalieva, A.V.; Rasskazov, V.A. Enzymatic methods for the determination of pollution in seawater using salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius. Mar. Pollut. Bull. 2014, 79, 188–195. [CrossRef] [PubMed] 14. Seitkalieva, A.V.; Menzorova, N.I.; Rasskazov, V.A. Application of different enzyme assays and biomarkers for pollution monitoring of the marine environment. Environ. Monit. Assess. 2016, 188, 1–13. [CrossRef] [PubMed] 15. Ducharme, J.; Auclair, K. Use of bioconjugation with cytochrome P450 enzymes. Biochim. Et Biophys. Acta (BBA) Proteins Proteom. 2018, 1866, 32–51. [CrossRef] [PubMed] 16. Rudeck, J.; Bert, B.; Marx-Stoelting, P.; Schönfelder, G.; Vogl, S. Liver lobe and strain differences in the activity of murine cytochrome p450 enzymes. Toxicology 2018, 404, 76–85. [CrossRef] [PubMed] 17. Brummund, J.; Müller, M.; Schmitges, T.; Kaluzna, I.; Mink, D.; Hilterhaus, L.; Liese, A. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro. J. Biotechnol. 2016, 233, 143–150. [CrossRef] 18. Sharma, B.; Dangi, A.K.; Shukla, P. Contemporary enzyme based technologies for bioremediation: A review. J. Environ. Manag. 2018, 210, 10–22. [CrossRef] [PubMed] 19. Zheng, S.; Chen, B.; Qiu, X.; Lin, K.; Yu, X. Three novel cytochrome P450 genes identified in the marine polychaete Perinereis nuntia and their transcriptional response to xenobiotics. Aquat. Toxicol. 2013, 134, 11–22. [CrossRef] 20. Han, J.; Won, E.-J.; Kang, H.-M.; Lee, M.-C.; Jeong, C.-B.; Kim, H.-S.; Hwang, D.-S.; Lee, J.-S. Marine copepod cytochrome P450 genes and their applications for molecular ecotoxicological studies in response to oil pollution. Mar. Pollut. Bull. 2017, 124, 953–961. [CrossRef] 21. Basilone, G.; Gargano, A.; Corriero, A.; Zupa, R.; Santamaria, N.; Mangano, S.; Ferreri, R.; Pulizzi, M.; Mazzola, S.; Bonanno, A. Liver melanomacrophage centres and CYP1A expression as response biomarkers to environmental pollution in European anchovy (Engraulis encrasicolus) from the western Mediterranean Sea. Mar. Pollut. Bull. 2018, 131, 197–204. [CrossRef] [PubMed] 22. Mundle, S.O.; Spain, J.C.; Lacrampe-Couloume, G.; Nishino, S.F.; Lollar, B.S. Branched pathways in the degradation of cDCE by cytochrome P450 in Polaromonas sp. JS666. Sci. Total Environ. 2017, 605, 99–105. [CrossRef] [PubMed] 23. Gunasekaran, V.; Donmez, E.; Girhard, M.; Urlacher, V.B.; Constantí, M. Biodegradation of fuel oxygenates and their effect on the expression of a newly identified cytochrome P450 gene in Achromobacter xylosoxidans MCM2/2/1. Process Biochem. 2014, 49, 124–129. [CrossRef] 24. Wu, R.-R.; Dang, Z.; Yi, X.-Y.; Yang, C.; Lu, G.-N.; Guo, C.-L.; Liu, C.-Q. The effects of nutrient amendment on biodegradation and cytochrome P450 activity of an n-alkane degrading strain of Burkholderia sp. GS3C. J. Hazard. Mater. 2011, 186, 978–983. [CrossRef] [PubMed] 25. Pandey, A.V.; Flück, C.E. NADPH P450 oxidoreductase: Structure, function, and pathology of diseases. Pharmacol. Ther. 2013, 138, 229–254. [CrossRef] [PubMed] 26. Klotz, A.V.; Stegeman, J.J.; Walsh, C. An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish Stenotomus chrysops. Arch. Biochem. Biophys. 1983, 226, 578–592. [CrossRef] 27. Sen, A.; Arinc, E. Purification and characterization of cytochrome P450 reductase from liver microsomes of feral leaping mullet (Liza saliens). J. Biochem. Mol. Toxicol. 1998, 12, 103–113. [CrossRef] 28. Arinç, E. Characterization of cytochrome P450 dependent mixed-function oxidase system of gilthead seabream (Sparus aurata; Sparidae) liver. Comp. Biochem. Physiol. Part B Comp. Biochem. 1993, 104, 133–139. [CrossRef] 29. Kojima, H.; Takahashi, K.; Sakane, F.; Koyama, J. Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes. J. Biochem. 1987, 102, 1083–1088. [CrossRef] 30. Kubota, S.; Yoshida, Y.; Kumaoka, H.; Furumichi, A. Studies on the microsomal electron-transport system of anaerobically grown yeast: V. Purification and characterization of NADPH-cytochrome c reductase. J. Biochem. 1977, 81, 197–205. [CrossRef] 31. Shen, A.L.; Porter, T.; Wilson, T.; Kasper, C. Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis. J. Biol. Chem. 1989, 264, 7584–7589. [CrossRef] [PubMed] 32. Kuzu, M.; Ciftci, M. Purification and characterization of NADPH-cytochrome P450 reductase from Lake Van fish liver microsomes and investigation of some chemical and metals’ effects on the enzyme activity. Turk. J. Chem. 2015, 39, 149–158. [CrossRef] 33. Kuwahara, T.; White, R.A., Jr.; Agosin, M. A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties. Arch. Biochem. Biophys. 1985, 239, 18–28. [CrossRef] [PubMed] 34. Tsou, C.-Y.; Matsunaga, S.; Okada, S. Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race. J. Biosci. Bioeng. 2018, 125, 30–37. [CrossRef] 35. Milhim, M.; Gerber, A.; Neunzig, J.; Hannemann, F.; Bernhardt, R. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase. J. Biotechnol. 2016, 231, 83–94. [CrossRef] 36. Lee, G.-Y.; Kim, H.M.; Ma, S.H.; Park, S.H.; Joung, Y.H.; Yun, C.-H. Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum. Plant Physiol. Biochem. 2014, 82, 116–122. [CrossRef] 37. Takahashi, N.; Saito, T.; Goda, Y.; Tomita, K. Characterization of microsomal NADPH-dependent aldehyde reductase from rat brain. J. Biochem. 1986, 99, 513–519. [CrossRef] 38. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef] 39. Guengerich, F.P.; Martin, M.V.; Sohl, C.D.; Cheng, Q. Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase. Nat. Protoc. 2009, 4, 1245. [CrossRef] 40. Williams, J.; Kamin, H. The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. J. Biol. Chem. 1962, 237, 587–595. [CrossRef] 41. Yonetani, T. Studies on cytochrome c peroxidase II. Stoichiometry between enzyme, H2O2, and ferrocytochrome c and enzymic determination of extinction coefficients of cytochrome c. J. Biol. Chem. 1965, 240, 4509–4514. [CrossRef] [PubMed] 42. Swift, M.L. GraphPad prism, data analysis, and scientific graphing. J. Chem. Inf. Comput. Sci. 1997, 37, 411–412. [CrossRef] 43. Arrhenius, S. On the reaction velocity of the inversion of cane sugar by acids. J. Phys. Chem 1889, 4, 226. 44. Copeland, R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis; John Wiley & Sons: Hoboken, NJ, USA, 2004. 45. Lamb, S.B.; Lamb, D.C.; Kelly, S.L.; Stuckey, D.C. Cytochrome P450 immobilisation as a route to bioremediation/biocatalysis. FEBS Lett. 1998, 431, 343–346. [CrossRef] |
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© 2023 by the authors. Licensee MDPI, Basel, Switzerland. |
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Atribución 4.0 Internacional (CC BY 4.0) |
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https://creativecommons.org/licenses/by/4.0/ |
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Atribución 4.0 Internacional (CC BY 4.0) © 2023 by the authors. Licensee MDPI, Basel, Switzerland. https://creativecommons.org/licenses/by/4.0/ http://purl.org/coar/access_right/c_abf2 |
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Atribución 4.0 Internacional (CC BY 4.0)© 2023 by the authors. Licensee MDPI, Basel, Switzerland.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Bahramian Nasab, SoudehHomaei, AhmadFERNANDEZ LAFUENTE, ROBERTODel Arco, JonFernández-Lucas, Jesús2023-08-14T20:26:09Z2023-08-14T20:26:09Z2023-01-29Bahramian Nasab, S.; Homaei, A.; Fernandez-Lafuente, R.; Del Arco, J.; Fernández-Lucas, J. A Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver. Mar. Drugs 2023, 21, 99. https://doi.org/10.3390/ md21020099https://hdl.handle.net/11323/1038510.3390/ md210200991660-3397Corporación Universidad de la CostaREDICUC - Repositorio CUChttps://repositorio.cuc.edu.co/The use of marine enzymes as catalysts for biotechnological applications is a topical subject. Marine enzymes usually display better operational properties than their animal, plant or bacterial counterparts, enlarging the range of possible biotechnological applications. Due to the fact that cytochrome P450 enzymes can degrade many different toxic environmental compounds, these enzymes have emerged as valuable tools in bioremediation processes. The present work describes the isolation, purification and biochemical characterization of a liver NADPH-dependent cytochrome P450 reductase (CPR) from the marine fish Liza klunzingeri (LkCPR). Experimental results revealed that LkCPR is a monomer of approximately 75 kDa that is active in a wide range of pH values (6–9) and temperatures (40–60 °C), showing the highest catalytic activity at pH 8 and 50 °C. The activation energy of the enzyme reaction was 16.3 kcal mol−1 K−1. The KM values for cytochrome C and NADPH were 8.83 μM and 7.26 μM, and the kcat values were 206.79 s−1 and 202.93 s−1, respectively. LkCPR displayed a specific activity versus cytochrome C of 402.07 µmol min−1 mg1, the highest activity value described for a CPR up to date (3.2–4.7 times higher than the most active reported CPRs) and showed the highest thermostability described for a CPR. Taking into account all these remarkable catalytic features, LkCPR offers great potential to be used as a suitable biocatalyst.12 páginasapplication/pdfMultidisciplinary Digital Publishing Institute (MDPI)Switzerlandhttps://www.mdpi.com/1660-3397/21/2/99A novel, highly potent nadph-dependent cytochrome P450 reductase from waste liza klunzingeri liverArtículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a85Marine Drugs1. Hale, S.S.; Buffum, H.W.; Hughes, M.M. Six decades of change in pollution and benthic invertebrate biodiversity in a southern New England estuary. Mar. Pollut. Bull. 2018, 133, 77–87. [CrossRef] [PubMed]2. Gaur, N.; Narasimhulu, K.; PydiSetty, Y. Recent advances in the bio-remediation of persistent organic pollutants and its effect on environment. J. Clean. Prod. 2018, 198, 1602–1631. [CrossRef]3. Ismail, M.; Akhtar, K.; Khan, M.; Kamal, T.; Khan, M.A.; Asiri, A.M.; Seo, J.; Khan, S.B. Pollution, toxicity and carcinogenicity of organic dyes and their catalytic bio-remediation. Curr. Pharm. Des. 2019, 25, 3645–3663. [CrossRef]4. Razzaghi, M.; Homaei, A.; Vianello, F.; Azad, T.; Sharma, T.; Nadda, A.K.; Stevanato, R.; Bilal, M.; Iqbal, H. Industrial applications of immobilized nano-biocatalysts. Bioprocess Biosyst. Eng. 2022, 45, 237–256. [CrossRef] [PubMed]5. Ranjbari, N.; Razzaghi, M.; Fernandez-Lafuente, R.; Shojaei, F.; Satari, M.; Homaei, A. Improved features of a highly stable protease from Penaeus vannamei by immobilization on glutaraldehyde activated graphene oxide nanosheets. Int. J. Biol. Macromol. 2019, 130, 564–572. [CrossRef]6. Yan, H.; Wu, L.; Yu, J. The environmental impact analysis of hazardous materials and the development of green technology in the shipbreaking process. Ocean Eng. 2018, 161, 187–194. [CrossRef]7. Homaei, A. Purification and biochemical properties of highly efficient alkaline phosphatase from Fenneropenaeus merguiensis brain. J. Mol. Catal. B Enzym. 2015, 118, 16–22. [CrossRef]8. Ufarte, L.; Laville, E.; Duquesne, S.; Potocki-Veronese, G. Metagenomics for the discovery of pollutant degrading enzymes. Biotechnol. Adv. 2015, 33, 1845–1854. [CrossRef]9. Kadri, T.; Rouissi, T.; Brar, S.K.; Cledon, M.; Sarma, S.; Verma, M. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J. Environ. Sci. 2017, 51, 52–74. [CrossRef]10. Homaei, A. Immobilization of Penaeus merguiensis alkaline phosphatase on gold nanorods for heavy metal detection. Ecotoxicol. Environ. Saf. 2017, 136, 1–7. [CrossRef]11. Ahuja, S.K.; Ferreira, G.M.; Moreira, A.R. Utilization of enzymes for environmental applications. Crit. Rev. Biotechnol. 2004, 24, 125–154. [CrossRef]12. Nikolaivits, E.; Dimarogona, M.; Fokialakis, N.; Topakas, E. Marine-derived biocatalysts: Importance, accessing, and application in aromatic pollutant bioremediation. Front. Microbiol. 2017, 8, 265. [CrossRef] [PubMed]13. Menzorova, N.I.; Seitkalieva, A.V.; Rasskazov, V.A. Enzymatic methods for the determination of pollution in seawater using salt resistant alkaline phosphatase from eggs of the sea urchin Strongylocentrotus intermedius. Mar. Pollut. Bull. 2014, 79, 188–195. [CrossRef] [PubMed]14. Seitkalieva, A.V.; Menzorova, N.I.; Rasskazov, V.A. Application of different enzyme assays and biomarkers for pollution monitoring of the marine environment. Environ. Monit. Assess. 2016, 188, 1–13. [CrossRef] [PubMed]15. Ducharme, J.; Auclair, K. Use of bioconjugation with cytochrome P450 enzymes. Biochim. Et Biophys. Acta (BBA) Proteins Proteom. 2018, 1866, 32–51. [CrossRef] [PubMed]16. Rudeck, J.; Bert, B.; Marx-Stoelting, P.; Schönfelder, G.; Vogl, S. Liver lobe and strain differences in the activity of murine cytochrome p450 enzymes. Toxicology 2018, 404, 76–85. [CrossRef] [PubMed]17. Brummund, J.; Müller, M.; Schmitges, T.; Kaluzna, I.; Mink, D.; Hilterhaus, L.; Liese, A. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro. J. Biotechnol. 2016, 233, 143–150. [CrossRef]18. Sharma, B.; Dangi, A.K.; Shukla, P. Contemporary enzyme based technologies for bioremediation: A review. J. Environ. Manag. 2018, 210, 10–22. [CrossRef] [PubMed]19. Zheng, S.; Chen, B.; Qiu, X.; Lin, K.; Yu, X. Three novel cytochrome P450 genes identified in the marine polychaete Perinereis nuntia and their transcriptional response to xenobiotics. Aquat. Toxicol. 2013, 134, 11–22. [CrossRef]20. Han, J.; Won, E.-J.; Kang, H.-M.; Lee, M.-C.; Jeong, C.-B.; Kim, H.-S.; Hwang, D.-S.; Lee, J.-S. Marine copepod cytochrome P450 genes and their applications for molecular ecotoxicological studies in response to oil pollution. Mar. Pollut. Bull. 2017, 124, 953–961. [CrossRef]21. Basilone, G.; Gargano, A.; Corriero, A.; Zupa, R.; Santamaria, N.; Mangano, S.; Ferreri, R.; Pulizzi, M.; Mazzola, S.; Bonanno, A. Liver melanomacrophage centres and CYP1A expression as response biomarkers to environmental pollution in European anchovy (Engraulis encrasicolus) from the western Mediterranean Sea. Mar. Pollut. Bull. 2018, 131, 197–204. [CrossRef] [PubMed]22. Mundle, S.O.; Spain, J.C.; Lacrampe-Couloume, G.; Nishino, S.F.; Lollar, B.S. Branched pathways in the degradation of cDCE by cytochrome P450 in Polaromonas sp. JS666. Sci. Total Environ. 2017, 605, 99–105. [CrossRef] [PubMed]23. Gunasekaran, V.; Donmez, E.; Girhard, M.; Urlacher, V.B.; Constantí, M. Biodegradation of fuel oxygenates and their effect on the expression of a newly identified cytochrome P450 gene in Achromobacter xylosoxidans MCM2/2/1. Process Biochem. 2014, 49, 124–129. [CrossRef]24. Wu, R.-R.; Dang, Z.; Yi, X.-Y.; Yang, C.; Lu, G.-N.; Guo, C.-L.; Liu, C.-Q. The effects of nutrient amendment on biodegradation and cytochrome P450 activity of an n-alkane degrading strain of Burkholderia sp. GS3C. J. Hazard. Mater. 2011, 186, 978–983. [CrossRef] [PubMed]25. Pandey, A.V.; Flück, C.E. NADPH P450 oxidoreductase: Structure, function, and pathology of diseases. Pharmacol. Ther. 2013, 138, 229–254. [CrossRef] [PubMed]26. Klotz, A.V.; Stegeman, J.J.; Walsh, C. An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish Stenotomus chrysops. Arch. Biochem. Biophys. 1983, 226, 578–592. [CrossRef]27. Sen, A.; Arinc, E. Purification and characterization of cytochrome P450 reductase from liver microsomes of feral leaping mullet (Liza saliens). J. Biochem. Mol. Toxicol. 1998, 12, 103–113. [CrossRef]28. Arinç, E. Characterization of cytochrome P450 dependent mixed-function oxidase system of gilthead seabream (Sparus aurata; Sparidae) liver. Comp. Biochem. Physiol. Part B Comp. Biochem. 1993, 104, 133–139. [CrossRef]29. Kojima, H.; Takahashi, K.; Sakane, F.; Koyama, J. Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes. J. Biochem. 1987, 102, 1083–1088. [CrossRef]30. Kubota, S.; Yoshida, Y.; Kumaoka, H.; Furumichi, A. Studies on the microsomal electron-transport system of anaerobically grown yeast: V. Purification and characterization of NADPH-cytochrome c reductase. J. Biochem. 1977, 81, 197–205. [CrossRef]31. Shen, A.L.; Porter, T.; Wilson, T.; Kasper, C. Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis. J. Biol. Chem. 1989, 264, 7584–7589. [CrossRef] [PubMed]32. Kuzu, M.; Ciftci, M. Purification and characterization of NADPH-cytochrome P450 reductase from Lake Van fish liver microsomes and investigation of some chemical and metals’ effects on the enzyme activity. Turk. J. Chem. 2015, 39, 149–158. [CrossRef]33. Kuwahara, T.; White, R.A., Jr.; Agosin, M. A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties. Arch. Biochem. Biophys. 1985, 239, 18–28. [CrossRef] [PubMed]34. Tsou, C.-Y.; Matsunaga, S.; Okada, S. Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race. J. Biosci. Bioeng. 2018, 125, 30–37. [CrossRef]35. Milhim, M.; Gerber, A.; Neunzig, J.; Hannemann, F.; Bernhardt, R. A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase. J. Biotechnol. 2016, 231, 83–94. [CrossRef]36. Lee, G.-Y.; Kim, H.M.; Ma, S.H.; Park, S.H.; Joung, Y.H.; Yun, C.-H. Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum. Plant Physiol. Biochem. 2014, 82, 116–122. [CrossRef]37. Takahashi, N.; Saito, T.; Goda, Y.; Tomita, K. Characterization of microsomal NADPH-dependent aldehyde reductase from rat brain. J. Biochem. 1986, 99, 513–519. [CrossRef]38. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef]39. Guengerich, F.P.; Martin, M.V.; Sohl, C.D.; Cheng, Q. Measurement of cytochrome P450 and NADPH–cytochrome P450 reductase. Nat. Protoc. 2009, 4, 1245. [CrossRef]40. Williams, J.; Kamin, H. The preparation and properties of microsomal TPNH-cytochrome c reductase from pig liver. J. Biol. Chem. 1962, 237, 587–595. [CrossRef]41. Yonetani, T. Studies on cytochrome c peroxidase II. Stoichiometry between enzyme, H2O2, and ferrocytochrome c and enzymic determination of extinction coefficients of cytochrome c. J. Biol. Chem. 1965, 240, 4509–4514. [CrossRef] [PubMed]42. Swift, M.L. GraphPad prism, data analysis, and scientific graphing. J. Chem. Inf. Comput. Sci. 1997, 37, 411–412. [CrossRef]43. Arrhenius, S. On the reaction velocity of the inversion of cane sugar by acids. J. Phys. Chem 1889, 4, 226.44. Copeland, R.A. Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis; John Wiley & Sons: Hoboken, NJ, USA, 2004.45. Lamb, S.B.; Lamb, D.C.; Kelly, S.L.; Stuckey, D.C. Cytochrome P450 immobilisation as a route to bioremediation/biocatalysis. FEBS Lett. 1998, 431, 343–346. [CrossRef]121221Marine enzymesCytochrome P450 reductasePurificationBiotechnologyPublicationORIGINALA Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.pdfA Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.pdfArtículoapplication/pdf2421223https://repositorio.cuc.edu.co/bitstreams/1a424b3f-b7e6-4717-b992-e17ada84e742/downloadf8c224fd1ab81545bb0ac9edf55d1a6aMD51LICENSElicense.txtlicense.txttext/plain; charset=utf-814828https://repositorio.cuc.edu.co/bitstreams/71ab8af0-f799-4c71-99bb-5bfe5cfcc3e2/download2f9959eaf5b71fae44bbf9ec84150c7aMD52TEXTA Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.pdf.txtA Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.pdf.txtExtracted texttext/plain44865https://repositorio.cuc.edu.co/bitstreams/696cf3ca-05e0-401b-ad00-4744c8b5d174/download20e2a7135fd606724de85d8e624144a9MD53THUMBNAILA Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.pdf.jpgA Novel, Highly Potent NADPH-Dependent Cytochrome P450 Reductase from Waste Liza klunzingeri Liver.pdf.jpgGenerated Thumbnailimage/jpeg16230https://repositorio.cuc.edu.co/bitstreams/b0b19aa0-8d8e-4805-96ce-9cd0b7778db3/downloadf8bb9c672fdd47233c3c15e9d51b0746MD5411323/10385oai:repositorio.cuc.edu.co:11323/103852024-09-17 14:08:32.953https://creativecommons.org/licenses/by/4.0/© 2023 by the authors. Licensee MDPI, Basel, Switzerland.open.accesshttps://repositorio.cuc.edu.coRepositorio de la Universidad de la Costa CUCrepdigital@cuc.edu.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