International Journal of Research and Scientific Innovation (IJRSI) | Volume IX, Issue VII, July 2022 | ISSN 2321–2705
Effect of pH and Temperature on Recombinant CYP450
Giwa, O. E*, Arotupin, D. J and Akinyosoye, F. A.
Department of Microbiology, Federal University of Technology Akure, P.M.B. 704 Akure, 340001, Nigeria
*Corresponding Author
Abstract: Cytochromes P450 (CYPs) has been studied to be of great importance to humans, plants and environment as a whole. Hence it has been recognised as GRAS i.e., generally regarded as safe status. The optimum pH and temperature for the production and stability of recombinant CYP450 proteins was evaluated after expression using Escherichia coli BL21 (DE3) with the catalytic module of cytochrome P450 gene (CYP102A2) containing pET-28a and elution of the recombinant protein of interest (recombinant CYP450 proteins) from the expressed total recombinant proteins. Highest biomass yield of the recombinant organism expressing the POI was observed at 30 ºC followed by declination with further increase in temperature. Total protein yield of 1.5 M was observed at 25 ºC while after elution for the POI, highest estimated concentration was observed to be 2.4 mM concentration at optimum temperature of 30 ºC. The highest recombinant POI concentration 2.4 mM after elution was observed at optimum purification elution buffer concentration of 150 mM 30 ºC however the temperature that showed the highest purification of the eluted protein was 37 ºC at elution buffer concentration of 150 mM. The pH also showed broad influence on the enzymatic activity of the recombinant POI ranging from acidic to alkaline. The optimum pH of recombinant CYP450 activity was near neutrality where maximum activity was recorded. However, when pH was at both extremes, the biological activity was still visible. The biological activity increased significantly from pH 2 till neutral pH but decline at pH 9. Crystallization result reveals the presence of rod cluster crystals shape, in the well with 0.3 M, Na acetate salt 25% PEG and 2 K MME.
Keywords: Cytochromes P450, optimum, pH and temperature
I.INTRODUCTION
Cytochromes P450 (CYPs) has been studied to belong to a very bulky group of enzyme superfamilies and are commonly found in all living organisms like bacteria, fungi, plants and animals (Werck-Reichhart, and Feyereisen, 2000; Nelson, 2018) hence are regarded as generally safe (GRAS). This group of enzymes work in synergistic cascade process leading to catalyzing more than 20 known types of reactions in regio- and stereoselective manners such as hydroxylation, dealkylation, epoxidation, oxidation, dehalogenation, dehydrogenation and reduction (Nguyen et al., 2020). It has also been studied to effectively catalyse the breaking down of complex substrates, toxic substances and xenobiotic (Bernhardt, 2006) hence has been a potential bioremediation prospect.