Improving the operational stability of the alkane hydroxylating cytochrome P450: CYP153A6
Jacobs, Chéri Louise
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CYP153 hydroxylases have been found to be a very diverse group of cytochrome P450 enzymes because they have been isolated from a many different genera of alkane-degrading bacteria collected from various environments. This family of enzymes were the first bacterial monooxygenases identified that could catalyse terminal hydroxylation of aliphatic alkanes by regio-selectively inserting a single oxygen atom into the terminal C-H bond. Currently, there are no synthetic chemical catalysts capable of performing such a reaction. Therefore, CYP153s have attracted great interest from the petrochemical industry for the production of value added chemicals. In addition, the cost of using biological catalysts is much less than chemical catalysts and they promote green chemistry. However, many CYP153s are unstable biocatalysts therefore one of the long-term goals of our research group is to create a stable CYP153 hydroxylase for large-scale application. This study was a first attempt at achieving this goal. The first aim of this study was to establish factors affecting the operational stability of CYP153A6 - a well-characterised member of the CYP153 family – in order to establish conditions suitable for challenging mutants. Our research group had previously cloned the complete operon from Mycobacterium sp. HXN-1500 encoding CYP153A6, Ferredoxin reductase and Ferredoxin into pET-28b (+) and expressed it in E. coli BL21 (DE3). When evaluating the operational stability of CYP153A6 (wild type), we observed that enzyme activity decreased when glucose dehydrogenase was added to biotransformations to facilitate cofactor regeneration. Glucose dehydrogenase oxidises glucose to glucono-lactone using NAD+ or NADP+ as cofactor. In the presence of water, glucono-lactone is converted to gluconic acid causing acidification which caused the activity of CYP153A6 to decrease. In cell free extracts limited interaction with FdR and Fdx limited 1-octanol production, so that with additional FdR and Fdx, 1-octanol production increased significantly. We also determined that the wild-type CYP153A6 could efficiently take care of cofactor regeneration using glucose and glycerol as a source of electrons when biotransformations are carried out 20°C. When storing the enzyme overnight, adding additional NADH to biotransformations appeared to have a stabilising effect on CYP153A6. Currently it appears as though CYP153A6 is most stable when biotransformations are carried out using a 300 mM Tris-HCl (pH 8) buffer containing 100 mM glucose and glycerol. We observed that higher buffer concentrations such as 400 mM Tris significantly decreased enzyme activity as well as when the buffer pH was lower than 7 or higher than 8. When evaluating the oxidative stability of the CYP153A6, an increase in the initial enzyme activity was observed when glucose oxidase was added to biotransformations for in situ generation of H2O2. However, reactions containing H2O2 levelled off after 8 h whereas reactions without H2O2 did not, indicating that H2O2 was having a negative effect on the stability of CYP153A6. In addition, glucose oxidase also produces gluconic acid in the presence of water which could have simultaneously contributed to the decrease in enzyme activity as observed in the reactions containing glucose dehydrogenase. The reason for evaluating the operational stability of the CYP153A6 under various conditions was in order to select a set of conditions that would not only optimise reaction conditions for evaluating CYP153A6 mutants but the conditions should challenge the mutants as well. The second aim of the study was to design and construct mutants of CYP153A6 by substituting specific cysteine and methionine residues with more oxidatively stable amino acids. Mutants were designed using the 3DM Bio-Prodict database in conjunction with Yasara. Site-directed mutagenesis was used to construct the mutants using the Quickchange PCR method as well as the Megaprimer PCR method. Once DNA sequencing confirmed that mutagenesis was successful, the plasmid DNA was transformed into E. coli BL21 (DE3). A total of 14 CYP153A6 mutants were created in this study. For the third and final aim of this study, the operational stability of these 14 mutants together with 11 mutants that had previously been constructed by Dr. Opperman was evaluated by performing biotransformations of n-octane using cell free extracts. The conditions for the biotransformation were based on the set of conditions that had been selected for the mutants in the first aim of this study. The mutants were thus evaluated using a 300 mM Tris-HCl buffer (pH 8) containing 100 mM glucose and glycerol; additional FdR and Fdx were added to all reactions and as well additional 1 mM NADH. Biotransformations were carried out at 30°C and the mutants were tested in the presence of 800 and 1600 U/L (final concentration) glucose oxidase. After evaluation, mutants were divided into five groups based on expression levels and activity. From these results, it was clear that mutants with mutations M18I, M56G, M241L, M292F and M231L_M232L, all on the periphery of the enzyme, displayed significantly improved activity over the wild type in the presence of H2O2. Mutant M231L_M232L displayed the most improved activity indicating that combinatorial mutants will improve enzyme stability more significantly than single mutants.