Biocatalyst cytochromes

The abihty of iron to exist in two stable oxidation states, ie, the ferrous, Fe ", and ferric, Fe ", states in aqueous solutions, is important to the role of iron as a biocatalyst (79) (see Iron compounds). Although the cytochromes of the electron-transport chain contain porphyrins like hemoglobin and myoglobin, the iron ions therein are involved in oxidation—reduction reactions (78). Catalase is a tetramer containing four atoms of iron peroxidase is a monomer having one atom of iron. The iron in these enzymes also undergoes oxidation and reduction (80). 

Enzyme-mediated chiral sulfoxidation has been reviewed comprehensively in historical context [188-191]. The biotransformation can be mediated by cytochrome P-450 and flavin-dependent MOs, peroxidases, and haloperoxidases. Owing to limited stability and troublesome protein isolation, a majority of biotransformations were reported using whole-cells or crude preparations. In particular, fungi have been identified as valuable sources of such biocatalysts and the catalytic entities have not been fully identified in all cases. 

Cytochrome P450 is considered the most versatile biocatalyst known. The actual reaction mechanism is complex and has been briefly described previously (Figure 11-6). It has been shown by the use of that one atom of oxygen enters R—OH and one atom enters water. This dual fate of the oxygen accounts for the former naming of monooxygenases as mixed-function oxidases. The reaction catalyzed by cytochrome P450 can also be represented as follows ... 

All cytochrome P450s are hemoproteins and generally have a wide substrate specificity, acting on many exogenous and endogenous substrates. They represent the most versatile biocatalyst known. 

Table 9.4 Metabolite production of selected drugs using human cytochrome biocatalysts available from Codexis, Inc"... 

Bernhardt, R. (2006) Cytochromes P450 as versatile biocatalysts. Journal of Biotechnology, 124, 128-145. 

The improvement of its activity and stability has been approach by the use of GE tools (see Refs. [398] and [399], respectively). A process drawback is the fact that the oxidation of hydrophobic compounds in an organic solvent becomes limited by substrate partition between the active site of the enzyme and the bulk solvent [398], To provide the biocatalyst soluble with a hydrophobic active site access, keeping its solubility in organic solvents, a double chemical modification on horse heart cytochrome c has been performed [400,401], First, to increase the active-site hydrophobicity, a methyl esterification on the heme propionates was performed. Then, polyethylene glycol (PEG) was used for a surface modification of the protein, yielding a protein-polymer conjugates that are soluble in organic solvents. 

Improving functionality may involve a complex biocatalytic system including more than one biocatalyst, as it is the case in BDM reactions. Additionally to the BDS for instance, another biocatalyst active for BDM [395,406], which consists of a heme oxygenase or a Cytochrome reductase could be used to widen up the functionality. 

Although mammalian CYPs are attractive candidates for use as commercial biocatalysts, many functional characteristics limit the opportunities to exploit such a system. Association of the enzymes with membranes prevents easy extraction and purification and limits the opportunities to produce useful recombinant enzymes by cloning the relevant genes for expression in microbial systems. All P450s have a porphyrin-haem active site that requires a second protein to reduce the iron component, often cytochrome P450 reductase or... 

In a recent investigation to develop novel cytochrome P450 biocatalysts, DNA shuffling was used to produce chimeric cytochrome P450s mutants with enhanced biocatalytic activities, which were then co-expressed with NADPH-cytochrome reductase in E. coli to form an efficient system, in this case demonstrated to be effective for indole oxidation [69]. 

Purified cytochrome P450 polypeptide CYP76B1 from Helianthus tuberosus and its application as biocatalyst in particular for the degradation of environmental pollutants and for altering the resistance of plants sensitive to the phenylurea family of herbicides Patent number US6376753 (2002)... 

As an example, it has been estimated that for the industrial production of fine chemicals, biotransformations should accomplish a minimum space-time yield of 0.1 g l-1 h-1 and a minimum final product concentration of 1 g l-1, while for pharmaceuticals, the minimum requirements are 0.001 g l-1 h-1 and 0.1 g l1 for volumetric productivity and product concentration, respectively [6]. Analysis of enzymes with recognized industrial potential, such as cytochrome P450, showed that some of the parameters are already within industrially relevant ranges [7]. The improvements achieved with these biocatalysts through protein, cell, and process engineering are based on the understanding of their molecular arrangement and catalytic mechanisms. 

Vazquez-Duhalt R (1999) Cytochrome c as a biocatalyst. J Mol Catal B Enzym 7 241-249... 

---