Recombinantly expressed enzymes

The most important enzyme converting isoprene to both monoepoxides and also to the diepoxide is CYP2E1, as shown by both recombinant expressed enzymes and in human liver microsomes (Bogaards et al., 1996). 

Gene transfection and expression Enzyme specific Suitable only for proof-of-concept experiments also no inducibility Cell lines with recombinantly expressed enzymes... 

This approach is not restricted to bacterial or viral cells. Mammalian cells under highly proliferating conditions can be cultured at increasing exposure to a compound in attempts to create resistant mutants. Alternatively, one can sometimes use a structural biology approach to predict amino acid changes that would abrogate inhibitor affinity from study of enzyme-inhibitor complex crystal structures. If the recombinant mutant enzyme displays the diminished inhibitor potency expected, one can then devise ways of expressing the mutant enzyme in a cell type of interest and look to see if the cellular phenotype is likewise abolished by the mutation. 

Rodriguez, S., Schroeder, K.T., Kayser, M.M. and Stewart, J.D. (2000) Asymmetric synthesis of /3-hydroxy esters and alpha-alkyl-beta-hydroxy esters by recombinant Escherichia coli expressing enzymes from baker s yeast. The Journal of Organic Chemistry, 65 (8), 2586-2587. 

The properties listed are for recombinant DszB enzyme expressed in E. coli. The original enzyme from R. erythropolis could not be isolated due to its low yield from the native host. 

The detailed characterization of hydroxynitrile lyases from Sorghum hicolor (E.C. 4.1.2.11) and Linum usitassimum (E.C. 4.1.2.37) has been hampered for a long time due to the lack of a recombinant expression system. Therefore our studies were focused on cloning of the coding genes, recombinant expression, and characterization of these enzymes. 

HNLs comprise a heterogenous enzyme family, since hydroxynitrile lyase activity has evolved in different structural frames by convergent evolution [17, 18]. Thus, (S) -specific HNLs based on an a/P-hydrolase fold framework from Manihot esculmta (cassava) [19-21], Hevea hrasilensis (rubber tree) [22-26], and Sorghum hicolor (millet) [27-33] have been described. (R)-specific HNLs based on the structural framework of oxidoreductases were isolated from Linum usitatissimum (flax) [30, 34-37] and Rosaceae (e.g., bitter almonds) [31, 38]. Despite their potential in biocatalysis only few HNLs (from cassava and rubber tree) are available by recombinant gene expression, which is a prerequisite for their technical application [20, 24]. Thus, cloning, recombinant expression, and... 

Cytochrome b5 affects the kinetics of drug metabolism by certain CYP enzymes hence, coexpression of this microsomal hemoprotein (together with NADPH-CYP reductase) can affect the catalytic efficiency of certain recombinant CYP enzymes (76,109). For example, the presence of cytochrome b5 tends to increase Fmax for reactions catalyzed by CYP3 A4, whereas it tends to decrease Km for reactions catalyzed by CYP2E1. In both cases, cytochrome b5 increases Vmax/Km, which is a measure of in vitro intrinsic clearance. The fact that some commercially available recombinant CYP enzymes are expressed with cytochrome b5 while others are not complicates the interpretation of results of studies performed with recombinant human CYP enzymes. 

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