Functional expression in Bacillus subtilis of mammalian NADPH-cytochrome P450 oxidoreductase and its spore-display.
The technology for over-expressing NADPH-cytochrome P450 reductase (CPR), a diflavin-containing enzyme, offers the opportunity to develop enzymatic systems for environmental detoxication and bioconversions of drugs, pesticides and fine chemicals. In this study, Bacillus subtilis was chosen to express rat CPR (rCPR) because of its capacities for high protein production and spore formation. rCPR was expressed in B. subtilis DB104 under the transcriptional control of an IPTG-inducible fusion promoter of P(groE) and P(tac). The expressed rCPR was released into the culture medium after sporulation by autolysis of the host cell. It was associated with and displayed on the spore surfaces; this was confirmed by measuring rCPR activity in purified spores and analyzing its accessibility to anti-rCPR antibodies using flow cytometry. The spore-displayed rCPR was able to reduce cytochrome c and ferricyanide, and also assisted in the O-deethylation of 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin (EFC) by human cytochrome P450 1A2, indicating that it was functionally active. Spore surface display of rCPR in B. subtilis appears to be useful for preparing cytochrome P450-related enzymes, and spore biocatalysts of rCPR are likely to have wide biotechnological applications.
Yim
Sung-Kun
SK
Systems Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 111 Gwahangno, Yuseong-gu, Daejeon 305-806, Republic of Korea.
Jung
Heung-Chae
HC
Yun
Chul-Ho
CH
Pan
Jae-Gu
JG
D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D019149:Bioreactors; D015153:Blotting, Western; D003001:Cloning, Molecular; D019388:Cytochrome P-450 CYP1A2 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005292:Ferricyanides / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D008860:Microscopy, Ultraviolet; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013171:Spores, Bacterial / Q000201:enzymology
D005292:Ferricyanides; D011994:Recombinant Proteins; C007931:hexacyanoferrate III; D045304:Cytochromes c; D019388:Cytochrome P-450 CYP1A2; D009251:NADPH-Ferrihemoprotein Reductase