title: The co-immobilization of P450-type nitric oxide reductase and glucose dehydrogenase for the continuous reduction of nitric oxide via cofactor recycling.
abstract: The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres. Successful co-immobilization of a fungal nitric oxide reductase (a member of the cytochrome P450 enzyme family) and a bacterial glucose dehydrogenase was obtained with the carboxyl-functionalized microspheres, with enzyme activity maintenance of 158% for nitric oxide reductase and 104% for glucose dehydrogenase. The optimal stoichiometric ratio of these two enzymes was subsequently determined to enable the two independent chemical reactions to be catalyzed concomitantly, allowing for near-synchronous cofactor conversion rates. This dual-enzyme system provides a novel research tool with potential for in vitro investigations of nitric oxide, and further demonstrates the successful immobilization of a P450 enzyme with potential application towards the immobilization of other cytochrome P450 enzymes.
authors.affiliation: CSIR Biosciences, Building 20, Meiring Naudé Road, Brummeria, Pretoria, 0001, South Africa; Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Private Bag, X20, Hatfield 0028 Pretoria, South Africa. Electronic address: seikegarny1@gmail.com.
authors.forename: Seike
authors.initials: S
authors.lastname: Garny
authors.affiliation: CSIR Biosciences, Building 20, Meiring Naudé Road, Brummeria, Pretoria, 0001, South Africa. Electronic address: nbeetonkempen@csir.co.za.
authors.forename: Natasha
authors.initials: N
authors.lastname: Beeton-Kempen
authors.affiliation: CSIR Biosciences, Building 20, Meiring Naudé Road, Brummeria, Pretoria, 0001, South Africa. Electronic address: igerber@csir.co.za.
authors.forename: Isak
authors.initials: I
authors.lastname: Gerber
authors.affiliation: Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, Private Bag, X20, Hatfield 0028 Pretoria, South Africa. Electronic address: jan.verschoor@up.ac.za.
authors.forename: Jan
authors.initials: J
authors.lastname: Verschoor
authors.affiliation: ReSyn Biosciences (Pty) Ltd., CSIR Biosciences, Building 20, Meiring Naudé Road, Brummeria, Pretoria, 0001, South Africa, P.O. Box 2860, Edenvale, Johannesburg, Gauteng, South Africa. Electronic address: jjordaan@resynbio.com.
authors.forename: Justin
authors.initials: J
authors.lastname: Jordaan
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000378:metabolism*; D008863:Microspheres; D009097:Multienzyme Complexes / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D009569:Nitric Oxide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D042843:Glucose 1-Dehydrogenase; C034747:nitric-oxide reductase
keywords: Cofactor recycling; Cytochrome P450; Enzyme immobilization; Glucose dehydrogenase; Nitric oxide; Nitric oxide reductase