This work has been supported by grant CTQ2009-07568 from Spanish Ministerio de Ciencia e Innovacion. A. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovacion for a Ramon y Cajal fellowship (RyC-2009-03813). Mr. Hernandez is a holder of a MAEC-AECID fellowship.

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Hydrogen Peroxide in Biocatalysis. A Dangerous Liaison

Oxidoreductases are a group of enzymes that have been regarded uneconomical for industrial processes due to their dependence on cofactors or prosthetic groups for activity and the difficulties of regenerating these. Especially, flavoproteins have long been neglected for biocatalytical applications. The prosthetic group of some of these enzymes, but not all, can be regenerated by oxygen, resulting in hydrogen peroxide formation, which is detrimental to enzyme stability. As a contribution to alleviating this problem, a novel concept for the regeneration of electron acceptors (redox mediators) for flavoenzymes is described. Flavin-containing enzymes such as cellobiose dehydrogenase (CDH) or pyranose oxidase (P2O) are used in conjunction with laccases and a redox mediator. The flavin of the synthetic enzyme is reduced while the oxidized product of interest is formed, in turn, the flavin is reoxidized with the help of an electron acceptor, which then is regenerated using a laccase. Laccases are copper containing phenol oxidases that can transfer four electrons to oxygen, producing two molecules of water. Preliminary screening experiments with different redox mediators, and a coupled enzyme system of CDH and laccase, showed that a wide variety of different substances can efficiently shuttle electrons between these two enzymes. Among them are substituted and unsubstituted ortho - and para -quinones, benzoquinone imines, cation radicals such as 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), redox dyes such as phenothiazines or phenoxazines, as well as iron complexes. Experiments in which CDH completely oxidizes lactose to lactobionic acid and P2O entirely converts glucose to 2-keto-glucose are presented. Catalytic amounts of redox mediators are used and continuously regenerated by a laccase. Specific productivities of up to 19.3 g·(h·kU) −1 and 72 g·(h·kU) −1 for CDH and P2O, respectively, were found. The total turnover numbers (TTNs) for the two enzymes used were in the range of 10 5 –10 6 . Oxygen supply for the laccase is a crucial factor in avoiding rate limitation. Undeniably, this system facilitates the efficient use of a hitherto underexploited group of enzymes for preparative purposes.

Continuous enzymatic regeneration of redox mediators used in biotransformation reactions employing flavoproteins

Electrochemical study of sulphonated ferrocenes as redox mediators in enzyme electrodes

Glucose oxidase was immobilized onto poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two methods: by covalent bonding through epichlorohydrin and by entrapment between pHEMA membranes. The highest immobilization efficiency was found to be 17.4% and 93.7% for the covalent bonding and entrapment, respectively. The K m values were 5.9 mmol dm -3 , 8.8 mmol dm -3 and 12.4 mmol dm -3 for free, bound and entrapped enzyme, respectively. The V max values were 0.071 mmol dm -3 min -1 , 0.067 mmol dm -3 min -1 and 0.056 mmol dm -3 min -1 for free, bound and entrapped enzyme

Immobilization of glucose oxidase: a comparison of entrapment and covalent bonding.

Enzymatic electrocatalysis as a strategy for electrochemical detection in heterogeneous immunoassays.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/bit.260271119

C. Hervagault

Papers

Increase in operational stability of immobilized glucose oxidase by the use of an artifical cosubstrate