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Biotechnology and Applied Biochemistry (1999) 29, (207–212) (Printed in Great Britain)

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Cofactor recycling in a coupled enzymeoxidation–reduction reaction: conversion of -oxo-fatty acids into -hydroxy and dicarboxylic acids

Alberto Nuñez¹, Thomas A. Fogliaand George J. Piazza

U.S. Department of Agriculture, Eastern Regional ResearchCenter, Agricultural Research Service, USDA, 600 East MermaidLane, Wyndmoor, PA 19038, U.S.A.

Abbreviations used: ADH, alcohol dehydrogenase; AldDH, aldehyde dehydrogenase; GC/MS; gas chromatography–MS; HPLS, hydroperoxide lyase; LOX, lipoxygenase.

¹ To whom correspondence should be addressed.

Aldehydes are reduced to alcohols by the enzyme alcohol dehydrogenase (ADH), whereas the enzyme aldehyde dehydrogenase (AldDH) oxidizes aldehydes to carboxylic acids. ADH and AldDH require, respectively, the reduced and oxidized forms of the cofactor NAD (NAD⁺/NADH). By combining both oxidation and reduction reactions into one process, it is possible to produce alcohols and carboxylic acids simultaneously from aldehydes by continuous recycling of the NAD⁺/NADH cofactor. However, both enzymes need to be active within the same pH region and buffer system. To test this hypothesis, the pH profile (V_max and V_max/K_m) as well as the pK_a of the prototropic groups involved in catalysis for both dehydrogenases were determined using (Z,Z)-nona-2,4-dienal as a model substrate. The pH profile (V_max and V_max/K_m) of both enzymes overlapped in the pH range of 6–8 in potassium phosphate buffer. When the coupled enzyme system was used at pH 7 with 10% NAD⁺ cofactor, over 90% of the starting aldehyde was converted to its corresponding acid and alcohol derivatives in a 1:1 ratio. The sequential action of the enzymes lipoxygenase and hydroperoxide lyase converts polyunsaturated fatty acids to aldehydic fatty acids. The products arising from the oxidation or reduction of the aldehydic functionality are of industrial interest. It was found that 13-oxo-9-(Z),11-(E)-tridecadienoic acid, the product of the sequential reaction of soya bean lipoxygenase and hydroperoxide lyase from Chlorella pyrenoidosa on linoleic acid, is also a substrate in this coupled enzyme system.

Received 12 August 1998/27 October 1998; accepted 10 November 1998

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