Preparation of an Alcohol‐Dehydrogenase–NAD(H)–Sepharose Complex Showing No Requirement of Soluble Coenzyme for Its Activity

Abstract

1 Horse liver alcohol dehydrogenase and an NADH analogue, N⁶-[(6-aminohexyl)carbamoylmethyl]-NADH, have been co-immobilized to Sepharose 4B under conditions permitting binary complex formation between the enzyme and the cofactor. 2 The enzyme-coenzyme-matrix preparations were assayed with a coupled oxidoreduction reaction and showed activities, prior to addition of coenzyme, that were up to 40% of that obtained in excess of free coenzyme. 3 A molar ratio of 1:1 between the amount of bound nucleotide and bound enzyme was sufficient to obtain high activities in the absence of free coenzyme. 4 The highest recycling rate obtained for the immobilized nucleotide was 3400 cycles per hour. 5 Both thermal and storage stability of alcohol dehydrogenase was increased when the enzyme was co-immobilized with the NADH analogue. 6 The efficiency of the immobilized preparations (measured as product formation per minute and per assay volume) was higher (1.4 to 5 times in our assays) than the corresponding systems of free enzyme (in total enzyme units) and nucleotide in an identical assay volume.