The co-immobilization of NAD and dehydrogenases and its application to bioreactors for synthesis and analysis.

A polymerizable NADderivative, Ar6-[A^-[A^-(2-hydroxy-3-methacrylamidopropyl)carbamoylmethyl]carbamoylmethyl]-NAD, formate dehydrogenase, and malate dehydrogenase were entrapped all together in polyacrylamide gels.The entrapment was carried out by radical copolymerization, and consequently NADwas bound on the matrix which enclosed the enzymes.These gels had the function of producing L-malate from oxalacetate and formate.The L-malate production was also continuously done in a column reactor for 3 days.Another gel was similarly prepared with A^6-[A^-(6-methacrylamidohexyl)carbamoylmethyl]-NAD, horse liver alcohol dehydrogenase, and diaphorase.This gel was shown to catalyze the formation of resorufin from resazurin and ethanol.This gel was applicable to ethanol analysis using a fluorescence spectrophotometer to determine resorufin.The analyzer was usable for one week.Recycling of NADis a prerequisite for the industrial application of dehydrogenases.The most effective way of realizing this is to maintain both NADand the coupled dehydrogenases in one reactor system (bioreactor) and recycle NADin situ.The following methods have been developed for this purpose: NADis boundto water-soluble polymersand then the polymer-bound NADand the dehydrogenases are placed all together in an ultra filtration apparatus,1'2) enclosed with a semipermeable membrane,3'40 microencapsulated,5) or immobilized in a collagen membrane.6)Nowwe report another method in which the dehydrogenases and a polymerizable NADderivative are co-entrapped in a polyacrylamide gel.The gel is prepared by radical copolymerization, and consequently NADis bound on the matrix which encloses the dehydrogenases.By this co-immobilization method, two kinds of gels were prepared: FDH-MDH-NAD gel and LADH-diaphorase-NAD gel.*1The present paper deals with the catalytic function of the gels and their application to a column reactor producing L-malate and to an analytical system for ethanol.MATERIALS AND METHODS Materials.NAD, NADH,glutathione (reduced form), yeast ADH, horse liver ADH, pig heart MDH, yeast FDH, and pig heart diaphorase were purchased from Boehringer-Mannheim-Yamanouchi Ltd. (Tokyo).The following reagents and others were obtained from the chemical companies listed below: oxalacetic acid and dllipoic acid-Nakarai Chemicals Ltd. (Kyoto); resazurin sodium salt and nitro blue tetrazolium-Tokyo Kasei Kogyo Co., Ltd.(Tokyo); acrylamide and methylenebisacrylamide-Wako Pure Chemical Co.(Osaka); bovine serum albumin-Seikagaku Kogyo Co., Ltd.(Tokyo); cellulose-coated glass plates (Avicel SF)-Funakoshi Pharmaceutical Co. Ltd. (Tokyo).N6-[N-[N-(2-hydroxy-3-methacrylamidopropyl) carbamoylmethyl] carbamoylmethyl]-NAD and N6-[N-(6-methacrylamidohexyl)carbamoylmethyl]-NAD were synthesized by the methods described previously.7 ) Acrylamide was recrystallized from benzene before use.Analysis.A Hitachi 124 spectrophotometer with a ther-