Selective enzyme amplification of NAD +/NADH using coimmobilized glycerol dehydrogenase and diaphorase with amperometric detection

Abstract

A flow system for substrate recycling of NAD +/NADH was set up with an enzyme reactor containing coimmobilized glycerol dehydrogenase (GDH) and diaphorase. The product from the diaphorase catalysis, hexacyanoferrate(II), aws detected amperometrically at a glassy carbon electrode. The amplification factor was 150 for a reactor volume of 100 μ l at a flow-rate of 0.5 ml/min. With a stopped flow of four minutes, the signal increased another 88 times, resulting in a signal amplification of 13 300 times. Equations are derived for the amplification factor and used for a discussion of the optimization of amplification systems. The K m for GDH with glycerol as a substrate was found to be 5 × 10 −3 M at pH 8.0. GDH from Cellulomonas sp. was purified on a gel filtration column and the purified enzyme showed a specificity toward NAD +, compared to NADP +, that was higher than 99.9%. Due to the NAD + specificity of the purified GDH, the enzyme amplification system reported here could be used in detection systems for enzyme immunoassays when using alkaline phosphatase as a label and NADP + as a substrate. The stability of immobilized GDH and diaphorase is several orders of magnitude better than that of alcohol dehydrogenase, which is the enzyme commonly used for NAD +-specific detection in these applications.