Coimmobilization of malate dehydrogenase and formate dehydrogenase in polyethyleneglycol(#4000)diacrylate gel by droplet gel‐entrapping method

Abstract

A new immobilization technique suitable for coupled enzymes requiring cofactors was established. This is a droplet gel‐entrapping method in which many small droplets including the enzymes are fixed in the gel. The first emulsion was prepared by mixing of a solution containing thermostable malate dehydrogenase (MDH) and formate dehydrogenase (FDH) with benzene containing a surfactant. The first emulsion was added to a solution containing polyethyleneglycol(#4000)diacrylate and N,N′‐methylenebisacrylamide to prepare the second emulsion (w/o/w). After the second emulsion was gelled by addition of potassium persulfate and 3‐dimethylaminopropionitrile, the benzene was removed. The expressed MDH and FDH activities of the MDH–FDH immobilized gel were 7.1 and 13.9% of the initial activities, respectively. The K_m values of the gel were 0.60mM for formate and 1.5μM for NAD, respectively. The K_m for formate and NAD were found to be extremely low. By using the column packed with 30 g gel having the MDH activity of 41.7 units and the FDH activity of 11.1 units, 13.8mM oxalacetate was completely converted to malate at 30°C. The malate production rate was not affected by the concentration of more than 50mM formate, more than 2mM oxalacetate, and more than 0.1 mM NAD, respectively. Long‐term malate production was demonstrated at 30°C by passing the substrate solution containing the two substrates and NAD through the column. The maximum conversion ratio (7.8%) was obtained at the fifth day, and 83% of maximum productivity was maintained even after 3 weeks. The expressed FDH activity at the fifth day was calculated to be 20.5% of the initial activity.