Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration

Cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD+ in enzyme-catalyzed reactions. Herein, glycerol dehydrogenase (GDH) and NADH oxidase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for effective regeneration of NAD+. After optimization, the activity of combi-CLEAs and separate CLEAs mixtures were 950 and 580 U/g, respectively. And the catalytic stability of combi-CLEAs against pH and temperature was superior to the free enzyme mixture. After ten cycles of reuse, the catalytic efficiency could still retain 63.3% of its initial activity, indicating that the constructed combi-CLEAs system had excellent reusability. Also, the conversion of glycerol to 1,3-dihydroxyacetone (DHA) was improved by the constructed NAD+ regeneration system, resulting in 4.6%, which was 2.5 times of the free enzyme system. Thus, wide applications of this co-immobilization method in the production of various chiral chemicals could be expected in the industry for its high efficiency at a low cost.