Immobilization of multienzymes and cofactors within lipid-polyamide membrane microcapsules for the multistep conversion of lipophilic and lipophobic substrates

Abstract

Cofactors cannot be retained within polyamide membrane microcapsules unless the cofactors have been covalently linked to macromolecules. In this paper, a new approach using lipid-polyamide membrane microcapsules has resulted in the retention of unmodified cofactors. Lipid-polyamide microcapsules can be made to contain urease (urea amidohydrolase, EC 3.5.1.5), glutamate dehydrogenase (NAD(P) +) [ l-glutamate: NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3], alcohol dehydrogenase (alcohol:NAD + oxidoreductase, EC 1.1.1.1), NAD +, NADH and α-ketoglutarate. Lipophilic substrates like ammonia can equilibrate rapidly into the microcapsules. The rate of conversion of ammonia into glutamate was studied. NAD + retained in the microcapsules was effectively recycled into NADH and 0.25 μmol NAD + converted 10 μmol ammonia into glutamate. Without cofactor recycling, 10 μmol NADH had to be microencapsulated to convert the same amount of ammonia into glutamate. By adjusting the ratio of cholesterol and lecithin in the lipid component of the membrane, it was also possible to achieve a good urea-permeable membrane without any leakage of cofactor or α-ketoglutarate. This way urea permeated through the lipid-polyamide membrane microcapsules was sequentially converted into ammonia and then glutamate.