IMMOBILIZATION OF GAMMA-GLUTAMYL-TRANSPEPTIDASE, A MEMBRANE ENZYME, IN GEL BEADS VIA LIPOSOME ENTRAPMENT

Bovine kidney gamma-glutamyl transpeptidase, a membrane enzyme, was im mobilized in gel beads by application of the method of Wallsten et al. (Biochim. Biophys. Acta, 982, 47-52, 1989). The gel beads were equili brated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed against a buffer for reconstitution and immobil ization of enzyme-bound liposomes in the pores of the beads. From the standpoints of the immobilized contents of protein and phospholipids a nd of the reactivity of gamma-glutamyl transpeptidase, a dialysis buff er of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in t he enzyme-phospholipidcholate dispersion, and the use of Sepharose CL- 6B as the support gel were found to be most appropriate for the immobi lization of gamma-glutamyl transpeptidase. gamma-Glutamyl transpeptida se was activated and stabilized by reconstitution in liposomes. In ope ration with a packed bed reactor, liposome-bound gamma-glutamyl transp eptidase immobilized in Sepharose CL-6B exhibited relatively stable an d constant activity for 12 h. In addition, it was found that enzyme su bstrates were able to pass through the pores of the gel beads to inter act with the enzyme present on the outer surface of the liposome membr ane in the gel beads. These results thus indicated that a novel suppor t made up of liposomes and Sepharose CL-6B would permit efficient immo bilization of lipid-requiring and/or membrane enzymes.