Immobilization of γ-Glutamuyl Transpeptidase, as Membrane Enzyme, in Gel Beads via Liposome Entrapment

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Bovine kidney γ-glutamyl transpeptidase, a membrane enzyme, was immobilized in gel beads by application of the method of Wallsten et al. (Biochim. Biophys. Acta, 982,47-52,1989). The gel brads were equilibrated with a dispersion of the enzyme, phospholipids, and cholate and subsequently dialyzed aginast a buffer for reconstitution and immobilization of enzyme-bound liposomes in the pores of the beads. From the srandpoints of the immobilized contents of protein and phospholipids and of the reactivity of γ-glutamyl tranpeptidase, a dialysis buffer of Tris-HCl (pH 7.5), a phospholipid concentration of 45 mg/ml in the enzyme phospholipid-cholate dispersion, and the use of Sepharose CL-6B as the support gel were found to be most appropriate for the immobilization of γ-glutamyl transpeptidase. γ-Glutamyl transpeptidase was activated and stabilized by reconstitution in liposomes. In operation with a packed bed reactor, liposome-bound γ-glutamyl transpeptidase immobilized in Sepharose CL-6B exhibited relatively stable and constant activity for 12 h. In addition, it was found that enzyme substrates were able to pass through the pores of the gel beads to interact with the enzyme present on the outer surface of the liposome membrane in the gel beads. These results thus indicated that a novel support made up of liposomes and Sepharose CL-6B would permit efficient immobilization of lipid-requiring and/or membrane enzymes.
社団法人日本生物工学会の論文
1994-03-25

Immobilization of γ-Glutamuyl Transpeptidase, as Membrane Enzyme, in Gel Beads via Liposome Entrapment

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