Immobilization of galactosyltransferase and continuous galactosylation of glycoproteins in a reactor.
We immobilized human milk galactosyltransferase covalently to CNBr- and tresylchloride-activated Sepharose. The enzyme was also immobilized non-covalently to Concanavalin A-Sepharose and to monoclonal anti-galactosyltransferase antibodies which were bound via their Fc-fragment to Protein G-Sepharose. With the covalent methods, up to 72% of the enzyme could be bound to the carrier, but more than 90% of the specific activity was lost. In contrast, non-covalent immobilization yielded only about 50% immobilization efficiency, but 21% and 25% of specific activity, respectively, could be recovered. The stability of immobilized galactosyltransferase as compared to native enzyme was considerably increased: at room temperature, 55% of initial immobilized activity was lost after 65 hours compared to 95% of loss of soluble enzyme activity. Immobilized galactosyltransferase was then used for continuous galactosylation of the glycoproteins ovalbumin, endo H-treated yeast invertase and bovine serum albumin-N-acetylglucosamine in a "slurry" reactor. 55%, 35% and 25%, respectively, of all acceptor sites on these glycoproteins could be galactosylated by this method.
Schneider
R
R
Central Research Laboratories, Ciba-Geigy AG, Basel, Switzerland.
Hammel
M
M
Berger
E G
EG
Ghisalba
O
O
Nueesch
J
J
Gygax
D
D
D002240:Carbohydrate Sequence; D004800:Enzymes, Immobilized; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D007700:Kinetics; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D006023:Glycoproteins; D005700:Galactosyltransferases; D005690:Galactose