Characteristics of an immobilized form of transglutaminase: A possible increase in substrate specificity by selective interaction with a protein spacer

Transglutaminase was covalently immobilized on poly(lysyl)-alpha(s)-casein which was covalently attached to 3-aminopropyl porous glass. Attachment of polylysine to the protein spacer, using soluble transglutaminase as a catalyst, greatly increased the concentration of immobilization sites, resulting in a 74% yield of immobilized protein. Catalytic efficiencies, kcat/Km, for the immobilized form were 12-13% of that for the soluble enzyme with either alpha(s)-casein or beta-lactoglobulin as substrate. Also, the catalytic efficiency was 5-fold greater with alpha(s)-casein than with beta-lactoglobulin using either soluble or immobilized enzyme. Selective interaction of the protein substrate with the protein spacer resulted in selective catalysis of isopeptide bond formation in alpha(s)-casein due to increased local concentrations of this protein in the immobilized enzyme matrix. When a mixture of alpha(s)-casein and beta-lactoglobulin was used as substrate, preferential cross-linking of alpha(s)-casein occurred with the immobilized enzyme as compared with the soluble enzyme