Influence of gelatin matrices cross-linked with transglutaminase on the properties of an enclosed bioactive material using beta-galactosidase as model system.
Transglutaminase (protein-glutamine: amine gamma-glutamyltransferase, EC 2.3.2.13) from Streptoverticillium mobaraense has been used to stabilize immobilisates produced with beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae and acid-processed gelatins of different qualities as support. The isopeptide level of N epsilon-(gamma-L-glutamyl)-L-lysine bonds formed by transglutaminase was determined to estimate their influence on the kinetic properties of the enclosed beta-galactosidase. An HPLC procedure using precolumn derivatization of the gelatin hydrolysates with FMOC-chloride was chosen which permits the analysis of cross-linked lysine with satisfactory precision. Depending on the gelatin quality, the degree of cross-links necessary for the transformation of gelatin into an insoluble protein was in the range 0.3-32.3% of the available lysine residues. beta-Galactosidase was entrapped in the gelatin matrices with a yield of 8-46% of the initial activity. Long reaction times for cross-linking were due to low yields rather than to the number of isopeptide bonds. Repeated use of the immobilisates did not lead to an appreciable loss of activity. The Vmax of beta-galactosidase were diminished by immobilization caused by a tighter package of the protein chains rather than by the extent of cross-links, while the obtained Km values of the free enzyme and the immobilisates were quite similar. Also, the pH and temperature of optima of the free enzyme and the gelatin immobilisates differ only slightly. The data suggest that the immobilization procedure only moderately affects the activity of enzymes catalysing the reaction of a small compound if gelatin with high jelly strength is cross-linked in a 10% solution with transglutaminase.
Fuchsbauer
H L
HL
Fachbereich Chemische Technologie, Fachhochschule Darmstadt, Germany.
Gerber
U
U
Engelmann
J
J
Seeger
T
T
Sinks
C
C
Hecht
T
T
D001236:Aspergillus oryzae / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003094:Collagen; D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005780:Gelatin; D007202:Indicators and Reagents; D007700:Kinetics; D013306:Streptomycetaceae / Q000201:enzymology; D011503:Transglutaminases / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D005780:Gelatin; D003094:Collagen; D011503:Transglutaminases; D001616:beta-Galactosidase