A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor.
We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.
Stempfer
G
G
Boehringer Mannheim Therapeutics, Pennzberg, Germany.
Höll-Neugebauer
B
B
Kopetzki
E
E
Rudolph
R
R
D019149:Bioreactors; D001709:Biotechnology; D002384:Catalysis; D015195:Drug Design; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012995:Solubility; D000520:alpha-Glucosidases / Q000737:chemistry / Q000378:metabolism
D004800:Enzymes, Immobilized; D011993:Recombinant Fusion Proteins; D000520:alpha-Glucosidases