Crosslinking of imprinted proteases to maintain a tailor-made substrate selectivity in aqueous solutions.
A covalent method to keep imprinted properties of proteins stable in aqueous as well as in organic environment is described. To stabilize the ligand induced acceptance for D-configured substrates by alpha-chymotrypsin or subtilisin Carlsberg, each protein was first vinylated by acylation with itaconic anhydride. Then, the tailoring of the derivatized proteins by precipitation in the presence of N-acetyl-D-tryptophan from an aqueous medium with 1-propanol, and the subsequent crosslinking of the enzyme preparations with ethylene glycol dimethacrylate in cyclohexane was carried out. The crosslinked imprinted proteins (CLIPs) obtained catalyzed the hydrolysis of N-acetyl-D-tryptophan ethyl ester in phosphate buffer and the corresponding back reaction in cyclohexane, respectively. The repeated use of CLIP-alpha-chymotrypsin in D-ester hydrolysis was demonstrated. Furthermore, this particular CLIP-alpha-chymotrypsin showed no loss in activity when it subsequently was used in the synthesis of N-acetyl-D-tryptophan ethyl ester in cyclohexane again. In the case of D-ester hydrolysis the reaction rate acceleration (k(enz)/k(nonenz)) was in the same order of magnitude of about 10(4)-10(5) mM(-1) for the two CLIP-proteases. The results suggest that enzymes tailored by imprinting technique do not lose their induced "new" property in the presence of water when they are prepared according to the described vinylation/crosslinking method (CLIP technique).
Peissker
F
F
Institute of Biochemistry and Biotechnology, Braunschweig, Germany.
Fischer
L
L
D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry*; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011480:Protease Inhibitors / Q000737:chemistry; D012996:Solutions; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry; D014867:Water / Q000737:chemistry
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011480:Protease Inhibitors; D012996:Solutions; D014867:Water; C006392:N-acetyltryptophan; D014364:Tryptophan; D010450:Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; C117608:alpha-chymotrypsin