Functional properties and application in peptide synthesis of trypsin modified with cyclodextrin-containing dicarboxylic acids

摘要

Bovine pancreatic trypsin was chemically modified with several beta-cyclodextrin (CD) derivatives containing adipic, pimelic and codecanodioic acids using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) as coupling agent. The modifying agents used were mono-6-(4-carboxybutane-l-carboxamidoyl)-6-deoxy-beta-cyclodextrin, mono-6-(5-carboxypentane-l-carboxamidoyl)-6-deoxy-beta-cyclodextrin and mono-6-(10-carboxydecane-1-carboxamidoyl)-beta-cyclodextrin. The modified enzymes contained about 6 mol of oligosaccharide per mol of trypsin. The catalytic and stability properties of trypsin were improved after the chemical modification. The thermostability profile of CD-modified trypsins was increased by about 10-14degreesC. The conjugates were also more stable against thermal incubation at different temperatures ranging from 45 to 60degreesC. In comparison with native trypsin, the cyclodextrin-enzyme conjugates were markedly more resistant to autolytic degradation at pH 9.0. Furthermore, the CD-trypsin conjugates gave higher aminolysis rates in kinetically controlled peptide synthesis reactions. The results reported here suggest that conjugation of enzymes with beta-cyclodextrin derivatives is a useful method for improving the stability and the catalytic properties of these biocatalysts. (C) 2004 Elsevier B.V All rights reserved.