Thermal stabilization of trypsin by enzymic modification with β-cyclodextrin derivatives

Streptoverticillum sp. transglutaminase was used as catalyst for the attachment of several beta-cyclodextrin derivatives to the glutamine residues in bovine pancreatic trypsin. The modifying agents used were mono-6-ethylenediamino-6-deoxy-beta-cyclodextrin, mono-6-propylenediamino-6-deoxy-beta-cyclodextrin, mono-6-butylenediamino-6-deoxy-beta-cyclodextrin and mono-6-hexylenediamino-6-deoxy-beta-cyclodextrin. The transformed trypsin preparations contained about 3 mol of oligosaccharides/mol of protein. The specific esterolytic activity of trypsin was increased by about 4-21 after conjugation. The Km values for cyclodextrintrypsin complexes represented about 58-87% of that corresponding to the native enzyme. The optimum temperature for esterolytic activity of trypsin was increased by about 5-10 degreesC after enzymic modification with the cyclodextrin derivatives. The thermostability was increased by 16 degreesC for the modified trypsin. Thermal inactivation at different temperatures ranging from 45 to 60 degreesC was markedly increased for the oligosaccharide-trypsin complexes. This modification also protected the enzyme against autolysis at alkaline pH.