Transglutaminase-catalysed glycosidation of trypsin with aminated polysaccharides

Abstract

Dextran (MW=7.2x10(4)), carboxymethylcellulose (MW=2.5x10(4), substitution degree=0.7) and Ficoll (MW=6.9x10(4)) were derivatized with 1,4-diaminobutane and covalently attached to bovine pancreatic trypsin through a transglutaminase-catalysed reaction. The conjugates contained an average of 0.7-1.8 mol of polymers per mol of protein, and retained about 61-82% of the original esterolytic activity of trypsin. The optimum pH for trypsin was shifted to alkaline values after enzymatic glycosidation. The thermostability of the polymer-enzyme complexes was increased in about 13.7-50.0 degrees C over 10 min incubation. The prepared conjugates were also more stable against thermal incubation at different temperatures ranging from 50 degrees C to 60 degrees C. In comparison with native trypsin, the enzyme-polymer complexes were about 22- to 48-fold more resistant to autolytic degradation at pH 9.0. Transglutaminase-catalysed glycosidation also protected trypsin against denaturation in surfactant media, with 9- to 68-fold increased half-life times in the presence of 0.3% (w/v) sodium dodecylsulfate

Amination of enzymes to improve biocatalyst performance: coupling genetic modification and physicochemical tools

“…Table 1 shows a resume of the main examples. [67][68][69][70][71][72][73][74][75][76][77] The objectives could be enzyme crosslinking (analyzing the effect of the crosslinking size), 67 or just to check the effect of the general modications. Stabilities or activities could be improved in some cases.…”

“…In a quite sophisticated strategy, several polysaccharides were derivatized with 1,4-diaminobutane and covalently attached to bovine pancreatic trypsin through a transglutaminase-catalyzed reaction. 77 Thus, amination of free enzymes, even although not very utilized, has been used in diverse examples with good results.…”

Polymer–protein conjugates: an enzymatic activity perspective

“…In addition, the resulting conjugates had similar pH-activity profiles to the native trypsin. In another example, Villalonga et al 82 have covalently modified the glutamine residues on trypsin with 1,4-diaminobutanederivatized dextran, carboxymethyl cellulose (CMC), and polymeric sucrose by means of the enzyme transglutaminase (TGase). The thermal stabilization was ascribed to the co-operative contribution of both covalent coupling and the formation of new hydrogen bonds between the covalently linked polysaccharide and the hydrophilic groups at the surface of the enzyme.…”

Simultaneously and separately immobilizing incompatible dual-enzymes on polymer substrate via visible light induced graft polymerization

“…Trypsin is one specific digestive enzyme which can cut arginine or lysine on the carboxyl end of proteins or peptides [36][37][38]. When the two enzymes were mixed together, TGase may crosslink the trypsin and at the same time trypsin may hydrolyze TGase [39]. Therefore, completely separate immobilizing them on one substrate can not only retain their activities but also allow them to catalyze multi-steps reaction effectively.…”