Chemically stabilized trypsin used in dipeptide synthesis.
Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30 degrees and 70 degrees C: T50 values were 59 degrees C and 46 degrees C, respective. EG trypsin's half-life of 25 min at 55 degrees C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-L-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-L-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin.
Murphy
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A
School of Biological Sciences, Dublin City University, Dublin 9, Republic of Ireland.
O Fágáin
C
C
D000818:Animals; D002417:Cattle; D004151:Dipeptides / Q000138:chemical synthesis*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology; D013388:Succinimides; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism*
D004151:Dipeptides; D007202:Indicators and Reagents; D013388:Succinimides; D014357:Trypsin