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° and 70°C: T 50 values were 59°C and 46°C, respective. EG trypsin's half‐life of 25 min at 55°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 K m values for both amide and ester substrates; its k cat values for two amides (benzoyl‐ l ‐arginine p ‐nitroanilide and benzyloxycarbonyl glycyl‐glycyl‐arginyl‐7‐amino‐4‐methyl coumarin) increased, whereas its k cat value for an ester (thiobenzoyl benzoyloxycarbonyl‐ l ‐lysinate) decreased slightly. The specific activity ( k cat / K m ) 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. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:366–373, 1998.