Chemical modification of bovine trypsin for use in peptide synthesis
Murphy, Ann (1996) Chemical modification of bovine trypsin for use in peptide synthesis. PhD thesis, Dublin City University.
Full text available as:
The first synthesis reaction achieved via reversal of the catalytic reaction of a hydrolytic enzyme was reported at the end of the last century. There has been a renewed interest in the use of proteases in peptide synthesis in the last twenty years. Proteases offer numerous advantages over conventional chemical methods, including reduction of racemization and side reactions. However, there are drawbacks associated with their use, including destabilization and loss of activity both at high temperatures and at high concentrations of organic solvents. Another major problem is autolysis which can occur when proteases are in solution. It is, therefore necessary to modify the enzyme so as to improve its stability.
The aim of this project was to chemically modify bovine trypsin in an attempt to enhance the stability of the enzyme and subsequently to use the stabilized forms in peptide synthesis. Initially, assay systems were developed and studies on native trypsin with respect to thermostability, organotolerance and stability to dénaturants were undertaken. Chemical modification of bovine trypsin was carried out using carbodiimides and N-hydroxy-succinimide esters. Stabilized derivatives were produced using acetic acid N-Hydroxy-succinimide ester (AANHS) and ethylene glycol bis (succinic acid N-Hydroxy-succinimide ester) or EGNHS for short. AA-NHS neutralizes the positive charge on lysine residues. Trypsin stabilized with EG-NHS a crosslinking reagent contained no dimers or higher order derivatives suggesting that intramolecular crosslinking had occurred. Approximately 8 out of 14 lysine residues per trypsin molecule were modified with AA-NHS and EG-NHS. The AA- and EG-NHS treated trypsins showed enhanced thermostability between 30 and 70°C compared with the native. T50 values for native, AA-NHS and EG-NHS trypsin were 46°C, 51°C and 59°C respectively. At 55°C, the AA- and EG-NHS modified trypsins' half-lives are 8.7 and 25 minutes versus 4.3 minutes for the native enzyme. Modified forms of trypsin exhibited decreased rates of autolysis. EG-NHS trypsin showed enhanced stability in aqueous mixtures of organic solvents, while AA-NHS trypsin showed enhanced stability at 65°C in aqueous mixtures o f the following organic solvents: 1,4-dioxan, dimethy lformamide, dimethylsulphoxide and acetonitrile.
Kinetic studies showed that both modified forms had decreased Km and increased kcat values compared with native trypsin for ester and amide substrates. The substrate specificity of modified trypsin was also enhanced. Catalytic activity of AA-trypsin in aqueous-organic mixtures was found to be higher than that of native in a range of organic solvents, while little or no difference between native and EG-trypsin was observed.
Each form of trypsin was used to prepare the dipeptide benzoyl arginyl leucinamide from derivatives of its constituent amino acids. The product yield increased with increasing organic solvent concentration and decreasing temperature. Maximum product yield was obtained in 95% acetonitrile at 4°C. The rate of peptide synthesis of modified trypsin was greater than native in 95% t-butanol and in 95% acetonitrile, with the highest rate being observed for EG-trypsin.
Archive Staff Only: edit this record