The use of chemically stabilised proteolytic enzymes in peptide synthesis
Colleary, Sandra (2003) The use of chemically stabilised proteolytic enzymes in peptide synthesis. PhD thesis, Dublin City University.
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The aim of this project was to study various serine proteases, both native and chemically modified, with a view to their application in peptide synthesis. Various chemical modifications of these were carried out to improve their stability before peptide synthesis.
Porcine trypsin was stabilised by reaction with ethylene glycol bis-(succinic acid Nhydroxy-succinimide ester) (EG). The enhanced stability is likely due to intramolecular crosslink(s) being formed in the enzyme. EG-tiypsin retained 100% amidase activity at 55°C for up to 150 minutes compared to 10 minutes for the native. EG-trypsin also had a 5-fold greater esterase/amidase kcat/Km value than the native, in an aqueous solution (Esterase/amidase ratio indicates potential usefulness in peptide synthesis).
Literature reports indicate that formylation of trypsin's tryptophan residues retains esterase activity while eliminating amidase activity. Modification of the tryptophan residues in porcine trypsin to 1-formyltryptophan was undertaken. The formylated trypsin retained 100% esterase activity up to 50°C. The T5() value was found to be 55°C (compared to 45°C for the native porcine trypsin). This increased thermal stability was unexpected. As had previously been shown, no amidase activity was detected but the residual esterase activity was too low for practical use.
Next to be investigated were subtilisin Carlsberg and alcalase, an industrial alkaline protease. Alcalase had a 20-fold higher esterase/amidase ratio than subtilisin Carlsberg in an aqueous solution whereas in a 50% (v/v) DMF solution the subtilisin Carlsberg demonstrated a 3-fold higher ratio compared to the alcalase. These were also stabilised by reaction with EG. Subtilisin Carlsberg demonstrated 50% amidase activity after 12.5 minutes at 65°C whereas the modified form retained the same activity for up to 56 minutes. Alcalase was more stable, with the native showing 50% amidase activity after 56 minutes and the modified showing the same activity at 100 minutes.
Both the native and EG-modified forms of alcalase and subtilisin Carlsberg were then used to make the tripeptide Tyr-Gly-GIy. Native subtilisin Carlsberg showed the greatest rate of peptide synthesis with the optimum temperature being 25°C. Native and EGtrypsin were used in the synthesis of the peptide Bz-A rg-Leu-Ni I2 with the EG-trypsin synthesising twice as much peptide as the native.
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