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Chemically stabilized trypsin used in dipeptide synthesis.
1School of Biological Sciences, Dublin City University, Dublin 9, Republic of Ireland.
Biotechnology and Bioengineering
|April 1, 1999
Summary
Chemical modification of bovine trypsin with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) created a more stable enzyme derivative (EG trypsin). EG trypsin demonstrated enhanced thermal stability, reduced autolysis, and improved performance in organic solvents for peptide synthesis.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Protein Chemistry
Background:
- Bovine pancreatic trypsin is a widely used serine protease.
- Native trypsin can exhibit limited stability in organic solvents and at elevated temperatures.
- Modifications to enzyme structure can enhance stability and catalytic properties.
Purpose of the Study:
- To chemically modify bovine trypsin to improve its stability and catalytic efficiency.
- To characterize the properties of the modified trypsin derivative (EG trypsin).
- To evaluate the performance of EG trypsin in peptide synthesis reactions.
Main Methods:
- Bovine trypsin was modified using ethylene glycol bis(succinic acid N-hydroxysuccinimide ester).
- Thermal stability was assessed by T50 values and half-life measurements.
- Enzyme activity and kinetic parameters (Km, kcat) were determined for amide and ester substrates.
- Peptide synthesis yields were compared between native and modified trypsin in various organic solvents.
Main Results:
- Approximately 8 of 14 lysines were modified, yielding EG trypsin.
- EG trypsin showed significantly increased thermal stability (T50 = 59°C vs. 46°C) and a fivefold longer half-life at 55°C.
- EG trypsin exhibited reduced autolysis and retained higher activity in organic solvents like acetonitrile and DMSO.
- Kinetic analysis revealed lower Km and increased kcat for amide substrates, resulting in higher specific activity.
- EG trypsin achieved higher yields and faster reaction rates in kinetically controlled peptide synthesis, particularly in 95% acetonitrile.
Conclusions:
- Chemical modification of bovine trypsin with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester) yields a more stable and catalytically efficient enzyme.
- EG trypsin is a promising biocatalyst for peptide synthesis, especially in organic media.
- The enhanced properties of EG trypsin offer advantages for enzymatic synthesis applications requiring improved stability and activity.