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Chemically stabilized trypsin used in dipeptide synthesis.

A Murphy1, C O Fágáin

  • 1School of Biological Sciences, Dublin City University, Dublin 9, Republic of Ireland.

Biotechnology and Bioengineering
|April 1, 1999
PubMed
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.

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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.

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  • 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.