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Related Experiment Videos

Silanized silica bound trypsin as analytical probe.

D Bharadwaj1, A Mukherjee, M S Roy

  • 1Indian Institute of Chemical Biology, Calcutta.

Indian Journal of Biochemistry & Biophysics
|August 1, 1992
PubMed
Summary

Immobilized trypsin on silanized silica offers enhanced activity and stability for protein digestion. This reusable enzyme matrix is effective for peptide mapping and limited proteolysis, simplifying analytical procedures.

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Area of Science:

  • Biochemistry
  • Enzyme immobilization
  • Protein chemistry

Background:

  • Soluble trypsin is widely used for protein digestion but lacks stability and reusability.
  • Enzyme immobilization is a key strategy to improve enzyme properties and facilitate separation.
  • Silanized silica is a common support material for enzyme immobilization.

Purpose of the Study:

  • To immobilize trypsin onto silanized silica via covalent coupling.
  • To evaluate the activity, stability, and reusability of the immobilized trypsin.
  • To assess the suitability of the immobilized enzyme for limited proteolysis and peptide mapping.

Main Methods:

  • Covalent coupling of trypsin to silanized silica.
  • Enzymatic activity assays using albumin as a substrate.

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  • Thermostability assessments at elevated temperatures.
  • Electrophoretic analysis of protein digestion products after repeated use.

    • Main Results:

    • Immobilized trypsin exhibited significant activity (30-38%) and enhanced thermostability compared to soluble trypsin.
    • The enzyme matrix efficiently processed albumin for limited proteolysis, yielding similar products upon repeated use.
    • Electrophoretic analysis confirmed consistent digestion patterns with the immobilized enzyme, comparable to the soluble form.

    Conclusions:

    • Covalent immobilization of trypsin on silanized silica yields a robust and reusable biocatalyst.
    • The immobilized enzyme is suitable for efficient limited proteolytic processing and peptide mapping applications.
    • Easy removal of the enzyme matrix simplifies downstream processing in analytical workflows.