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Stabilization of invertase by molecular engineering.

Pattamawadee Tananchai1, Yusuf Chisti

  • 1School of Engineering, Massey University, Palmerston North, New Zealand.

Biotechnology Progress
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Crosslinking Saccharomyces cerevisiae extracellular invertase with diisocyanates significantly enhanced its thermal stability. This molecular engineering approach improved enzyme resistance to denaturation, showing potential for industrial applications.

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Area of Science:

  • Biochemistry
  • Enzyme Engineering
  • Protein Stabilization

Background:

  • Extracellular invertase from Saccharomyces cerevisiae is crucial for sucrose hydrolysis.
  • Thermal denaturation limits the industrial application of enzymes.
  • Protein crosslinking is a strategy to improve enzyme stability.

Purpose of the Study:

  • To stabilize extracellular invertase from Saccharomyces cerevisiae against thermal denaturation.
  • To investigate the effect of diisocyanate crosslinking on enzyme thermostability.
  • To quantify the changes in denaturation kinetics after crosslinking.

Main Methods:

  • Enzyme purification and characterization of extracellular invertase.
  • Chemical crosslinking using homobifunctional diisocyanates of varying lengths (n=4, 6, 8).
  • Thermal denaturation assays and kinetic analysis (denaturation constant, activation energy).
  • Confirmation of crosslinking via SDS-PAGE.

Main Results:

  • Crosslinking with 1,4-diisocyanatobutane (n=4) was most effective in enhancing thermostability.
  • Optimized crosslinking (0.5 mg/mL protein, 30 μmol/mL reagent) dramatically improved stability.
  • The first-order thermal denaturation constant at 60°C decreased from 1.567 min⁻¹ (native) to 0.437 min⁻¹ (stabilized).
  • Activation energy for denaturation increased from 391 kJ/mol (native) to 466 kJ/mol (stabilized).

Conclusions:

  • Intermolecular and intramolecular crosslinking effectively stabilizes extracellular invertase against thermal denaturation.
  • Diisocyanate crosslinking is a viable method for protein molecular engineering to enhance enzyme thermostability.
  • The stabilized enzyme exhibits significantly improved resistance to heat, broadening its potential for biotechnological uses.