Steady-state generation of hydrogen peroxide: kinetics and stability of alcohol oxidase immobilized on nanoporous alumina

Biotechnol Lett. 2013 Apr;35(4):585-90. doi: 10.1007/s10529-012-1110-5. Epub 2012 Dec 15.

Abstract

Alcohol oxidase from Pichia pastoris was immobilized on nanoporous aluminium oxide membranes by silanization and activation by carbonyldiimidazole to create a flow-through enzyme reactor. Kinetic analysis of the hydrogen peroxide generation was carried out for a number of alcohols using a subsequent reaction with horseradish peroxidase and ABTS. The activity data for the immobilized enzyme showed a general similarity with literature data in solution, and the reactor could generate 80 mmol H2O2/h per litre reactor volume. Horseradish peroxidase was immobilized by the same technique to construct bienzymatic modular reactors. These were used in both single pass mode and circulating mode. Pulsed injections of methanol resulted in a linear relation between response and concentration, allowing quantitative concentration measurement. The immobilized alcohol oxidase retained 58 % of initial activity after 3 weeks of storage and repeated use.

MeSH terms

  • Alcohol Oxidoreductases / metabolism*
  • Alcohols / metabolism
  • Aluminum Oxide*
  • Bioreactors
  • Enzymes, Immobilized / metabolism*
  • Hydrogen Peroxide / metabolism*
  • Kinetics
  • Pichia / enzymology*

Substances

  • Alcohols
  • Enzymes, Immobilized
  • Hydrogen Peroxide
  • Alcohol Oxidoreductases
  • alcohol oxidase
  • Aluminum Oxide