Improving dioxygenase stability by gene chromosome insertion: implementation in immobilized-cell systems

Area of Science:

• Biotechnology
• Environmental Microbiology
• Enzyme Engineering

Background:

• The enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase is crucial for degrading aromatic compounds but can be unstable.
• Recombinant cell immobilization is a strategy to enhance enzyme stability and activity in bioreactors.

Purpose of the Study:

• To investigate the immobilization of recombinant Escherichia coli CC118-D cells expressing 3,4-dihydroxyphenylacetate 2,3-dioxygenase.
• To compare the stability and activity of immobilized cells versus cells in suspension.
• To evaluate the potential of this system for bioremediation.

Main Methods:

• Recombinant Escherichia coli CC118-D strain construction by inserting the Klebsiella pneumoniae hpaB gene.
• Immobilization of recombinant cells and comparison of dioxygenase activity and cell viability with suspension cultures.
• Testing stability and degradation efficiency over 15 days and implementation in a pilot-scale bioreactor.

Main Results:

• Immobilization significantly increased the stability of 3,4-dihydroxyphenylacetate 2,3-dioxygenase and improved the degradation of 3,4-dihydroxyphenylacetate.
• Recombinant cells and enzyme activity remained stable for at least 15 days post-immobilization.
• Pilot-scale bioreactor implementation achieved 100% stabilization of dioxygenase activity.

Conclusions:

• Immobilization of recombinant E. coli CC118-D is an effective strategy to stabilize 3,4-dihydroxyphenylacetate 2,3-dioxygenase.
• This approach offers a robust tool for efficient bioremediation of aromatic pollutants.