Determination of intracellular dehydrogenase activities using flow injection analysis

Area of Science:

• Biotechnology
• Analytical Chemistry
• Biochemistry

Background:

• Efficient cell disintegration is crucial for releasing intracellular components like enzymes.
• Traditional methods can be time-consuming and lack reproducibility.
• Miniaturized systems offer potential for faster and more automated analyses.

Purpose of the Study:

• To develop and compare reproducible, rapid methods for yeast cell disintegration in miniaturized flow cells.
• To integrate cell disintegration with automated flow injection analysis (FIA) for enzyme activity determination.
• To validate the system for sequential and automated analysis of intracellular enzymes.

Main Methods:

• Comparison of ultrasonics and ultraturrax disintegration in a flow-through chamber versus mixer mill.
• Integration of a sterile on-line sampling device with the flow-through chamber.
• Development of an automated flow injection analysis (FIA) setup for enzyme activity measurement.
• Sequential determination of malate dehydrogenase, formate dehydrogenase, and formaldehyde dehydrogenase.

Main Results:

• Reproducible and rapid cell disintegration was achieved using ultrasonics and ultraturrax in miniaturized flow cells.
• The integrated system allowed for automated, on-line sampling and analysis of released enzyme activities.
• The FIA setup demonstrated successful sequential determination of multiple enzymes.
• Automated determination of intracellular formate dehydrogenase from Hansenula polymorpha yeast cells was performed.

Conclusions:

• Developed miniaturized flow cell systems provide reproducible and rapid yeast cell disintegration.
• Automated FIA integration enables efficient on-line analysis of intracellular enzyme activities.
• The presented methods offer a significant advancement for high-throughput biochemical analysis.