Rapid assay for tryptophanase using reversed-phase high-performance liquid chromatography

Area of Science:

• Biochemistry
• Analytical Chemistry
• Enzymology

Background:

• Tryptophanase enzyme activity assays are crucial for understanding microbial metabolism.
• Existing spectrophotometric methods often suffer from limitations in speed, sensitivity, and sample preparation.

Purpose of the Study:

• To develop a rapid, sensitive, and reproducible high-performance liquid-chromatographic (HPLC) assay for tryptophanase.
• To enable accurate measurement of indole production, a product of tryptophanase activity.
• To overcome the drawbacks of conventional spectrophotometric assays.

Main Methods:

• Development of a reversed-phase HPLC method coupled with fluorometric detection.
• Enzymatic assay utilizing tryptophanase to liberate indole from tryptophan.
• Incubation and chromatographic separation optimized for speed and sensitivity.

Main Results:

• The developed HPLC assay provides a total incubation time of 20 minutes.
• Indole elution occurs rapidly within 8 minutes, demonstrating efficient separation.
• The method achieves sensitivity in the nanomole range.
• Assay of tryptophanase activity in E. coli yielded an average activity of 6589.6 U/g of cells.

Conclusions:

• The novel HPLC assay is a rapid, sensitive, and reproducible method for quantifying tryptophanase activity.
• This technique offers significant advantages over standard spectrophotometric methods, including minimal sample preparation.
• The assay is suitable for accurate determination of enzyme activity in biological samples like E. coli.