Automated Colorimetric Determination of Achromobacter lyticus Protease Activity in Fermentation Samples Using Tos–Gly–Pro–Lys–pNA as a Chromogenic Substrate

Abstract

An automated method (COBAS FARA // centrifugal analyzer) has been developed for the determination of the activity of Achromobacter lyticus lysyl-specific protease. The chromogenic substrate used was Tos–Gly–Pro–Lys–pNA. The enzyme catalyzes the liberation of p-nitroaniline from the peptide, forming a yellow color, which when recorded at 405 nm, is proportional to the protease activity. The general technical conditions as well as the general enzymatic kinetics have been optimized. The resulting method had a good precision, sensitivity and speed. The LOQ was 0.01 U ml ^{- 1} and the LOD was 0.0001 U ml ^{- 1} . The method was linear within the range of 0.010 U ml ^{- 1} to 0.125 U ml ^{- 1} with r = 0.9999 and with a small or no intercept. The repeatability was <0.7%. The reproducibility (day-to-day) was <1.3%. Robustness testing showed that the most critical parameters were wavelength, pH and temperature.