Azocasein assay for alkaline protease in complex fermentation broth

An azocasein assay has been developed for determination of alkaline protease in fermentation broth from a complex substrate containing ca. 50 g/l protein which to a high degree interferes with azocasein. Methods described in the literature have been found inaccurate as results deviate ca. 50% from true activity, so one of the methods is modified in order to eliminate interference. Proportionality between the relative azocasein concentration and the deviation from true activity is found. An azocasein concentration of 350 mg azocasein per ml sample gives satisfactory results with an accuracy of ±5%. Application of a standard addition method also improves the accuracy, but is laborious and less precise.

a :

true enzyme activity

a _s :

activity determined from standard curve

AU :

Anson unit

c _A/T :

relative azocasein concentration (mg azocasein per mg total protein)

r :

ratio between measured and true enzyme activity

RD :

relative deviation from true value (%)

RSD :

relative standard deviation (%)

TCA :

trichloroacetic acid

Authors and Affiliations

1. Center for Process Biotechnology, Department of Biotechnology, Building 223, Technical University of Denmark, DK-2800, Lyngby, Denmark

   Stig Lykke Iversen & Mogens Holger Jørgensen

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