Measurement of constitutive L-pyrrolidonyl peptidase activity from Streptococcus and Enterococcus using tetrazotized 0-dianisidine.

The detection of L-pyrrolidonyl peptidase activity is extremely useful for the differentiation of Enterococcus species and Streptococcus pyogenes from other members of the family Streptococaceae. This analysis has generally been performed utilizing the hydrolyzable substrate L-pyrrolidonyl beta-naphthylamine. After the substrate was hydrolyzed, free beta-naphthylamine has been detected utilizing the reagent para-dimethylaminocinnamaldehyde. The cinnamaldehyde and naphthylamine reagents combined to form an orange color, much like the indole reaction. The use of the cinnamaldehyde reagent had several drawbacks however: color development was not sharp, and the reagent was difficult to produce, and it was not stable. A new indicator system employing tetrazotized 0-dianisidine was developed. An extremely deep burgundy colored complex resulted from the reaction between the new indicator and B-Naphthylamine. This diazo reagent showed excellent correlation with results obtained with para-dimethylaminocinnamaldehyde and yielded more objective, distinct endpoints. This inexpensive reagent can be utilized either in a liquid form or dried on paper discs.