Protease (EC 3.4.21.62)

In the current application an authorisation is sought under Article 4 for protease (subtilisin) under the category / functional group 4(a) "zootechnical additives / digestibility enhancers", according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, the authorisation is sought for all growing poultry species. 

The feed additive is intended to be marketed under the trade name PRoAct 360 as a solid formulation with a minimum protease activity of 600000 NFP / g feed additive. 

According to the Applicant, one protease unit (NFP) is defined as the amount of enzyme that releases 1 μmol of p-nitroaniline from 1 mM substrate (N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide) per minute at pH 9.0 and 37°C. 

The feed additive is intended to be used directly into feedingstuffs or through premixtures at a recommended minimum protease activity of 30000 NFP / kg complete feedingstuffs. 

For the quantification of the protease activity in the feed additive, premixtures and feedingstuffs, the Applicant submitted methods based on the quantification of the para-nitroaniline (pNA) released by the action of the protease on the N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide substrate. These methods have been single-laboratory validated and further verified and the Applicant reported relative standard deviations for repeatability (RSDr) and intermediate precision (RSD_ip) ranging from 1.9 to 14.9 %, and recovery rates (R_rec) ranging from 74 to 113 %. 

Based on the overall available performance data, the EURL recommends for official control the above mentioned single-laboratory validated and further verified colorimetric methods for the quantification of the protease (subtilisin) activity in the feed additive, premixtures and feedingstuffs. 

Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761) is not considered necessary.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2024/778

For the determination of the protease activity in the feed additive, premixtures and compound feed: 

• Colorimetric methods based the enzymatic reaction of protease on the N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide substrate