Preparation of endo-1,4-beta-xylanase (EC 3.2.1.8) - 4a36

In the current application, an authorisation of a preparation of endo-1,4-beta-xylanase (EC 3.2.1.8) is sought under Article 4(1) for all poultry, under the category/functional group 4 (a) "zootechnical additives"/"digestibility enhancers" according to Annex I of Regulation (EC) No 1831/2003. 

According to the Applicant, the active agent of the feed additive is endo-1,4-beta-xylanase, produced by fermentation with the genetically modified strain Komagaetella phaffii ATCC KMI000000. The activity of endo-1,4-beta-xylanase is expressed in xylanase units (U). One U unit is the amount of enzyme that releases 0.0067 micromoles of reducing sugar (xylose equivalent) per minute and per gram of enzyme product at 50 °C and pH 5.3. 

The feed additive is intended to be marketed as a solid formulation having a guaranteed minimum endo-1,4-beta-xylanase activity of 3000000 U / g and to be incorporated directly into complete feedingstuffs or through premixtures to obtain a minimum xylanase activity of 30000 or 45000 U / kg feedingstuffs depending on the target species. 

For the quantification of the xylanase activity in the feed additive the Applicant submitted a single-laboratory validated and further verified method based on the enzymatic hydrolysis of xylanase on the beechwood xylan substrate and the colour formation of the released reducing sugar with arsenomolybdate. 

Furthermore, for the quantification of endo-1,4-beta-xylanase in premixtures and feedingstuffs the Applicant submitted another single-laboratory validated and further verified colorimetric method, based on the measurement on the quantification of water soluble dyed fragments produced by the action of xylanase on a commercially available (Xylazyme AX®, Megazyme) azurine cross-linked wheat arabinoxylan substrate. 

Based on the acceptable performance characteristics the EURL recommends for official control (i) the single-laboratory validated and further verified colorimetric (arsenomolybdate) method for the determination of endo-1,4-beta-xylanase in the feed additive and (ii) the colorimetric (Megazyme) method for the determination of 1,4-beta-xylanase in 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) 2023/668

For the determination of endo-1,4-beta- xylanase in the feed additive:

— colorimetric method based on the enzymatic hydrolysis of endo-1,4-beta-xylanase on the beechwood xylan substrate.