Preparation of endo-1,4-beta-xylanase (EC 3.2.1.8) - 4a35

In the current application, an authorisation of a preparation of endo-1,4-beta-xylanase (EC 3.1.3.28) is sought under Article 4(1) for chickens and turkeys for fattening, chickens reared for laying, turkeys reared for breeding and minor avian species (including also sporting, ornamental and exotic birds), 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 of the genetically modified strain Komagaetella phaffii xyl-2 (DSM 33574). The activity of endo-1,4-beta-xylanase is expressed in xylanase units (XU). One XU unit is the amount of enzyme which releases 1 nano-mol of reducing sugar (xylose equivalent) per second from xylan of beechwood at 50 °C and pH 6.0. The feed additive is intended to be marketed as a solid formulation, having a guaranteed minimum endo-1,4-beta-xylanase activity of 15000 XU / g and to be incorporated directly into complete feedingstuffs to obtain minimum xylanase activity of 10000 XU / kg feedingstuffs.

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 the beechwood xylan substrate and the colour formation of the released reducing sugar with 3,5-dinitrosalicylic acid (DNS). 

Furthermore, for the quantification of endo-1,4-beta-xylanase in feedingstuffs the Applicant submitted another single-laboratory validated and further verified colorimetric method, based on the measurement of 4-nitrophenol group produced by the action of xylanase on a commercially available substrate (Megazyme). 

Based on the acceptable performance characteristics the EURL recommends for official control (i) the single-laboratory validated and further verified colorimetric (DNS) 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 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) 2023/669

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

colorimetric (DNS) method based on the enzymatic hydrolysis of the beechwood xylan substrate.