Endo-1,4- beta-xylanase EC 3.2.1.8

In the current application authorisation is sought under article 13(3) for Ronozyme® WX under the category/functional "zootechnical additives"/"digestibility enhancers". Specifically, authorisation is sought for the use of a new Ronozyme® WX produced from an optimized Aspergillus oryzae strain. The authorisation is sought for the use of the feed additive for poultry for fattening, piglets (weaned) and pigs for fattening. 

According to the Applicant, endo-1,4-beta-xylanase is the active agent of Ronozyme® WX. The Applicant expresses the xylanase enzymatic activity in xylanase units (FXU), defined as "the amount of enzyme which liberates 7.8 micromoles of reducing sugars (xylose equivalents) from azo-wheat arabinoxylan per minute at pH 6.0 and 50°C". 

The product is intended to be marketed as solid and liquid formulations having a guaranteed minimum xylanase activity of 1000 FXU/g and 650 FXU/ml respectively. The feed additive formulations are intended to be included through premixtures (solid) or directly in feedingstuffs (solid and liquid) to obtain a minimum activity of 100 or 200 FXU/kg feedingstuffs, depending on the target species. 

For the quantification of the xylanase activity in the feed additive the Applicant proposed a single-laboratory validated and further verified colorimetric method that requires the use of an automatic device (Konelab Analyzer). Upon request of the EURL of a manual method, the Applicant proposed to apply a ring-trial validated colorimetric method based on the quantification of the coloured compounds produced by the dinitro salicylic acid (DNSA) and the xylosylic moieties released by the action of endo-1,4-beta-xylanase on arabinoxylan. Furthermore, for the quantification of the xylanase activity in premixtures and feedingstuffs the Applicant proposes a single-laboratory validated and further verified colorimetric method based on the quantification of the water soluble dyed fragments produced by the action of endo-1,4-beta-xylanase on oat spelt azo-xylan. In both methods the calibration is conducted using a reference standard with a known enzyme activity expressed in FXU. 

Based on the satisfactory performance characteristics, recalculated from the experimental data available, the EURL recommends for official control the colorimetric methods mentioned above for the quantification of the xylanase 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) is not considered necessary.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2024/1058

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

• Colorimetric method measuring coloured compound produced by the dinitro salicylic acid (DNSA) and the xylosylic moieties released by the action of xylanase on arabinoxylan

For the quantification of endo–1,4-beta-xylanase in premixtures and compound feed:

• Colorimetric method measuring water soluble dye released by action of xylanase from dye-labelled oat spelt azo-xylan