For the measurement of endo-1,4-?-D-xylanase in enzyme preparations, bread improver mixtures and animal feeds. Contains Xylazyme AX Tablets and xylanase enzyme controls (A. niger and Trichoderma longibrachiatum).
INTRODUCTION:
Arabinoxylan is the major endosperm cell-wall polysaccharide of
wheat and rye and is found in significant proportions in most cereal
solutions and slurries of high viscosity, and in animal nutrition it
reduces the rate of nutrient absorption from the gut.
endo-β-D-Xylanase (xylanase) is added to feeds to catalyse
depolymerisation of this polysaccharide. It can be demonstrated
that endo-cleavage by xylanase of just one bond per thousand in the
arabinoxylan backbone can significantly remove viscosity properties.
Of the carbohydrase enzymes used as feed supplements, one of the
most difficult to measure has been xylanase. These problems are
attributed to several factors, including the low levels of enzyme
added to the feed, inactivation of enzyme during pelleting, binding
of the enzyme to feed components and the presence of specific
xylanase inhibitors.
The only biochemical methods which are sufficiently sensitive, specific
and robust to measure xylanase in feeds are viscometric assays and
those employing dyed xylan or arabinoxylan polysaccharides.
Viscometric assays are tedious, whereas assays employing dyed
xylan substrates are rapid, reproducible and simple to perform.
We recommend the use of either Xylazyme AX tablets or Azo-
Wheat Arabinoxylan (Azo-WAX). Xylazyme AX based assays
are about 5-fold more sensitive than assays employing Azo-WAX.
However, this latter substrate does have sufficient sensitivity in most
applications, and results are slightly more reproducible than with
Xylazyme AX.
It is generally accepted that xylanase enzymes which are best suited
to feed applications have optimal activity at pH 6.0. Consequently,
these enzymes are generally assayed at this pH in 100 mM sodium
phosphate buffer. In recovery experiments, however, we found that
sodium phosphate buffer extracts only a small proportion (< 20%)
of the amount of enzyme added to the feed. Thus a wide range of
alternative extractants and extraction conditions have been evaluated.
For feeds containing Trichoderma sp. xylanases, the best and most
consistent results have been obtained using 100 mM acetic acid
or 100 mM sodium acetate buffer (pH 4.7) at room temperature.
Optimal extraction of Humicola sp. xylanases was achieved with a
buffer containing 100 mM MES buffer (pH 6.0) and 1 % w/v sodium
dodecyl sulphate (SDS).
KITS:
Kits containing the required reagents to measure xylanase in animal
feeds are available from Megazyme. These kits contain:
1. Xylazyme AX test tablets (200 tablets).
2. A. niger control xylanase (~ 295 mU/mL at 40°C and pH 4.7)
in 50 % (v/v) glycerol (activity stated on vial).
3. T. longibrachiatum control xylanase (~ 386 mU/mL at 40°C and
pH 6.0) in 50% (v/v) glycerol (activity stated on vial).
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