Abstract
Feed additive enzymes are used in animal nutrition for better utilization of animal diets. As all other feed additives, xylanase and/or (1-3,1-4)-
β
-glucanase enzymes must be detectable in animal feed. The activity of non starch polysaccharide degrading enzymes can be detected by hydrolysation of specific substrates. The detection limit and -range depend on substrate characteristics and the biochemical characteristics of active enzymes. To increase the detection limit substrates were physically, chemically and biologically modified and tested. The commercial enzyme preparations Avizyme
®
1300 (T. longibrachiatum, T. viride), Biofeed plus
®
(H. insolens), Biofeed Wheat
®
(A. oryzae), Lyxasan
®
(A. niger I), Roxazyme
®
(T. viride), Barlican
®
(T. reesei I), Biopract
®
(T. reesei II), Energex
®
(A. niger II), Novozyme 104
®
(no declaration), and Hostazym X
®
(no declaration) were purified, their activities and substrate specificities determined. They contained at least two xylanase activities except Biofeed Wheat
®
. The enzyme preparations consisted of mixtures of different enzymes, differing in isoelectric point and pH behaviour. The analysis of hydrolytic products showed a profile for each enzyme preparation which was independent from the enzyme concentration. The different distribution of hydrolytic products influenced the activity estimations. Thus, it is generally necessary to generate a separate calibration curve for each enzyme preparation. Chromogenic substrates were xylans or
β
-glucans, ionically or covalently coupled with a dye. The enzymatic hydrolysation frees the ionic dye or produces hydrolytic products with covalent coupled dye. Different dyes (covalently or ionically coupled) and different substrates (ultrasonicated, autoclaved, carboxymethylated) were combined and tested in enzyme assays. These preparations were not superior to commercial preparations. Ultrasonicated oat spelt xylan allowed the xylanase activity to be measured as viscosity reduction without measuring the initial viscosity of the substrate solution, because no initial increase of the viscosity was observed after addition of enzyme. In an agar diffusion assay enzyme solutions were incubated in wells of an agar plate containing substrate. The plate was stained with congo red. Congo red is bound by highmolecular polysaccharide. Enzymatic hydrolysis is shown after red staining as clear lysis zones. The range and detection limit was improved using modified substrates (ultrasonicated or autoclaved) and a digital photodocumentation system. The dye reaction is a new method for the determination of xylanase activities. Soluble ultrasonicated oat spelt xylan was hydrolysed and hydrolytic products bound reactive green. In sodium acetate buffer the detection limit of enzyme activity was 0.005 IE/ml, in feed stuff extract about 0.05 IE/ml. |
