Study On Endogenous Arabinoxylans Degrading Enzymes From Wheat Malts

Now wheat and wheat malts are commonly used as main raw material in the brewing industry. Arabinoxylans(AXs) is the main non-starch polysaccharide consisting of wheat cell walls, and its reasonable degradation affects viscosity, turbidity and filtration rate. Nevertheless, AXs also affects the foam property and mellowness of beer. Because of its complex structure, its degradation needs a series of enzymes. There are endogenous AXs degrading enzymes in wheat marts, including endo-β-1-4-xylanase(EC 3.2.1.8, endoxylanase), β-D-xylosidase(EC 3.2.1.37) and α-L-arabinofuranosidase(EC 3.2.1.55, arabinosidase). In this study, we purified endoxylanase which had single electrophoretic band from wheat marts named Yannong 24, and investigated its enzymatic characteristics; we also investigated enzymatic characteristics of β-D-xylosidase from wheat marts; the change of enzyme activity of endoxylanase, β-D-xylosidase and arabinosidase were comfirmed during malting; the differences in WEAXs content and molecular weight of wheat malts were compared during malting. The main conclusions were listed as following:(1)Endoxylanase was purified from wheat marts, by ammonium sulfate precipitation, Q-Sepharose Fast Flow anion exchange chromatography and Phenyl-Sepharose 6 F F hydrophobic chromatography. Finally, electrophoretically pure endoxylanase was obtained. The enzyme was purified 11.72 fold with specific activity of 4.30u/mg. The molecular weight was 27.8kDa. The properties of endoxylanase were investigated. The enzyme exhibited optimum activity at pH6.0, 40°C. The Na+ had almost no effect on the activity, while Ca2+, Mg2+ and K+ activated the enzyme obviously. The Cu2+ showed obvious inhibitory effect.(2)β-D-xylosidase was extracted and characterized from wheat malts. The optimal pH of the enzyme was 3.5 and 4.5. It can be inferred that there was the presence of isoenzymes. The enzyme was comparatively stable at pH 3.0-6.0 and the relative enzyme activity was 26.2% at pH7.0. The optimum temperature of the enzyme was 70°C. β-D-xylosidase showed better thermal stability at 35-60°C, while it showed worse thermal stability at 70-80°C. The enzyme activity was lost for 20 min at 80°C. Moreover, the activity of wheat malt β-D-xylosidase was severely inhibited by Cu2+. The Km and Vmax values of β-D-xylosidase against p-NPX were 1.74 mM and 0.76 mM·min-1, respectively.(3)The endoxylanase activity increased rapidly and increased by 507.09% at the end of steeping; at the start of germination, the enzyme activity decreased slightly, and then increased gradually. After reaching a peak value of 10.10 u at day 4, the enzyme activity was stable; the deactivation rate of endoxylanase was 65.82% during drying. The arabinosidase activity increased steadily and reached the maximum value of 40.07 u at day 5, the enzyme activity was stable at the fifth day of germination; the deactivation rate of arabinosidase was 69.58% during drying. From steeping to the fifth day of germination, the β-D-xylosidase activity increased gradually and reached the maximum value of 170.31 u at day 5, the enzymatic activity still had tendency to increase at day 5; the deactivation rate of β-D-xylosidase was 18.74%. Comparing with the first two enzymes, the deactivation rate of β-D-xylosidase was less.(4)The content of wheat malts WEAXs increased by 0.75% and increased by 163.04% when comparing with that of wheat. The change of WEAXs content focused on steeping and the first day of germination, and especially the day 1. The content of WEAXs increased by 0.46% at day 1 and was 100% more than that of wheat. The molecular weight of 0.4-6.2kDa was the main constituents of WEAXs, its ratio in wheat was 56.80% and increased by 18.87% during malting. From the start of malting to the first day of germination, the content of large molecular weight constituents which were higher than 35.4kDa increased gradually, then the constituents content reduced gradually, while the content of small molecular weight constituents which were 0.4-35.4kDa increased all the time and increased by 218.81%. The content of large molecular weight constituents reduced which may be caused by its degradation into small molecular weight constituents.