Enzymatic Properties of endo-1,4-β-xylanase from Wheat Malt

Background: Arabinoxylan (AX) is the main non-starch polysaccharide in wheat. Wheat malts are traditional raw materials for beer brewing. AX is divided into water-soluble arabinoxylan (WEAX) and waterinsoluble arabinoxylan (WUAX). In the mashing stage of beer production, WUAX in malt is degraded by arabinoxylanase to WEAX, which is further degraded to smaller molecules and retained in the final beer. The viscosity of WEAX is related to its molecular weight. WEAX with higher molecular weight and viscosity can increase viscosity and turbidity and reduce filtration speed of wort and beer; WEAX with moderate molecular weight and viscosity contributes to the foaming characteristics and foam stability, and promotes the taste and texture of a beer; WEAX with small molecular weight has the functions of anti-tumor and lowering blood pressure and is regarded as a prebiotic. Because WEAXs with different molecular weight and properties have different impacts on the beer brewing process and qualities of the final beer, it becomes more important to control the degradation of AX during the brewing process of a beer. Endo-1,4-β-xylanase (EC 3.2.1.8) is the most important AX degrading enzyme, which cleaves the β -xylosidic bond between two d-xylopyranosyl residues linked in β-(1,4). The study of enzymatic properties of endo-1,4-β-xylanase from wheat malt is very important for the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. Objective: In this article, our motivation is to study the enzymatic properties (including optimum pH and temperature, pH and temperature stability, the effect of inhibitors) of wheat malt endo-1,4-β-xylanase. Methods: In this article, we prepared crude enzyme according to the method of Guo with minor modifications. The endo-1,4-β-xylanase activity was determined according to the method of Biely in the previous report with minor modifications. The 0.5 mL crude enzyme sample was mixed with 0.5 mL 1 mg/mL 4-O-methyl-dglucurono- d-xylan dyed with Remazol Brilliant Blue R (RBBR-Xylan) solution, intensively mixed, and incubated at 40 °C for exactly 90 min. The reaction was stopped by precipitation using 2 mL absolute ethanol, and the reaction mixture was stirred acutely and placed at room temperature for 30 min. Then, the mixture was mixed again and centrifuged at 6000 g for 10 min. The supernatant was collected and the absorbance was measured at 590 nm. Absolute ethanol and RBBR-Xylan were added to the control tubes first, and after the reaction was completed, the crude enzyme sample was added. One unit of endo-1,4-β-xylanase was defined as at pH 5.5 and 40 °C liberate 1 μmol xylose equivalents in 1 min per g dry wheat malt. Results: The results showed that the optimal activity of endo-1,4-β-xylanase was achieved at pH 5.5-6.0, and the enzyme was extremely stable at pH 4.5, 5.5 and 6.5 after incubation for 30, 50 and 60 min, respectively. The optimal temperature was 40-45 °C and the deactivation temperature was 75 °C. Endo-1,4-β-xylanase was stable at 20 °C and 40 °C; the stability was slightly decreased at 50 °C and rapidly decreased at 55 °C. The enzyme activity was mildly inhibited by K+, Na+, and Pb2+, moderately inhibited by Ca2+, Mg2+ and Mn2+ and severely inhibited by Cu2+, Ag+ and EDTA. Conclusion: We have got the enzymatic properties of endo-1,4-β-xylanase from wheat malt, so during wort mashing, we could apply this research result to carry out the rational formulation of the content and molecular weight of WEAX in wort and beer during the mashing procedure when using wheat malt as the main raw materials. Expected to solve the technical problems such as high viscosity, slow filtration speed and so on, but also highlight the typical flavors of WEAX such as rich and persistent foam and mellow texture during the brewing process of a beer.