| Wheat and wheat malt are traditional raw materials for beer production.Arabinoxylan?AX?is the main non-starch polysaccharide?NSP?in wheat and wheat malt.In the beer production process,the molecular size and content of AX have an important influence on the viscosity,turbidity,filtration speed,foaming characteristics of wort and beer.Endo-?-1,4-xylanase is the key AX degrading enzyme.In this study,enzymatic properties,including optimum pH,optimum temperature,pH stability,temperature stability and effects of metal ions on wheat malt endo-?-1,4-xylanase were studied;an endo-?-1,4-xylanase was isolated,purified,and proteomics identified;and the degradation characteristics of the purified enzyme on wheat-derived AX were described.The main findings were as follows:?1?It was found that the optimum pH of the wheat malt endo-?-1,4-xylanase was 5.5 and6.0;the enzyme was stable at pH 4.5,5.5 and 6.5 after incubation for 30,50,and 60 min,respectively;the optimum temperature was 40°C and 45°C and the deactivation temperature was 75°C;the endo-1,4-?-xylanase was stable at 20°C and 40°C for 120 min,but the activity only remained 43.91%when incubated at 50°C for 60 min,the enzyme showed a great sensitivity at 55°C and 60°C.When the concentration was 10 mM/L,Cu2+,Ag+and EDTA had a severe inhibitory effect on the enzyme activity,Ca2+,Mg2+,Mn2+had a moderate inhibitory effect,and K+,Na+and Pb2+had a slightly inhibited effect.?2?A novel endo-1,4-?-xylanase was purified from wheat malt following the procedures of crude enzyme extraction,ammonium sulfate precipitation,cation-exchange chromatography,two-step anion-exchange chromatographies.The purified endo-1,4-?-xylanase had a specific activity of 3.94 u/mg,and the apparent relative molecular weight?Mr?of which was approximately 58000 Da.The purification fold was 9 and the recovery was 0.7%.The content of exo-xylanases,that were arabinofuranosidase and?-D-xylosidase,was very low.After LC-MS/MS identification,the purified enzyme had the highest matching degree with a Glycoside Hydrolase 10?GH10?domain-containing protein A0A3B6MY89 from wheat,there were 30 match peptides with a score above the threshold and the Prot-cover was 45.5%.?3?The purified endo-?-1,4-xylanase was a true endo-acting enzyme and had a significant degradation effect on the high viscosity wheat derived water extracted arabinoxylan?WEAX?.After 24 h degradation,the WEAX with a weight average molecular weight of 346 kDa was degraded to 126 kDa and the viscosity of the degradation system dropped from 1.30 mPa·s to 1.12 mPa·s.The average degree of polymerization?avDP?of WEAX decreased from 25.29?2 h?to 16.22.After scanning by electron microscope,WEAX was irregular in shape and compact in structure before degradation,and gradually became fluffy and loose after degradation,forming more flocculent substances.In addition,during the degradation process,a small amount of WEAX was degraded to arabinoxylan-oligosaccharides?AXOS?with a degree of polymerization of 26,but no monosaccharides were produced.The degradation rate was highest in the first 3.5 h.?4?The purified endo-?-1,4-xylanase also could degrade wheat-derived water-unextracted arabinoxylan?WUAX?.After 6 h of degradation,the content of WEAX in the reaction solution increased from 1.41?g/mL to 13.56?g/mL.Along with the degradation time,a relatively high amount of AXOS was produced,and it was speculated that the enzyme also had a degradation effect on oligosaccharides.The molecular weight of WEAX in the enzymatic hydrolysis product was between 23 and 27 kDa.After 2 h of reaction,the viscosity?1.06 mPa·s?of the degradation product increased significantly,and then the viscosity of the reaction solution decreased with the increase of degradation time.With the increase of shear rate,the viscosity of WEAX solution decreased and remained almost unchanged under the condition of shear rate after 30 s-1,which indicated that WEAX solution was a pseudoplastic fluid.The degradation rate was highest in the first 2 h. |
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