Purification And Characterisation Of β-Xylosidase In Wheat Bran

β-Xylosidase is one of the important enzyme of xylanase family, it has been widely usedin many fields. The research about it mostly concentrated on microrganisms source, this studyanalysised and determined the endogenous β-xylosidase in the Chinese wheat bran bypurificating and determining its enzymatic properties, which can provide a theoreticalreference for the mechanism of xylanase to AX and dough.First, the enzyme activity and enzymatic properties of β-xylosidase in different varietiesof wheat bran had been determined. Results showed that: the enzyme activity was different indifferent varieties of wheat bran, enzyme activity between coarse bran and fine bran in thesame kind wheat was also different; the enzyme activity in fine bran was higher than those incoarse bran. The optimal temperature and optimal pH value were50℃or60℃,5,respectively; it had a better thermal stability between2050℃or2060℃; In addition, theresidual activity was80%during the pH value in the range56. Ca2+, Co2+promoted theenzyme activity, however, Ag+inhibited the enzyme activity.Second, this article optimized the extraction conditions of β-xylosidase by use ofresponse surface methodology. Based on the single factor experiments, it designed theresponse surface experiments according to Box-Benhnken Design method by takingextraction pH, material-to-water ratio, extraction time as independent variables and the yieldof endogenous β-xylosidase as response value, carried out significance and interactionanalysis to determine the optimum extraction conditions, and verified the results. The resultsshowed that the yield of endogenous β-xylosidase could reach84.19mU/g under the optimumextraction conditions by pH value4.93, material-to-water ratio1:9.85(g/mL), extractiontime36.85min, and the result was very close to the predicted value optimized by responsesurface fitting.Third, the β-xylosidase of coarse wheat bran of Zhengmai8998was purified by fivepurification steps including ammonium sulfate precipitation, heat treatment, CM SepharoseFF gel filtration, Mono-S cation exchange chromatography, Mono-Q anion exchangechromatography. The enzyme activity reached230.75mU per mg protein and the recovery was2.74%by purifieding480.82fold. The molecular weight of the β-xylosidase was about40kD by SDS-PAGE.Fourth, the analysis of the enzymatic properties of the purified β-xylosidase showed thatthe optimal temperature and optimal pH were60℃and5, respectively, and it had a betterthermal stability between2050℃, the residual activity can be maintained more than70%, inaddition, the residual activity was80%during the pH value in the range56. Ca2+, Co2+ionspromoted the enzyme activity of5.83%and9.51%, respectively; Cu2+, Ni+, SDS inhibited theenzyme activity, Ag+decreased the enzyme activity to18.94%. The kinetic parameters KmandVmaxwere4.518mg/mL,0.392μmol/min·g-1respectively.Finally, it explored the changing of endogenous β-xylosidase activity in wheat branunder wheat germination. The study found that with increasing germination time, total AXcontent of different varieties coarse wheat bran and fine bran displayed a downward trend, theβ-xylosidase activity showed first increase and then decline. After germination84or96h,β-xylosidase had a highest enzyme activity, while after germination84or96h, β-xylosidaseactivity began to decline.