小麦芽β-1,4-木聚糖内切酶的分离纯化及其对植物源阿拉伯木聚糖的降解作用

发布时间：2018-01-16 00:12

  本文关键词：小麦芽β-1,4-木聚糖内切酶的分离纯化及其对植物源阿拉伯木聚糖的降解作用　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 小麦芽 β-1 4-木聚糖内切酶 分离纯化 LC-MS/MS 阿拉伯木聚糖 粘度

【摘要】：小麦芽富含阿拉伯木聚糖(AX),AX及酶解产物分子大小及结构会对麦汁及啤酒的粘度、浊度、过滤速度等产生重要影响,还具有抗肿瘤、提高免疫力等保健作用。β-1,4-木聚糖内切酶是降解AX主链改变AX溶解度、粘度、浊度等特性的关键酶。本文对小麦芽中β-1,4-木聚糖内切酶进行了分离纯化,并研究了β-1,4-木聚糖内切酶对植物源阿拉伯木聚糖的降解作用。主要研究结果如下:(1)经硫酸铵沉淀(ASI)、阴离子交换层析、疏水交换层析(HICIII)、凝胶过滤层析,获得了分子量为27.8 kDa,SDS-PAGE电泳条带单一的β-1,4-木聚糖内切酶(E-WMA)。纯化倍数为12.08倍,比活力为4.47 u/mg,回收率为1.45%。(2)液相串联质谱分析发现分子量为14.0 kDa的SDS-PAGE电泳条带也存在与β-1,4-木聚糖内切酶相匹配的肽段,小麦芽中可能存在分子量为14.0 kDa的β-1,4-木聚糖内切酶。(3)在40℃将硫酸铵沉淀后的β-1,4-木聚糖内切酶(简称ASI)作用于纯小麦芽麦醪,与对照相比,麦汁浊度明显降低,在30 min、90 min、120 min分别降低了7.16%、38.01%和22.22%;水溶性阿拉伯木聚糖(WEAX)含量增高;重均分子量为1.85×10~3 Da的组分含量变化最显著,作用时间低于30 min时将水不溶的阿拉伯木聚糖(WUAX)转化为重均分子量为1.85×10~3 Da的WEAX的速率占主导,高于30 min降解1.85×10~3Da的WEAX的速率占主导作用。(4)在纯小麦芽协定糖化初始阶段加入ASI,降低了终麦汁过滤时间;但麦汁粘度没有明显变化;ASI使浊度降低30.36%~50.00%。ASI添加量低于0.9 mg/mL时,将WUAX转化为重均分子量1.92×10~3 Da的WEAX的速率占主导;添加量高于0.9 mg/mL时,降解1.92×10~3 Da的WEAX的速率占主导。(5)疏水层析后得到的β-1,4-木聚糖内切酶(简称HICIII)可将玉米芯中WUAX转化成WEAX,主要产物分子量为1.54×106 Da。HICIII对甘蔗渣中AX有显著降解作用,可将甘蔗渣中WUAX转化为WEAX,还可将WEAX中组分1.20×106 Da降解为组分2.81×104 Da和组分2.18×10~3 Da。(6)E-WMA对低中高粘度的小麦源WEAX都有降解作用,对高粘度的WEAX降解效果最为显著。三种WEAX溶液的浓度为2 mg/m L,高粘度WEAX粘度为2.42 mPa·s,酶解后粘度下降9.92%,Mw由2.07×106 Da的组分降解为3.37×105 Da的组分;低粘度和高粘度WEAX粘度分别为1.77 mPa·s和2.09 mPa·s,酶解后粘度分别下降了5.65%和8.13%。此外,E-WMA可将小麦源的WUAX转化成WEAX,体系粘度增高了1.89%;E-WMA还可以将转化成的WEAX进一步降解,其Mw下降20.77%。
[Abstract]:Wheat sprouts are rich in arabinoxylum axon ax and the molecular size and structure of enzymatic hydrolysis products will have important effects on the viscosity turbidity and filtration speed of wort and beer. It also has anti-tumor effect. 尾 -1N 4-xylan endonuclease is the key enzyme to change the solubility, viscosity and turbidity of ax in the degradation of ax. 4xylan endonuclease was isolated and purified, and 尾 -1 was studied. The degradation effect of 4-xylan endonuclease on arabinoxylans from plants. The main results are as follows: 1) precipitation of ASIN by ammonium sulfate and anion exchange chromatography. Hydrophobic exchange chromatography (HICIII) and gel filtration chromatography were used to obtain a single 尾 -1 electrophoresis band with molecular weight of 27.8 kg / d SDS-PAGE. The purification times and specific activity of 4-xylan endonuclease E WMAA were 12.08 times and 4.47 u / mg respectively. The recovery of SDS-PAGE was 1.45 and 1.45%. The SDS-PAGE electrophoresis band with molecular weight of 14.0 kDa was also found to be 尾 -1 by tandem mass spectrometry (LC-MS). The peptide segment of 4-xylan endonuclease, 尾 -1N 4xylan4xylanase with molecular weight of 14.0 kDa, may exist in wheat bud. 尾 -1 after precipitation of ammonium sulfate at 40 鈩，

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