本文關(guān)鍵詞：小麥芽β-1,4-木聚糖內(nèi)切酶的分離純化及其對植物源阿拉伯木聚糖的降解作用　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小麥芽 β-1 4-木聚糖內(nèi)切酶 分離純化 LC-MS/MS 阿拉伯木聚糖 粘度

【摘要】：小麥芽富含阿拉伯木聚糖(AX),AX及酶解產(chǎn)物分子大小及結(jié)構(gòu)會對麥汁及啤酒的粘度、濁度、過濾速度等產(chǎn)生重要影響,還具有抗腫瘤、提高免疫力等保健作用。β-1,4-木聚糖內(nèi)切酶是降解AX主鏈改變AX溶解度、粘度、濁度等特性的關(guān)鍵酶。本文對小麥芽中β-1,4-木聚糖內(nèi)切酶進行了分離純化,并研究了β-1,4-木聚糖內(nèi)切酶對植物源阿拉伯木聚糖的降解作用。主要研究結(jié)果如下:(1)經(jīng)硫酸銨沉淀(ASI)、陰離子交換層析、疏水交換層析(HICIII)、凝膠過濾層析,獲得了分子量為27.8 kDa,SDS-PAGE電泳條帶單一的β-1,4-木聚糖內(nèi)切酶(E-WMA)。純化倍數(shù)為12.08倍,比活力為4.47 u/mg,回收率為1.45%。(2)液相串聯(lián)質(zhì)譜分析發(fā)現(xiàn)分子量為14.0 kDa的SDS-PAGE電泳條帶也存在與β-1,4-木聚糖內(nèi)切酶相匹配的肽段,小麥芽中可能存在分子量為14.0 kDa的β-1,4-木聚糖內(nèi)切酶。(3)在40℃將硫酸銨沉淀后的β-1,4-木聚糖內(nèi)切酶(簡稱ASI)作用于純小麥芽麥醪,與對照相比,麥汁濁度明顯降低,在30 min、90 min、120 min分別降低了7.16%、38.01%和22.22%;水溶性阿拉伯木聚糖(WEAX)含量增高;重均分子量為1.85×10~3 Da的組分含量變化最顯著,作用時間低于30 min時將水不溶的阿拉伯木聚糖(WUAX)轉(zhuǎn)化為重均分子量為1.85×10~3 Da的WEAX的速率占主導(dǎo),高于30 min降解1.85×10~3Da的WEAX的速率占主導(dǎo)作用。(4)在純小麥芽協(xié)定糖化初始階段加入ASI,降低了終麥汁過濾時間;但麥汁粘度沒有明顯變化;ASI使?jié)岫冉档?0.36%~50.00%。ASI添加量低于0.9 mg/mL時,將WUAX轉(zhuǎn)化為重均分子量1.92×10~3 Da的WEAX的速率占主導(dǎo);添加量高于0.9 mg/mL時,降解1.92×10~3 Da的WEAX的速率占主導(dǎo)。(5)疏水層析后得到的β-1,4-木聚糖內(nèi)切酶(簡稱HICIII)可將玉米芯中WUAX轉(zhuǎn)化成WEAX,主要產(chǎn)物分子量為1.54×106 Da。HICIII對甘蔗渣中AX有顯著降解作用,可將甘蔗渣中WUAX轉(zhuǎn)化為WEAX,還可將WEAX中組分1.20×106 Da降解為組分2.81×104 Da和組分2.18×10~3 Da。(6)E-WMA對低中高粘度的小麥源WEAX都有降解作用,對高粘度的WEAX降解效果最為顯著。三種WEAX溶液的濃度為2 mg/m L,高粘度WEAX粘度為2.42 mPa·s,酶解后粘度下降9.92%,Mw由2.07×106 Da的組分降解為3.37×105 Da的組分;低粘度和高粘度WEAX粘度分別為1.77 mPa·s和2.09 mPa·s,酶解后粘度分別下降了5.65%和8.13%。此外,E-WMA可將小麥源的WUAX轉(zhuǎn)化成WEAX,體系粘度增高了1.89%;E-WMA還可以將轉(zhuǎn)化成的WEAX進一步降解,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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