枯草芽孢杆菌对半纤维素的酶解和产酸性木寡糖研究

发布时间：2018-05-21 08:13

本文选题：枯草芽孢杆菌 + 木聚糖酶　；参考：《西北农林科技大学》2016年博士论文

【摘要】：枯草芽孢杆菌因具有较清晰的遗传背景和良好的胞外分泌性,并且其胞外分泌物无致病性,使其成为生物技术领域基础研究和工业应用研究中使用较多的重要菌种。枯草芽孢杆菌模式菌株168(Bacillus subtilis subsp.subtilis str.168)不仅能分泌木聚糖水解酶(Endo-1,4-β-xylanase A,Xyn A)和(Glucurono xylanase C,Xyn C),还能分泌阿拉伯呋喃糖水解酶(Arabinoxylan arabinofuranohydrolase,Axh43)。本文研究了枯草芽孢杆菌168分泌的水解酶Xyn A和Xyn C单独作用及协同作用下在枫香树甲基葡萄糖醛酸木聚糖(Methylglucuronoxylans,Me GXn)中的酶解作用;枯草芽孢杆菌衍生菌株(Bacillus subtilis MR42,MR44,MR45)在Me GXn中的生长情况及酶解产物,筛选出适合生产酸性木寡糖的菌株;Axh43在结构更为复杂的甜高粱秆阿拉伯糖甲基葡萄糖醛酸木聚糖(Methylglucuronoarabinoxylans,Me GAXn)中的酶解作用;筛选出的适合生产酸性木寡糖菌株在Me GAXn中的酶解产物结构。主要研究结果如下:1.重组表达的枯草芽孢杆菌168分泌的Xyn A和Xyn C在Me GXn中的酶解作用通过对重组表达的枯草芽孢杆菌168分泌的水解酶Xyn A和Xyn C单独作用及协同作用下在枫香树木聚糖(Me GXn)中的酶解作用的研究,结果表明:Xyn A和Xyn C由于自身酶解特性不一,对底物的结合特异性也有一定的差异,因此作用于Me GXn时可产生出不同长度的酸性木寡糖。借助薄层色谱法(TLC)、基质辅助激光解析电离飞行时间质谱法(MALDI-TOF-MS)与核磁共振氢谱法(1H NMR)对酶解得到的产物进行鉴定,结果表明Xyn A对底物的特异选择性较低,酶解出来的产物结构呈多样化,分别为中性木寡糖主要为木二糖(X2)和木三糖(X3)以及酸性木寡糖主要为甲基葡萄糖醛酸木四糖(Me GX4),并且甲基葡萄糖醛酸(Me G)侧链只连接在非还原端次位木糖上,证实Xyn A可以直接将木聚糖水解成短链的木二糖和木三糖,而当有Me G侧链出现的情况下,只能酶解特定的位点。Xyn C为具有高度特异性的酶,能识别Me G侧链,并只作用于连接着该基团木糖的靠近还原端的邻位木糖上,主要酶解产物为Me GX7,Me G侧链只连接在还原端次位木糖上,且不产生中性木寡糖,这也印证了其对底物的高度结合特异性,因此木聚糖水解酶Xyn C的酶解产物可控性较Xyn A的酶解产物可控性高。当Xyn A与Xyn C共同作用时,由Xyn A水解得到的Me GX4被Xyn C进一步水解而得到最终的酶解产物Me GX3,其中Me G侧链连接在中间的木糖上。2.生产酸性木寡糖菌株的筛选通过绘制枯草芽孢杆菌衍生菌株在Me GXn中的生长曲线并测定最终的总糖含量,借助TLC法、MALDI-TOF-MS法和1H NMR法对酶解产物进行鉴定,结果表明:枯草芽孢杆菌衍生菌株MR42和MR44在生长25 h后,菌液中总糖含量和主要产物有差异。菌株MR42(只分泌木聚糖水解酶Xyn A,xyn C基因受到干扰)在生长25 h后菌液中总糖含量为43%,其主要产物为Me GX4,Me G侧链连接在非还原端次位木糖上,与Xyn A酶解反应中的酶解产物一致;菌株MR44(只分泌木聚糖水解酶Xyn C,xyn A基因受到干扰)在生长25 h后总糖含量为75%,其主要产物为Me GX7,Me G侧链只连接在还原端次位木糖上,与Xyn C酶解反应中的酶解产物一致。对两个菌株总糖含量和产物结构的综合分析,筛选出菌株MR44用于酸性木寡糖的生产。3.重组表达的Xyn A,Xyn C和Axh43在Me GAXn中的酶解作用通过对重组表达的枯草芽孢杆菌168分泌的水解酶Xyn A,Xyn C和Axh43单独作用及协同作用下在甜高粱秆木聚糖(Methylglucuronoarabinoxylans,Me GAXn)中的酶解作用研究,利用TLC法和1H NMR法对酶解产物进行结构鉴定,结果表明:Axh43能直接释放出连接在木糖主链上的阿拉伯呋喃糖基团而不水解木糖主链,证实了其只具有水解阿拉伯呋喃糖侧链的酶解活性。而Xyn A与Axh43共同作用时,Axh43仍能释放阿拉伯呋喃糖,且Xyn A的水解产物则与以枫香树木聚糖为底物时的水解产物相似,主要为Me GX4,Me G侧链的连接位置在非还原端次位木糖。Xyn C与Axh43的共同水解产物则为分子量较大的酸性木寡糖,平均聚合度为12。4.枯草芽孢杆菌168衍生菌株MR44在Me GAXn中的酶解产物通过绘制MR44菌株在Me GAXn底物中的生长曲线并对最终产物进行总糖含量测定和结构鉴定,结果表明:在酶解反应48 h后,产物中总糖含量为70%,释放出的阿拉伯糖被细菌本身代谢。利用阴离子交换色谱柱(QAE Sephadex Q25)对菌液进行纯化得到酸性木寡糖,借助TLC法和1H NMR法对产物进行鉴定,发现产物的平均木糖单元长度约为12,平均每12个木糖单元只连接着一个Me G侧链,且Me G侧链只连接在还原端次位木糖上。本试验分析的木聚糖酶Xyn A和Xyn C以及阿拉伯呋喃糖水解酶Axh43的酶解作用及产物,有助于人们有针对性的应用它们对农林生物质进行降解而获得目的产物;筛选出的能生产特定长度的酸性木寡糖的枯草芽孢杆菌衍生菌株,为农林生物质的高值化利用提供了思路。
[Abstract]:Bacillus subtilis has a clear genetic background and good ecocytosis, and its exudates have no pathogenicity, making it a major strain used in basic research and industrial application research in the field of biotechnology. Bacillus subtilis model strain 168 (Bacillus subtilis subsp.subtilis str.168) can not only be divided into two strains. Endo-1,4- beta -xylanase A (Xyn A) and (Glucurono xylanase C, Xyn C) can also secrete the Arabia furanoglycidase (Arabinoxylan arabinofuranohydrolase). This paper studies the hydrolase of Bacillus subtilis 168 secreted by the hydrolase and its synergistic action in the maple tree methyl glucuronide The enzyme hydrolysis in Methylglucuronoxylans, Me GXn; the growth of Bacillus subtilis derived strain (Bacillus subtilis MR42, MR44, MR45) in Me GXn and the enzyme hydrolysates, screening the strain suitable for the production of acid oligosaccharides; Axh43 in the more complex structure of sweet sorghum stalk, Arabia sugar methyl glucuronide (Methylg) Enzyme hydrolysis in lucuronoarabinoxylans, Me GAXn, and the enzyme hydrolysate structure suitable for producing acid xylose oligosaccharides in Me GAXn. The main results are as follows: 1. the enzymatic hydrolysis of recombinant Bacillus subtilis 168 secreted by Bacillus subtilis and Xyn C in Me GXn through the water secreted by recombinant Bacillus subtilis 168 The enzymatic hydrolysis of enzyme Xyn A and Xyn C alone and in synergism in Maple gum tree polysaccharide (Me GXn) has been studied. The results show that Xyn A and Xyn C have some differences in the specificity of the substrate binding due to their different characteristics of their own enzymatic hydrolysis, so they can produce acid wood oligosaccharides with different lengths in Me GXn. Thin layer chromatography is used with the aid of thin layer chromatography. Method (TLC), matrix assisted laser analytical ionization time of flight mass spectrometry (MALDI-TOF-MS) and nuclear magnetic resonance spectroscopy (1H NMR) were used to identify the products obtained by enzymatic hydrolysis. The results showed that the specific selectivity of Xyn A to the substrate was low, and the structure of the hydrolysate was diversified, and the neutral wood oligosaccharides were mainly wood two sugar (X2) and wood three sugar (X3), respectively. And acid wood oligosaccharides are mainly methyl glucuronide four sugar (Me GX4), and methyl glucuronic acid (Me G) side chain is only connected to the non reductive secondary xylose. It is proved that Xyn A can hydrolyze xylan directly into short chain wood two sugar and wood three sugar. When the side chain of Me G appears, only the specific site.Xyn C is used as the tool. The highly specific enzyme can identify the Me G side chain and act only on the adjacent xylose adjacent to the reduced end of the group of xylose. The main hydrolysate is Me GX7, and the Me G side chain is connected only on the reducing end of the xylose, and does not produce neutral wood oligosaccharides, which also confirms the high binding specificity of the xylan to the substrate and thus the hydrolysis of xylan. The controllability of enzymatic hydrolysates of enzyme Xyn C is higher than that of Xyn A. When Xyn A is combined with Xyn C, the Me GX4, hydrolyzed by Xyn A, is further hydrolyzed to the final hydrolysate. The growth curve of bacteria derived strain in Me GXn and the final total sugar content were determined. The enzyme hydrolysates were identified by TLC, MALDI-TOF-MS and 1H NMR. The results showed that the total sugar content of Bacillus subtilis derived strain MR42 and MR44 was 25 h, and the total sugar content was different from that of the main products. Strain MR42 (only secreted xylan hydrolase Xyn) A, xyn C gene was disturbed) after growth of 25 h, the total sugar content was 43%, the main product was Me GX4, the Me G side chain was connected to the non reductive secondary xylose, which was consistent with the enzyme hydrolysis product in the Xyn A enzymatic hydrolysis reaction. The product is Me GX7, and the Me G side chain is connected only to the reduction of the secondary xylose, which is consistent with the hydrolysates in the enzymatic hydrolysis of Xyn C. A comprehensive analysis of the total sugar content and product structure of the two strains is used to screen out the Xyn A for the recombinant expression of.3. in the production of acid wood oligosaccharides. The enzymolysis of the hydrolase Xyn A, Xyn C and Axh43 secreted by Bacillus subtilis 168 in the sweet sorghum stalk xylan (Methylglucuronoarabinoxylans, Me GAXn) under the action and synergism of Bacillus subtilis 168 was studied. The structure of the hydrolysates was identified by TLC method and 1H NMR method. The results showed that Axh43 could directly release the main chain of xylose. The Arabia furan sugar group did not hydrolyze the main chain of xylose, which proved that it only had the enzymatic hydrolysis activity of the hydrolysis of Arabia furan side chain. While Xyn A and Axh43 combined, Axh43 could still release Arabia furan sugar, and the hydrolysates of Xyn A were similar to the hydrolysates of maple gum tree chitosan, mainly Me GX4, Me G side. The joint position of the chain at the non reductive end of the.Xyn C and Axh43 is a larger molecular weight of acid wood oligosaccharides. The average degree of polymerization is 12.4. of Bacillus subtilis 168 derived strain MR44 in Me GAXn by plotting the growth curve of the MR44 strain in the Me GAXn substrate and carrying out the total sugar content of the final product. The results showed that the total sugar content in the product was 70% after the enzymatic hydrolysis reaction of 48 h, and the released Arabia sugar was metabolize by bacteria itself. The acid wood oligosaccharides were purified by the anion exchange chromatography column (QAE Sephadex Q25). The products were identified by TLC and 1H NMR, and the average xylose of the products was found. The unit length is about 12, and the average 12 xylose units are only connected with one Me G side chain, and the Me G side chain is only connected to the reduction of the secondary xylose. The enzymatic hydrolysis of the xylanase Xyn A and Xyn C and the Axh43 hydrolysate of the Arabia furanolidase are analyzed by this test, which will help people to apply them to the agroforestry. The product of Bacillus subtilis, which can produce acid wood oligosaccharides of specific length, provides a way of thinking for the high value utilization of agroforestry.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93

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