本文選題：枯草芽孢桿菌 + 木聚糖酶��； 參考：《西北農(nóng)林科技大學(xué)》2016年博士論文

【摘要】：枯草芽孢桿菌因具有較清晰的遺傳背景和良好的胞外分泌性,并且其胞外分泌物無致病性,使其成為生物技術(shù)領(lǐng)域基礎(chǔ)研究和工業(yè)應(yīng)用研究中使用較多的重要菌種�？莶菅挎邨U菌模式菌株168(Bacillus subtilis subsp.subtilis str.168)不僅能分泌木聚糖水解酶(Endo-1,4-β-xylanase A,Xyn A)和(Glucurono xylanase C,Xyn C),還能分泌阿拉伯呋喃糖水解酶(Arabinoxylan arabinofuranohydrolase,Axh43)。本文研究了枯草芽孢桿菌168分泌的水解酶Xyn A和Xyn C單獨(dú)作用及協(xié)同作用下在楓香樹甲基葡萄糖醛酸木聚糖(Methylglucuronoxylans,Me GXn)中的酶解作用;枯草芽孢桿菌衍生菌株(Bacillus subtilis MR42,MR44,MR45)在Me GXn中的生長情況及酶解產(chǎn)物,篩選出適合生產(chǎn)酸性木寡糖的菌株;Axh43在結(jié)構(gòu)更為復(fù)雜的甜高粱稈阿拉伯糖甲基葡萄糖醛酸木聚糖(Methylglucuronoarabinoxylans,Me GAXn)中的酶解作用;篩選出的適合生產(chǎn)酸性木寡糖菌株在Me GAXn中的酶解產(chǎn)物結(jié)構(gòu)。主要研究結(jié)果如下:1.重組表達(dá)的枯草芽孢桿菌168分泌的Xyn A和Xyn C在Me GXn中的酶解作用通過對重組表達(dá)的枯草芽孢桿菌168分泌的水解酶Xyn A和Xyn C單獨(dú)作用及協(xié)同作用下在楓香樹木聚糖(Me GXn)中的酶解作用的研究,結(jié)果表明:Xyn A和Xyn C由于自身酶解特性不一,對底物的結(jié)合特異性也有一定的差異,因此作用于Me GXn時(shí)可產(chǎn)生出不同長度的酸性木寡糖。借助薄層色譜法(TLC)、基質(zhì)輔助激光解析電離飛行時(shí)間質(zhì)譜法(MALDI-TOF-MS)與核磁共振氫譜法(1H NMR)對酶解得到的產(chǎn)物進(jìn)行鑒定,結(jié)果表明Xyn A對底物的特異選擇性較低,酶解出來的產(chǎn)物結(jié)構(gòu)呈多樣化,分別為中性木寡糖主要為木二糖(X2)和木三糖(X3)以及酸性木寡糖主要為甲基葡萄糖醛酸木四糖(Me GX4),并且甲基葡萄糖醛酸(Me G)側(cè)鏈只連接在非還原端次位木糖上,證實(shí)Xyn A可以直接將木聚糖水解成短鏈的木二糖和木三糖,而當(dāng)有Me G側(cè)鏈出現(xiàn)的情況下,只能酶解特定的位點(diǎn)。Xyn C為具有高度特異性的酶,能識別Me G側(cè)鏈,并只作用于連接著該基團(tuán)木糖的靠近還原端的鄰位木糖上,主要酶解產(chǎn)物為Me GX7,Me G側(cè)鏈只連接在還原端次位木糖上,且不產(chǎn)生中性木寡糖,這也印證了其對底物的高度結(jié)合特異性,因此木聚糖水解酶Xyn C的酶解產(chǎn)物可控性較Xyn A的酶解產(chǎn)物可控性高。當(dāng)Xyn A與Xyn C共同作用時(shí),由Xyn A水解得到的Me GX4被Xyn C進(jìn)一步水解而得到最終的酶解產(chǎn)物Me GX3,其中Me G側(cè)鏈連接在中間的木糖上。2.生產(chǎn)酸性木寡糖菌株的篩選通過繪制枯草芽孢桿菌衍生菌株在Me GXn中的生長曲線并測定最終的總糖含量,借助TLC法、MALDI-TOF-MS法和1H NMR法對酶解產(chǎn)物進(jìn)行鑒定,結(jié)果表明:枯草芽孢桿菌衍生菌株MR42和MR44在生長25 h后,菌液中總糖含量和主要產(chǎn)物有差異。菌株MR42(只分泌木聚糖水解酶Xyn A,xyn C基因受到干擾)在生長25 h后菌液中總糖含量為43%,其主要產(chǎn)物為Me GX4,Me G側(cè)鏈連接在非還原端次位木糖上,與Xyn A酶解反應(yīng)中的酶解產(chǎn)物一致;菌株MR44(只分泌木聚糖水解酶Xyn C,xyn A基因受到干擾)在生長25 h后總糖含量為75%,其主要產(chǎn)物為Me GX7,Me G側(cè)鏈只連接在還原端次位木糖上,與Xyn C酶解反應(yīng)中的酶解產(chǎn)物一致。對兩個(gè)菌株總糖含量和產(chǎn)物結(jié)構(gòu)的綜合分析,篩選出菌株MR44用于酸性木寡糖的生產(chǎn)。3.重組表達(dá)的Xyn A,Xyn C和Axh43在Me GAXn中的酶解作用通過對重組表達(dá)的枯草芽孢桿菌168分泌的水解酶Xyn A,Xyn C和Axh43單獨(dú)作用及協(xié)同作用下在甜高粱稈木聚糖(Methylglucuronoarabinoxylans,Me GAXn)中的酶解作用研究,利用TLC法和1H NMR法對酶解產(chǎn)物進(jìn)行結(jié)構(gòu)鑒定,結(jié)果表明:Axh43能直接釋放出連接在木糖主鏈上的阿拉伯呋喃糖基團(tuán)而不水解木糖主鏈,證實(shí)了其只具有水解阿拉伯呋喃糖側(cè)鏈的酶解活性。而Xyn A與Axh43共同作用時(shí),Axh43仍能釋放阿拉伯呋喃糖,且Xyn A的水解產(chǎn)物則與以楓香樹木聚糖為底物時(shí)的水解產(chǎn)物相似,主要為Me GX4,Me G側(cè)鏈的連接位置在非還原端次位木糖。Xyn C與Axh43的共同水解產(chǎn)物則為分子量較大的酸性木寡糖,平均聚合度為12。4.枯草芽孢桿菌168衍生菌株MR44在Me GAXn中的酶解產(chǎn)物通過繪制MR44菌株在Me GAXn底物中的生長曲線并對最終產(chǎn)物進(jìn)行總糖含量測定和結(jié)構(gòu)鑒定,結(jié)果表明:在酶解反應(yīng)48 h后,產(chǎn)物中總糖含量為70%,釋放出的阿拉伯糖被細(xì)菌本身代謝。利用陰離子交換色譜柱(QAE Sephadex Q25)對菌液進(jìn)行純化得到酸性木寡糖,借助TLC法和1H NMR法對產(chǎn)物進(jìn)行鑒定,發(fā)現(xiàn)產(chǎn)物的平均木糖單元長度約為12,平均每12個(gè)木糖單元只連接著一個(gè)Me G側(cè)鏈,且Me G側(cè)鏈只連接在還原端次位木糖上。本試驗(yàn)分析的木聚糖酶Xyn A和Xyn C以及阿拉伯呋喃糖水解酶Axh43的酶解作用及產(chǎn)物,有助于人們有針對性的應(yīng)用它們對農(nóng)林生物質(zhì)進(jìn)行降解而獲得目的產(chǎn)物;篩選出的能生產(chǎn)特定長度的酸性木寡糖的枯草芽孢桿菌衍生菌株,為農(nóng)林生物質(zhì)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q93

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