【摘要】：木聚糖是一種天然的多糖,是纖維素的主要成分,在日常的生活中也比較常見的多糖,在農(nóng)作物如玉米芯中,玉米的秸稈等地方均有分布,是一種含量豐富,但是利用率還不是很好的多糖。其化學(xué)結(jié)構(gòu)相當(dāng)復(fù)雜,在不同物種中的結(jié)構(gòu)不同,甚至同一植株上的不同部位其結(jié)構(gòu)都是有所差異的。主要是木聚糖豐富的側(cè)鏈基團,也正是這些側(cè)鏈基團決定了其功能與結(jié)構(gòu)的多樣性,在側(cè)鏈基團取代的方面我們發(fā)現(xiàn),醚化,酯化,以及一些復(fù)合型的衍生物比較多。側(cè)鏈基團豐富,但其主鏈具有同源性,即其主鏈由β-1,4糖苷鍵連接D-木糖殘基而組成,一般情況下其主鏈的木糖殘基越少,支鏈越多,那么其溫度穩(wěn)定性越差,反之則穩(wěn)定性越好。木糖的溶解性方面比較特殊,一般情況下,木聚糖是溶于水的,但是有很多木聚糖并不溶于水,而是溶解于醇溶液中。這也是木聚糖分類的依據(jù)之一。作為一種天然的多糖,其水解產(chǎn)物的作用也越來越受到重視,特別是其徹底水解而生成的木糖分子,在醫(yī)療,食品等行業(yè)均有良好的發(fā)展前景,并且木聚糖相對易得,價格便宜,通常在一些農(nóng)作物的下腳料中即可獲得,是一種非常合適的生產(chǎn)木糖的原材料。木聚糖酶是一種糖苷水解酶,以GH10和GH11家族的木聚糖酶較多,也是現(xiàn)在普遍研究較多的。木聚糖酶是一種綜合的酶系,主要包括內(nèi)切β-1,4木聚糖酶,β-1,4木糖苷酶,以及可以使寡聚木糖徹底水解生產(chǎn)木糖的外切β1,4木聚糖酶,其中狹義的木聚糖酶就僅指內(nèi)切β-1,4木聚糖酶,作為糖苷水解酶家族中的一員,木聚糖酶有著很強的水解木聚糖的能力,在生產(chǎn)生活中的應(yīng)用十分廣泛,如造紙行業(yè),紙張的脫墨行業(yè),醫(yī)療行業(yè),食品加工行業(yè)等都有應(yīng)用,甚至在釀酒行業(yè)也有其用武之地,但是由于造價高,應(yīng)用受到了很大方面的限制�；蚬こ碳夹g(shù)對尋找良好的木聚糖酶野生菌是有利的條件,拿以往的科研工作來看,來源菌株大多是自然突變或者野生菌株,基因工程技術(shù)的引用使得可以利用人工的方法進行菌株的突變從而得到突變菌種進而增大產(chǎn)量的目的。本文實驗的目的是篩選出更加優(yōu)秀的菌株,希望能夠通過異源表達的過程提高木聚糖酶的產(chǎn)量,通過真核生物的蛋白修飾作用,其pH與溫度的穩(wěn)定性也能夠有所提高。本文的實驗方法是通過ICBI數(shù)據(jù)庫查閱資料,將來源于Halopiger xanaduesis的木聚糖酶基因通過PTDS法人工合成,利用畢赤酵母的密碼子偏愛性以及木聚糖酶的蛋白序列構(gòu)造表達載體。構(gòu)建的分泌表達載體經(jīng)過BglⅡ酶切,電擊轉(zhuǎn)化到畢赤酵母GS115中,并篩選得到陽性克隆。從最適pH、pH穩(wěn)定性、最適溫度、溫度穩(wěn)定性以及金屬離子和部分化合物對酶的性質(zhì)的影響等方面做了研究。實驗結(jié)果表明:木聚糖酶最大反應(yīng)速率為219μmol/min·mg,純化后酶液濃度為200μg/m L。酶液的Km值為2.2 mg/ml。重組木聚糖酶最適pH值為6.0,pH在4.5-8.0之間酶學(xué)性質(zhì)穩(wěn)定,酶的最適溫度是60℃,在溫度為20℃-65℃之間酶學(xué)性質(zhì)穩(wěn)定,金屬離子Mn2+與Cu2+對酶有抑制作用,Fe2+,Cr2+,和化合物SDS、DTT對酶有明顯促進作用。
[Abstract]:Xylan is a natural polysaccharide, is a main component of cellulose, and is also a common polysaccharide in daily life. The chemical structure of the plant is quite complex, and the structure in different species is different, and even the structure of different parts on the same plant is different. The major is the abundant side chain group of the xylan, and it is that these side chain groups determine the diversity of their function and structure, and in the aspect of the substitution of the side chain groups, we find that the etherification, the esterification, and some complex derivatives are more. The side chain group is rich, but its main chain has the homology, that is, its main chain is composed of the D-xylose residues linked by the 1-and 4-sugar chain bonds, the less the xylose residue in the main chain, the more the branched chain is, the worse the temperature stability, and the better the stability. The solubility of the xylose is more specific, in general, the xylan is water-soluble, but a lot of xylan is insoluble in water, but is dissolved in the alcohol solution. This is also a basis for the classification of xylan. As a natural polysaccharide, the effect of the hydrolysis product of the polysaccharide is also more and more important, in particular to the xylose molecule produced by the complete hydrolysis of the polysaccharide, and has a good development prospect in the fields of medical and food and the like, and the xylan is relatively easy to obtain and the price is low, It is usually obtained in the leftovers of some crops and is a very suitable raw material for the production of xylose. Xylanase is a glycosidase, and the xylanase of the family of GH10 and GH11 is more and more common. Xylanase is an integrated enzyme system, which mainly comprises endo-1,4-xylanase,1-1,4-xylanase, and exonuclease 1 and 4 xylanase capable of thoroughly hydrolyzing the oligoxylose to produce xylose, wherein the narrow xylanase is only the endo-1,4-xylanase, As a member of the family of the glyoxylases, the xylanase has strong ability to hydrolyze the xylan, and has wide application in the production and the life, such as the paper-making industry, the deinking industry of the paper, the medical industry, the food processing industry and the like, Even in the wine-making industry, there is a place to use it, but because of the high cost, the application is limited greatly. The genetic engineering technology is an advantageous condition for finding a good xylanase wild fungus, and in the past scientific research, the source strain is mostly a natural mutation or a wild strain, The reference of the gene engineering technique makes it possible to make the mutation of the strain by the artificial method so as to obtain the mutant strain and to increase the yield. The purpose of this paper is to screen out a more excellent strain, and it is hoped that the yield of the xylanase can be improved by the process of heterologous expression, and the stability of the pH and the temperature can be improved by the protein modification of the eukaryote. The experimental method of this paper is to check the data through the ICBI database, and the xylanase gene derived from Halopiger xanadu is artificially synthesized by the PTDS method, and the expression vector is constructed by the codon preference of the Pichia pastoris and the protein sequence of the xylanase. The constructed secretory expression vector was cut by Bgl II enzyme, and the electric shock was transformed into Pichia pastoris GS115, and a positive clone was obtained. The optimum pH, the stability of the pH, the optimum temperature, the temperature stability and the effect of the metal ions and some of the compounds on the properties of the enzyme were studied. The results showed that the maximum reaction rate of xylanase was 219. m u.mol/ min 路 mg, and the concentration of the enzyme solution after purification was 200. m u.g/ ml. The Km value of the enzyme solution was 2.2 mg/ ml. the optimum pH value of the recombinant xylanase is 6.0, the pH value is between 4.5 and 8.0, the enzymatic property is stable, the optimal temperature of the enzyme is 60 DEG C, the enzymatic property is stable between 20 DEG C and 65 DEG C, the metal ion Mn2 + and the Cu2 + have the inhibition effect on the enzyme, and the Fe2 +, Cr2 + and the compound SDS, DTT has a significant effect on the enzyme.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q78;Q55

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