来源于Halopiger xanaduensis的木聚糖醇基因在毕赤酵母中诱导表达及其酶学性质的研究

发布时间：2019-06-14 04:43

【摘要】：木聚糖是一种天然的多糖,是纤维素的主要成分,在日常的生活中也比较常见的多糖,在农作物如玉米芯中,玉米的秸秆等地方均有分布,是一种含量丰富,但是利用率还不是很好的多糖。其化学结构相当复杂,在不同物种中的结构不同,甚至同一植株上的不同部位其结构都是有所差异的。主要是木聚糖丰富的侧链基团,也正是这些侧链基团决定了其功能与结构的多样性,在侧链基团取代的方面我们发现,醚化,酯化,以及一些复合型的衍生物比较多。侧链基团丰富,但其主链具有同源性,即其主链由β-1,4糖苷键连接D-木糖残基而组成,一般情况下其主链的木糖残基越少,支链越多,那么其温度稳定性越差,反之则稳定性越好。木糖的溶解性方面比较特殊,一般情况下,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。这也是木聚糖分类的依据之一。作为一种天然的多糖,其水解产物的作用也越来越受到重视,特别是其彻底水解而生成的木糖分子,在医疗,食品等行业均有良好的发展前景,并且木聚糖相对易得,价格便宜,通常在一些农作物的下脚料中即可获得,是一种非常合适的生产木糖的原材料。木聚糖酶是一种糖苷水解酶,以GH10和GH11家族的木聚糖酶较多,也是现在普遍研究较多的。木聚糖酶是一种综合的酶系,主要包括内切β-1,4木聚糖酶,β-1,4木糖苷酶,以及可以使寡聚木糖彻底水解生产木糖的外切β1,4木聚糖酶,其中狭义的木聚糖酶就仅指内切β-1,4木聚糖酶,作为糖苷水解酶家族中的一员,木聚糖酶有着很强的水解木聚糖的能力,在生产生活中的应用十分广泛,如造纸行业,纸张的脱墨行业,医疗行业,食品加工行业等都有应用,甚至在酿酒行业也有其用武之地,但是由于造价高,应用受到了很大方面的限制。基因工程技术对寻找良好的木聚糖酶野生菌是有利的条件,拿以往的科研工作来看,来源菌株大多是自然突变或者野生菌株,基因工程技术的引用使得可以利用人工的方法进行菌株的突变从而得到突变菌种进而增大产量的目的。本文实验的目的是筛选出更加优秀的菌株,希望能够通过异源表达的过程提高木聚糖酶的产量,通过真核生物的蛋白修饰作用,其pH与温度的稳定性也能够有所提高。本文的实验方法是通过ICBI数据库查阅资料,将来源于Halopiger xanaduesis的木聚糖酶基因通过PTDS法人工合成,利用毕赤酵母的密码子偏爱性以及木聚糖酶的蛋白序列构造表达载体。构建的分泌表达载体经过BglⅡ酶切,电击转化到毕赤酵母GS115中,并筛选得到阳性克隆。从最适pH、pH稳定性、最适温度、温度稳定性以及金属离子和部分化合物对酶的性质的影响等方面做了研究。实验结果表明:木聚糖酶最大反应速率为219μmol/min·mg,纯化后酶液浓度为200μg/m L。酶液的Km值为2.2 mg/ml。重组木聚糖酶最适pH值为6.0,pH在4.5-8.0之间酶学性质稳定,酶的最适温度是60℃,在温度为20℃-65℃之间酶学性质稳定,金属离子Mn2+与Cu2+对酶有抑制作用,Fe2+,Cr2+,和化合物SDS、DTT对酶有明显促进作用。
[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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78;Q55

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