【作者】 徐顺清；

【导师】 张忠明；

【作者基本信息】 华中农业大学 ， 微生物学， 2009， 硕士

【摘要】 木聚糖广泛存在于植物细胞壁中,是自然界中含量仅次于纤维素的可再生生物资源。木聚糖的结构复杂,降解木聚糖酶需要多种水解酶的共同参与,其中木聚糖酶是降解木聚糖最关键的酶类,它能够作用于木聚糖分子中的β-1,4糖苷键,产生不同长度的木寡糖和少量的木糖,从而生成可以再次利用的小分子物质。为了利用丰富的木聚糖资源,就必须获得性质优良的木聚糖酶。本文以黑曲霉（Aspergillus niger）F19来源的野生型木聚糖酶基因xylB和突变的木聚糖酶基因ST7(Ser³³和Thr¹⁸⁶替换为为Cys,以此来引入1个二硫键,同时将酶表面的Ser³⁹、Ser⁵⁴、Thr¹⁰¹、Thr¹⁰⁵和Thr¹⁵¹替换为Arg)为研究对象,研究了xylB和ST7基因在毕赤酵母中的表达、重组木聚糖酶的纯化及其酶学性质,探索了突变体酶对热稳定性的影响,并对突变体木聚糖酶重组毕赤酵母的诱导条件进行了优化研究。主要结论如下:1.野生型木聚糖酶基因xylB和突变体木聚糖酶基因ST7在毕赤酵母中的表达将木聚糖酶基因xylB和ST7克隆至毕赤酵母表达载体pPIC9K,重组表达载体转化毕赤酵母GS115,经过甲醇诱导,木聚糖酶基因都获得特异性表达,而且木聚糖酶能够分泌到培养基中,筛选到两株高酶活性的重组毕赤酵母,野生型和突变体木聚糖酶重组毕赤酵母的酶活性分别达到82.9 U/ml和390.2 U/ml。2.野生型木聚糖酶和突变体木聚糖酶的纯化利用黑曲霉木聚糖酶可以与木聚糖进行特异性结合的特性,对木聚糖酶进行分离纯化,纯化的木聚糖酶在SDS-PAGE上均呈现出单一的蛋白条带,表明木聚糖酶得到纯化。3.野生型木聚糖酶和突变体木聚糖酶的酶学性质分析与野生型木聚糖酶相比,突变体木聚糖酶的最适反应温度从45℃提高到50℃,50℃时酶的半衰期从小于10 min提高到120 min,而且酶反应过程中生成物积累量的幅度从36.9%提高到276.3%,酶的热稳定性有了较大的提高。突变体木聚糖酶的比酶活性从2127.9±83.9 U/mg提高到3330.9±173.4 U/mg,K_m值从56.9 mg/ml降低到37.2 mg/ml,酶与底物的亲和性有较大提高。但V_max从82.9 mmol/ml.min.mg降低至27.0 mmol/ml.min.mg。4.突变体木聚糖酶重组酵母诱导条件的优化采用四因素三水平的正交设计试验研究了甲醇加入量、起始细胞浓度、pH和诱导时间四个因素对重组酵母产酶的影响,四个因素对产酶的影响大小为:甲醇加入量＞诱导时间＞pH＞起始细胞浓度。最佳的诱导条件为:BYPN培养基（pH 8.0）,每隔12 h加入1%甲醇,起始细胞浓度OD₆₀₀=3,诱导培养7d,在此条件下对重组酵母进行诱导培养,产酶水平可达到1850.6±25.2 U/ml。目前本实验室正在进行利用生物发酵罐对突变体木聚糖酶重组毕赤酵母进行诱导产酶的研究,以期提高木聚糖酶的产酶水平。更多还原

【Abstract】 Xylan which is mainly found in plant cell wall is the most abundant regenerate resource secondary to cellulose. For complicated structure of xylan , the degradation of it needs many kinds of hydrolases. Xylanase is the key hydrolases for degradation of xylan. Xylanase can catalyze the hydrolysis ofβ-1,4 xylose linkage of xylan into xylooligosaccharides and xylose. With this catalyze procedure, micromolecul carbohydrate which can be reused is produced. For exploitation of xylan resource, we must get xylanase with good property.In this dissertation, native xylanase xylB and mutational xylanase ST7(Ser³³ and Thr¹⁸⁶ were substituted with Cys. In order to introduce a disulfide bridge into catalytic domain. Ser³⁹、Ser⁵⁴、Thr¹⁰¹、Thr¹⁰⁵ and Thr¹⁵¹ were substituted with Arg.) were our research objects. xylB and ST7 gene were expressed in Pichia pastoris GS115. Native xylanase and mutational xylanase were purified. Characterization and thermostability of recombinant xylanase were researched. Inducing condition of mutational xylanase recombined Pichia pastoris was optimized, and the results showed that:1. Expression of native xylanase xylB and mutational xylanase ST7 in Pichia pastorisxylB and ST7 were cloned into Pichia pastoris expression vector pPIC9K, then transferd them into Pichia pastoris GS115. Xylanase were successfully expressed after inducing with methanol. Xylanase were secreted into medium. We got two recombinants with high xylanase activity, xylanase activity can reach 82.9 U/ml and 390.2 U/ml.2. Purification of native xylanase and mutational xylanaseWith the knowledge that some xylanase can bind xylan specific, Aspergillus niger xylanase were purified with xylan. Single protein band were found in the SDS-PAGE , demonstrated that xylanase were purified. 3. Characterization of native xylanase and mutational xylanaseCompared with native xylanase, optimal reaction temperature of mutational xylanase increased from 45℃to 50℃. Half-life of the mutational xylanase increased from lower than 10 min to 120 min. Accumulation amplitude of enzyme react product increased from 36.9% to 276.3%. These dates confirmed that thermostability of mutational xylanase was greatly improved. Specific activity of xylanase increased from 2127.9±83.9 U/mg to 3330.9±173.4 U/mg. K_m of xylanase decreased from 56.9 mg/ml to 37.2 mg/ml, affinity of xylanase was greatly improved. V_max of xylanase decresed from 82.9 mmol/ml.min.mg to 27.0 mmol/ml.min.mg.4. Optimization the induce condition of the mutational xylanase Pichia pastorisInfluence of methanol quantity、initial cell density、pH and inducing time to enzyme production were reseached with four factors and three levels Orthogonal design. The importance of these factors for enzyme production were : methanol quantity＞inducing time＞pH＞initial cell density. The best inducing condition were : BYPN medium（pH8.0）, added 1% methanol every 12 h, initial cell density was OD₆₀₀=3, induced 7 d. Activity of mutational xylanase can reached 1850.6±25.2 U/ml under the best inducing condition.With the purpose of improving the yield of xylanase, we are reseaching on the inducing condition of mutational xylanase production with bio-fermenter.更多还原