耐碱性纤维素酶的表达及其对纸浆改性和机制研究

发布时间：2018-01-09 07:12

本文关键词：耐碱性纤维素酶的表达及其对纸浆改性和机制研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：由于草浆等纸浆存在滤水性差、强度低等缺点,使其应用受到限制,一般生产过程中需要对其进行改性以改善滤水和强度性能。与化学法改性相比,采用纤维素酶(主要是内切酶EG)处理对纸浆改性是一种环境友好的技术,且已被证明在改善纸浆滤水性,提高成纸强度等方面具有良好效果。由于制浆造纸工艺往往在中、碱性条件下进行,这就要求使用的酶能够在高pH条件下维持酶活性,保证其催化作用有效进行。但市场上已有的商品纤维素酶大多为酸性酶,在碱性条件下酶活很低。虽然国内外对碱性纤维素酶的研究正逐渐深入,但目前仍存在很多问题,例如,已有产碱性纤维素酶菌株的产量低、酶系不合理、酶改性机理尚未完全清楚等。因此,设法提高碱性纤维素酶产量,合理优化酶系,以降低纤维素酶生产成本和提高对纸浆的改性效果,同时深入研究酶法改性机制等,对促进酶在造纸工业中的应用,降低生产成本等具有重要意义。基于以上背景,本论文在实验室前期研究基础上,通过构建耐碱性纤维素酶菌株,以希望提高纤维素酶产量;同时通过构建耐碱性纤维素酶与木聚糖酶共表达菌株,以达到优化改性酶系、提高酶法改性效果的目的;同时从不同角度研究了纸浆酶法改性的机制。本论文主要研究内容和结果如下:1.耐碱性纤维素酶的表达及其对纸浆改性效果研究为获得适合纸浆改性用的耐碱性纤维素酶,在实验室前期研究基础上,扩增来源于特异腐质霉的三个耐碱性内切纤维素酶H.EGⅠ,H.EGⅡ,H.EGⅤ的基因,并在毕赤酵母GS115中成功异源表达;同时在大肠杆菌BL21中成功异源表达了来源于不同芽孢杆菌的三个内切纤维素酶Y106-EG,z-16-EG及A30-EG。测定纯化后重组耐碱性纤维素酶的性质,发现这六种内切纤维素酶的最适pH在6.5-7.5左右,最适温度在50℃-60℃之间,各酶在pH 6.0-8.0条件下放置1h能够维持60%以上酶活性,其中Y106-EG、z-16-EG和H.EGV在pH 9.0条件下放置1 h可以维持60%以上酶活性。将六种耐碱性内切酶应用于麦草浆改性,发现Y106-EG在酶用量仅为0.2 IU/g条件下,与对照相比,能够降低12.5%的打浆度,抗张强度指数,耐破指数,撕裂指数分别提升14.6%、14.3%和10.7%。对纸浆的改性效果明显优于其它五种耐碱性内切葡聚糖酶,显示出良好的潜在应用前景。2.重组菌Y106OE-EG粗酶液对不同纸浆改性时的酶处理条件优化及与其它酶的协同作用研究为提高耐碱性纤维素酶Y106-EG的产量,利用基因工程方法,实现Y106-eg基因的同源过表达,经实验室前期优化的发酵培养基发酵,所得发酵液的CMCase酶活达到8.45 IU/ml,是出发菌株的8.28倍。利用工程菌Y106OE-EG粗酶液对杨木CMP(杨木化学机械浆),松木CMP(松木化学机械浆),麦草CP(麦草化学浆)进行改性研究发现,改性效果依次为麦草CP杨木CMP松木CMP。对酶法改性时的酶处理条件(处理温度、时间和pH等)进行了优化,发现当酶用量仅为0.2 IU/g浆,pH 7.0,温度55 ℃条件下对麦草浆处理2 h,与未用酶处理的对照相比,纸浆的抗张指数,耐破指数,撕裂指数分别增加了15.4%、16.9%和 11.8%。研究了工程菌Y106OE-EG粗酶液与其它酶的协同改性效果,发现Y106OE-EG的内切纤维素酶与纤维素膨胀因子SWO4的联合处理,与单独使用内切酶或SWO4相比,反而降低了纸浆的强度和纤维素结晶度,而当与β-葡萄糖苷酶(1.2 IU/g)或木聚糖酶(5IU/g)联合处理时,对纸浆的强度性质(抗张指数,撕裂指数,耐破指数)均有明显改善,在温度55 ℃C、pH 7.0条件下处理2 h,当Y106OE-EG粗酶液用量为0.2IU/g,也β-葡萄糖苷酶1.2IU/g时,与对照相比,麦草浆的抗张指数、撕裂指数、耐破指数分别提升23.68%、34.10%、20.82%。当木聚糖酶加量为5 IU/g时,与对照相比,麦草浆的抗张指数、撕裂指数、耐破指数分别提升32.65%、42.44%、25.00%。此外,木聚糖酶与SWO4或木糖苷酶的联合处理,对麦草浆的改性也具有良好的协同作用效果。综合而言,Y106OE-EG生产的内切纤维素酶与短小芽孢杆菌生产的木聚糖酶Xyn30的协同处理对纸浆的改性效果最佳,为后续的共表达菌株构建提供了理论依据。3.产耐碱性纤维素酶与木聚糖酶共表达菌株构建及产酶条件优化为实现耐碱性纤维素酶(Y106-EG)及木聚糖酶(Xyn30)在Y106-WT中的共表达,采用顺反子共表达,融合酶表达,串联共表达三种方式,意在提高枯草芽孢杆菌Y106-WT的产酶能力,结果表明,以串联共表达的方式能够实现两酶的共表达,对构建的共表达工程菌Y106OE-EG-Xyl进行液体发酵,发现该菌株在培养56 h后,CMCase酶活可达最高(8.20 IU/ml),木聚糖酶在菌株培养64 h时酶活达到最高(60.40 IU/ml),分析该菌株产粗酶液的酶系发现,其滤纸酶活(0.12IU/ml),外切纤维素酶活性(0.02IU/ml),β-葡萄糖苷酶(未测到)均较低,说明对应用于纸浆改性而言,共表达菌所产纤维素酶的酶系比较合理,在对纸浆进行改性的同时不损害纸浆纤维。利用工程菌Y106OE-EG-Xyl粗酶液在最佳处理pH 7.0,最佳处理温度55 ℃C,浆浓度10%(w/v)条件下处理麦草化学浆2 h,当酶用量为纤维素酶0.2 IU/g木聚糖酶活1.5IU/g时,与对照及单独添加纤维素酶(0.2IU/g)及木聚糖酶(1.5 IU/g)相比,纸浆白度和强度性能都有改善,其中与不加酶的对照浆处理相比,纸浆白度增加2.0%ISO,抗张指数、耐破指数、撕裂指数分别增加8.7%、16.6%和9.3%,而打浆度降低了 15%。利用混料设计优化了工程菌Y106OE-EG-Xyl发酵时培养基中的碳源及诱导剂比例,发现当麸皮,玉米芯,乳糖配比为3.33%、0.83%、0.83%时,液体发酵72 h,纤维素酶活可达7.9 IU/ml,木聚糖酶活可达74.5 IU/ml,木聚糖酶活比优化前提升了 14.1个酶活单位,且木聚糖酶与内切纤维素酶的比由原来的7.5提升至 9.4。4.纸浆的酶法改性机制研究通过对Y106OE-EG粗酶液处理前后纤维质量变化,纤维结晶度(XRD)及红外光谱(FITR-ATR)等的变化分析发现,经重组菌Y106OE-EG粗酶液处理后,杨木CMP和麦草CP的纤维质量得到明显改善,且改善效果好于松木CMP。Y106OE-EG粗酶液处理后,杨木CMP及麦草化学浆的结晶度与对照相比明显提高,但松木CMP结晶度则提高不明显,三种浆经酶处理后纸浆中的氢键强度均有所提升。分别提取了麦草CP、松木CMP和杨木CMP中的木质素组分,研究了木质素对Y106OE-EG的酶蛋白吸附特征,发现松木CMP和杨木CMP的木质素对纤维素酶蛋白的吸附能力明显高于麦草浆中的木质素,木质素对蛋白的不可逆吸附将影响酶对纤维底物的作用,因此相对而言,Y106OE-EG酶液更适合用于麦草浆的改性。分别利用Y106OE-EG和Y106OE-Xyl及共表达菌株Y106OE-EG-Xyl的粗酶液(EG 0.2 IU/g、Xylanase 1.5 IU/g、EG 0.2 IU/g-Xylanase 1.5 IU/g)处理纸浆,发现与对照或只添加EG或木聚糖酶的样品相比,共表达菌株的粗酶液处理后纸浆纤维的平均长度增加,宽度几乎不变,长宽比变大,细小纤维含量减少,卷曲指数及扭结指数均有所降低,且共表达菌株的粗酶液处理效果优于单独酶的处理效果。利用红外谱图对共表达菌株Y106OE-EG-Xyl粗酶液处理前后草浆的吸收峰相对强度进行分析,发现经共表达菌株粗酶液处理后,氢键强度明显提升。利用SEM观察了重组菌Y106OE-EG及共表达菌株Y106OE-EG-Xyl的粗酶液处理前后草浆纤维表面形态变化,发现经两菌株的粗酶液处理后,与对照相比,纸浆中的细小纤维含量明显减少,纤维变得平整。酶处理后纸浆在纤维质量、纤维素结晶度、氢键强度、表面形态等方面发生的上述变化,是酶处理改善纸浆强度和滤水性能的内在原因。分别从酶的结构和性质、蛋白表面电荷、蛋白的疏水性、蛋白在纸浆纤维上的吸附等角度,探讨了 Y106-EG所产粗酶液与来源于特异腐质霉的三个耐碱性内切纤维素酶H.EGⅠ、H.EGⅡ和H.EGⅤ以及来源于其它芽孢杆菌的两个耐碱性内切纤维素酶z-16-EG和A30-EG对麦草浆的改性差异及内在原因。研究发现,与其它五种耐碱性内切纤维素酶相比,Y106-EG由于酶蛋白对底物的亲和力相对较大,且在pH 7.0时,pH值低于其自身等电点使其氨基酸易被带上正电荷,另外,蛋白表面的Zeta电位与其它几种耐碱性内切酶相比负值较小,且蛋白的疏水性小,这些都使得Y106-EG酶蛋白更易于与带负电荷的细小纤维发生亲水性结合,从而有利于酶降解细小纤维。此外,Y106-EG酶蛋白是典型的(β/α)8桶状结构特征,结构稳定性较好且开口宽阔的活性中心使其可以容纳更多类型不同的多糖支链,在催化降解复杂底物时有较大优势,该酶的CBM属于A型CBM,易于结合于结晶型纤维素多糖上。这些特征使得Y106-EG在用于纸浆改性时表现出较好的优势。
[Abstract]:The straw etc. pulp drainability are poor, low intensity, so its application is limited, need to be modified to improve the drainage and strength properties of the general production process. Compared with chemical modification, cellulase (mainly enzyme EG) treatment on pulp modification is an environmental friendly the technology, and has been shown to improve pulp drainability, has good effect to improve paper strength and so on. Because of the pulping and papermaking process often in alkaline conditions, this requires the use of enzymes to maintain the enzyme activity in the condition of high pH, effectively guaranteeing the catalytic effect. But already on the market the goods are mostly acidic cellulase enzyme, in alkaline conditions, enzyme activity is very low. Although the domestic and foreign research on alkaline cellulase is deepening, but there are still many problems, for example, has a strain producing alkaline cellulase yield Low enzyme system is not reasonable, enzyme modification mechanism has not been fully understood. Therefore, to improve the yield of alkaline cellulase, optimizing cellulase enzyme system, to reduce production cost and improve the effect of modification of pulp, and studies on enzymatic modification mechanism, to promote the application of enzyme in papermaking industry, has important to reduce the production cost. Based on the above background, this paper based on the previous research in the laboratory, through the construction of alkaline cellulase strains, in the hope of improving yield of cellulase; at the same time through the construction of alkaline cellulase and xylanase co expression strain, to achieve the optimization of modified enzyme system, improve the effect of modified enzyme method; at the same time from different angles on the pulp enzymatic modification mechanism. The main research contents and results are as follows: 1. the expression of cellulase and alkali resistance of pulp modification effect for research For the pulp for modification of alkaline cellulase in the laboratory, based on the previous research of amplification from humicola insolens three alkaline endoglucanase H.EG I and H.EG II, H.EG V gene and heterologous expression in Pichia pastoris GS115; at the same time, the success of three endoglucanase Y106-EG heterologous expression. Different from Bacillus subtilis in Escherichia coli BL21, properties of z-16-EG and A30-EG. determination of purified recombinant alkaline cellulase, found that the optimum pH is about 6.5-7.5 in the six kinds of endoglucanase optimum temperature between 50, -60 DEG C, the enzyme 1h in pH placed under the condition of 6.0-8.0 can maintain more than 60% of the enzyme activity among them, Y106-EG, z-16-EG and H.EGV under the condition of pH 9 for 1 h can maintain more than 60% enzymes. Six kinds of alkali resistance enzymes used in wheat straw modified, Y106-EG found in the amount of enzyme is only 0.2 IU/g Under the condition, compared with the control, can reduce 12.5% of the beating degree, tensile index, burst index and tear index were increased by 14.6%, significantly higher than the other five kinds of alkaline endoglucanase 14.3% and 10.7%. of the pulp, showing good potential application prospect in recombinant.2. enzyme on Y106OE-EG optimization of enzyme treatment of different pulp modification and other enzymes to improve the synergistic effect of alkali cellulase Y106-EG production by genetic engineering method, the homology of Y106-eg gene overexpression, by fermentation laboratory optimization of fermentation medium, the enzyme activity reached 8.45 IU/ml CMCase fermentation liquor, is 8.28 times the starting strain. The engineering strain Y106OE-EG crude enzyme of poplar CMP (Yang Mu chemical mechanical pulp), CMP pine (pine chemical mechanical pulp (CP) of wheat straw, wheat straw chemical pulp) was modified to study found that modification effect The fruit followed by wheat straw CP poplar CMP pine CMP. on enzymatic modification of enzyme treatment conditions (temperature, time and pH) were optimized, when the enzyme dosage is 0.2 IU/g pulp, pH 7, temperature 55 degrees under the condition of wheat straw treatment for 2 h, compared with the control without enzyme the pulp tensile index, burst index and tear index were increased by 15.4%, 16.9% and 11.8%. to study the effect of modified engineering strain Y106OE-EG crude enzyme solution with other enzymes, it is found that the combined treatment with endo cellulase cellulose Y106OE-EG expansion factor SWO4, compared with the single use enzyme or SWO4, but reduce the strength and crystallinity of cellulose pulp, while beta glucosidase and glucose oxidase (1.2 IU/g) or xylanase (5IU/g) combined treatment, the strength properties of the pulp (tensile index, tear index, burst index) were significantly improved, at a temperature of 55 DEG C, pH 7 Under 2 h, when the Y106OE-EG crude enzyme dosage is 0.2IU/g, or beta glucosidase 1.2IU/g when compared with the control, the tensile index, tear index of wheat straw pulp, bursting index were increased by 23.68%, 34.10%, 20.82%. when xylanase dosage was 5 IU/g, compared with the control, anti the tensile index, tear index of wheat straw pulp, bursting index were increased by 32.65%, 42.44%, 25.00%. in the combined treatment of xylanase and xylosidase or SWO4, modification of wheat straw has good synergistic effect. In general, the production of Y106OE-EG fiber endo cellulase and Bacillus pumilus production xylanase Xyn30 co processing is the best modifier of pulp for strain construction provides a theoretical basis for.3. producing alkaline cellulase and xylanase expression strain construction and optimization of fermentation conditions for alkaline cellulase co expression of follow-up (Y106- EG) and xylanase (Xyn30) were expressed in Y106-WT, the cistron expression, fusion enzyme expression, series co expression in three ways, in order to improve the enzyme producing ability of Bacillus subtilis Y106-WT results show that co expression in series co expression way to achieve the two enzyme, liquid fermentation on the establishment of co expression engineering bacteria Y106OE-EG-Xyl, found that the strain in the culture after 56 h, CMCase activity was the highest (8.20 IU/ml), xylanase strain was cultured in enzyme activity reached the maximum of 64 H (60.40 IU/ml), found that the enzyme production of the strain analysis of crude enzyme, the filter paper activity (0.12IU/ml), cellobiohydrolase activity (0.02IU/ml), beta glucosidase (not detected) were low, indicating the corresponding modification for pulp, co expression of enzymes of bacteria producing cellulase is reasonable, in the pulp modified with no damage to the pulp fiber. By using engineering bacteria Y106 OE-EG-Xyl crude enzyme solution in the optimal treatment of pH 7, the optimum processing temperature is 55 DEG C, plasma concentration of 10% (w/v) under the condition of wheat straw chemical pulp processing 2 h, when the dosage of enzyme was 0.2 IU/g cellulase and xylanase activity of 1.5IU/g, and the control and the addition of cellulase and xylanase (0.2IU/g) (1.5 IU/g) compared have, improve pulp brightness and strength properties of the pulp, compared with the control treatment without enzyme, the whiteness of pulp increased 2.0%ISO, tensile index, burst index and tear index were increased by 8.7%, 16.6% and 9.3%, and the beating degree is reduced by using 15%. mixture design optimization of engineering bacteria Y106OE-EG-Xyl fermentation the medium carbon source and inducer ratio, when the wheat bran, corn cob, lactose ratio was 3.33%, 0.83%, 0.83%, 72 h liquid fermentation, cellulase activity was 7.9 IU/ml, the xylanase activity was up to 74.5 IU/ml, the activity of xylanase than before optimization improved 14.1 enzyme activity Unit, and xylanase and CMCase by enzyme method than the original 7.5 to 9.4.4. pulp modification mechanism through the study on the change of fiber quality of Y106OE-EG before and after enzyme treatment, the crystallinity of the fiber (XRD) and infrared spectroscopy (FITR-ATR) and other changes after analysis found that recombinant Y106OE-EG enzyme treatment after the fiber quality of poplar CMP and wheat straw CP was obviously improved, and the improvement effect is better than the pine CMP.Y106OE-EG crude enzyme solution after crystallization of poplar CMP and wheat straw pulp were significantly increased compared with the degree of crystallinity of CMP, but the pine increase is not obvious, three kinds of pulp by hydrogen bond strength were enzyme treatment after the upgrade. In the pulp were extracted from wheat straw CP, CMP and CMP in pine poplar lignin fractions, of lignin on Y106OE-EG enzyme protein adsorption characteristics, found that pine CMP and poplar CMP lignin on cellulase protein The adsorption capacity was significantly higher than that of wheat straw pulp lignin, lignin on protein irreversible adsorption will affect the effect of enzyme on fiber substrates, so relatively speaking, Y106OE-EG is more suitable for wheat straw pulp enzyme modification. Y106OE-EG enzyme and Y106OE-Xyl and co expression strain Y106OE-EG-Xyl (EG 0.2 IU/g, respectively, by Xylanase 1.5 IU/g, EG 0.2 IU/g-Xylanase 1.5 IU/g) treatment of pulp, we found that compared with the control or only adding EG or xylanase samples, the average length of total crude enzyme strains after the expression of pulp fibers increased, the width is almost unchanged, the ratio of length to width becomes larger, reduce the fines content, curl index and kink index decreased, and co expression of treatment effect of crude enzyme treatment effect is better than that of the strains. The infrared spectra of co expression strain Y106OE-EG-Xyl crude enzyme solution before and after the treatment of straw pulp and the absorption peak is relatively strong The degree of analysis, found by co expression strain of crude enzyme treatment, hydrogen bond strength significantly improved. Using SEM surface morphology changes were observed before and after the straw fiber and co expression of recombinant Y106OE-EG strain Y106OE-EG-Xyl crude enzyme treatment, found that the crude enzyme treatment of strain two, compared with the control, the content of fine fiber pulp the fiber becomes smooth. Significantly reduced after enzyme treatment in pulp fiber quality, cellulose crystallinity, hydrogen bond strength, the change of surface morphology occurred, is the inherent reason of enzyme treatment improve the pulp strength and drainage performance. From the structure and properties of the enzyme, protein surface charge, hydrophobic protein, protein in the pulp fiber adsorption angle, discusses the Y106-EG production and the crude enzyme from humicola insolens three alkaline endoglucanase H.EG 1, H.EG 2 and H.EG V and from the other The two Bacillus alkaline endoglucanase z-16-EG and A30-EG on wheat straw pulp modified difference and internal reasons. The study found that, compared with other five kinds of alkaline endoglucanase, Y106-EG as affinity enzyme protein of substrate is relatively large, and in pH 7, pH value is lower than the isoelectric point of the amino acid easy to be with a positive charge, in addition, Zeta protein negative surface potential is lower compared with other several alkali resistant enzymes, and protein hydrophobicity, which makes Y106-EG protein more easily and negatively charged fine fiber hydrophilic combination, which is beneficial to the enzymatic degradation of fine fiber. In addition, Y106-EG protein is a typical (alpha / beta) 8 barrel structure characteristics, good structure stability and wide opening activity center which can accommodate more different types of branched polysaccharides, in catalytic degradation of complex substrate is bigger The advantage of this enzyme, CBM belongs to A CBM, easy binding to crystalline cellulose polysaccharides. These characteristics make Y106-EG used in pulp modification showed better advantage.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS727;Q55

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