草酸青霉木质纤维素降解酶系的解析及主要相关蛋白组分的功能研究

发布时间：2018-06-18 02:11

  本文选题：草酸青霉 + 里氏木霉　； 参考：《山东大学》2016年博士论文

【摘要】：目前石油等化石资源日趋枯竭,雾霾、气候变化等环境问题日益严峻,急需寻找和开发可替代化石能源的新能源。可持续再生的木质纤维素资源储量非常丰富,而且这些资源并未得到充分的开发利用,有些还造成环境污染。利用微生物生产纤维素酶,进而将可持续再生的生物质转变为液体燃料和化学品,是解决资源、能源和环境问题的有效途径。丝状真菌通常缺乏自然木质纤维素降解酶系的某些组分,因此需要补充酶组分以完全水解复杂的木质纤维原料。木质纤维素降解酶系的组成应该根据不同组成的生物质材料而改变。优化的纤维素酶混合物的成分可以提高生物质的水解效率。然而,由于缺乏酶系组分的认识,优化设计纤维素酶系是有限的,应在蛋白质组学分析的基础上对酶系进行优化,进而开发出高效的生物转化过程。草酸青霉具有完整的纤维素酶系,纤维素降解能力强、生长迅速,其高产纤维素酶菌株JU-A10自从1996年就已用于工业化生产纤维素酶制剂。本论文在草酸青霉基因组学、转录组学前期研究的基础上,进一步通过蛋白质组学的方法,分析确定了草酸青霉纤维素酶系降解木质纤维素的潜力。并以此为依据,研究了草酸青霉内切葡聚糖酶Cel5B、外切葡聚糖酶Cel7A-2、超氧化物歧化酶和纤维素膨胀因子4个主要蛋白,为草酸青霉纤维素酶系的优化改良提供了思路和借鉴。本论文的主要研究内容和结果如下：1.在生物质糖化过程中草酸青霉与里氏木霉酶系的表现不同为了优化草酸青霉纤维素酶系,使用不同酶量进行糖化,比较分析了3种商业纤维素酶制剂的水解性能。结果表明,使用同等蛋白量的草酸青霉纤维素酶制剂糖化脱木素木糖渣(DCCR)比木糖渣(CCR)效率更高,20mg蛋白/g葡聚糖进行糖化时,DCCR的糖化率可以达到90%,而CCR的糖化率只有40%。另外,里氏木霉产纤维素酶对于CCR的糖化效率为72%,明显高于草酸青霉。表明具有物理屏障和非生产性吸附作用的木素会影响木质纤维素生物质的糖化,而且对于草酸青霉纤维素酶系的的影响更为显著。草酸青霉纤维二糖水解酶和木聚糖酶活较高,分别是里氏木霉的2.3和2.17倍,但具有较低的p-葡萄糖苷酶活性。通过添加p-葡萄糖苷酶,木糖渣转化率明显提高,当SP酶粉使用量为9 FPA/g葡聚糖时,按照FPA:BG=1:4额外添加BG,以DCCR为底物的葡聚糖转化率达到了92%,与单独使用SP进行糖化所需的酶量减少了64%。这证实了p-葡萄糖苷酶是草酸青霉纤维素酶糖化的主要瓶颈之一。里氏木霉纤维素酶的添加促进了草酸青霉纤维素酶降解木糖渣的效率,通过将分离的里氏木霉纤维素酶组分添加到草酸青霉纤维素酶中糖化的方法,初步确定了里氏木霉促进草酸青霉糖化的蛋白组分。这一发现有望用于革酸青霉纤维素酶系的改良。2.蛋白质组学结果表明草酸青霉与里氏木霉在酶系组成上不尽相同为了探究纤维素酶系组成对生物质糖化的影响,使用蛋白质组学工具解析了纤维素酶工业化生产菌株。研究结果表明草酸青霉能产生的纤维素酶和半纤维素酶的种类较多,包含多种作用于木质纤维素材料主链和侧链的酶,具有完全的木质纤维素降解酶系。SP纤维素酶制剂包含18个纤维素酶,其中包括3个纤维二糖水解酶(2个CBH I和1个CBHⅡ)和9个内切酶。SP包含两个BG,但是量比较低。草酸青霉纤维素酶系含有对生物质的糖化至关重要的裂解性多糖单加氧酶(LPMO)和膨胀蛋白(swollenin)。CBH、EG、BG、LPMO、几丁质酶和淀粉酶在ST中的蛋白比例分别为37.15%、15.03%、0.66%、2.11%、0.02%和0.01%,而在SP中的比例分别为39.00%、11.27%、0.25%、1.37%、0.19%和0.44%。与草酸青霉纤维素酶SP相比,在ST蛋白质组中检测到了SP所没有的几种蛋白,它们是纤维素诱导蛋白(Cip1和Cip2)、木葡聚糖酶、a-1,6-甘露聚糖酶、疏水蛋白和细胞壁蛋白。SP含有更多的碳水化合物结合模块第一家族(CBM1)的蛋白,这种差异可能造成了SP更大的底物吸附,从而导致酶解效率低于里氏木霉纤维素酶。采用蛋白质组学工具分析了吸附对纤维素酶系的影响,实验表明糖化过程中LPMOs和膨胀蛋白在糖化上清液中含量非常低,积极参与了生物质水解。含CBM1过多的蛋白组分可能会产生较多非生产性吸附,阻碍其对含复杂成分纤维素底物的降解。本研究提供了草酸青霉和里氏木霉全面的酶系组成和相对含量,为优化和合理设计高效低成本纤维素酶系奠定了基础。3.探索了草酸青霉内切酶Cel5B和外切酶Cel7A-2的酶学性质和糖化添加效果草酸青霉内切酶Cel5B和外切酶Cel7A-2是其纤维素酶系中含量第1和第8的蛋白,这两种蛋白已经在草酸青霉蛋白表达菌A11△中得到表达。为了研究草酸青霉的重要蛋白并确定糖化过程中这两种重要蛋白的作用,使用分子筛分离纯化了重组蛋白rCel5B和重组蛋白rCel7A-2。rCel5B为内切纤维素酶,与草酸青霉其他内切β-1,4-葡聚糖酶相比,rCel5B具有较好的热稳定性及pH耐受性。当温度为30～50℃时,保温6小时,酶活仍可保持在90%以上。当温度升高到60℃,保温6小时后,酶活降到80%。Cel5B在pH 4.2-6.0范围内可保存95%以上的酶活力。Cel5B对所用底物均有一定的水解能力,其中降解羧甲基纤维素钠的能力最强。重组蛋白rCel7A-2为外切型纤维素酶。Cel7A-2的最适作用温度为55℃。当温度为30～50℃时,保温6小时,酶活仍可保持在90%以上,当温度升高到60℃时,保温6小时后,酶活降到60%。Cel7A-2在pH3.6-4.8范围内可保存90%以上的酶活力,最佳pH为4.2,在pH 3可保存70%的酶活力,当pH升到5.4,酶活力迅速下降到20%,当pH为6.0、6.6时酶活力几乎消失。添加Cel5B和Cel7A-2对草酸青霉纤维素酶降解木质纤维素底物有一定的促进作用。单独加入Cel5B和Cel7A-2后,葡萄糖产量均有提高,葡聚糖转化率分别提高了2%和6%。使用中心组合实验设计研究Cel5B和Cel7A-2对于草酸青霉纤维素酶糖化木糖渣的影响,结果表明当酶用量为9 FPA/g纤维素时,添加13%Cel7A-2、21% Cel5B和29%BG时,葡萄糖转化率提高了33%。4.研究了草酸青霉Cu/Zn-SOD及膨胀蛋白对蛋白分泌、纤维素酶合成及木糖渣糖化的影响Cu/Zn-SOD和膨胀蛋白是草酸青霉分泌组中含量较高的纤维素酶辅助蛋白,为了研究这两种蛋白对于木质纤维素底物的作用,以纤维素酶高产菌株4-1为出发菌株,成功构建了二者的缺失菌株。结果表明缺失Cu/Zn-SOD影响了草酸青霉发酵液的纤维素酶酶活力与胞外蛋白浓度。而Cu/Zn-SOD本身对生物质糖化没有直接作用。通过表型及酶活测定分析,发现敲除膨胀蛋白也影响了草酸青霉发酵液的p-葡萄糖苷酶活力,敲除株表型没有发生明显变化。敲除株粗酶液等蛋白量糖化和等滤纸酶活糖化结果显示,敲除膨胀蛋白影响了木糖渣的糖化效率。以实验室保存的蛋白表达质粒为载体构建了膨胀蛋白表达菌株,用分离纯化后的膨胀蛋白进行外源添加糖化实验,发现膨胀蛋白与草酸青霉纤维素酶系有协同作用。在草酸青霉野生型菌株114-2中成功构建了膨胀蛋白过表达菌株,以优化草酸青霉纤维素酶系。
[Abstract]:At present, fossil resources such as oil are increasingly exhausted, fog and haze, climate change and other environmental problems are becoming increasingly severe. It is urgent to find and develop new energy sources that can replace fossil fuels. The reserves of sustainable regenerated lignocellulose resources are very rich, and these resources have not been fully exploited and utilized, and some also cause environmental pollution. Cellulase Producing, and then converting sustainable regenerated biomass into liquid fuel and chemicals, is an effective way to solve resources, energy and environmental problems. Filamentous fungi usually lack some components of natural lignocellulose degrading enzymes. Therefore, the enzyme components need to be supplemented to complete the complex lignocellulosic materials, lignocellulose, which is fully hydrolyzed. The composition of the degrading enzyme system should be changed according to the different biomass materials. The optimized composition of the cellulase mixture can improve the hydrolysis efficiency of the biomass. However, the optimization of the cellulase system is limited due to the lack of the understanding of the enzyme system components. An efficient biotransformation process has been developed. Penicillium oxalate has a complete cellulase system, cellulose degradation ability and rapid growth. The Cellulase Producing Strain JU-A10 has been used in the industrial production of cellulase preparations since 1996. This paper has been further passed on the basis of the prophase study of Penicillium oxalicum genomics and transcriptional studies. The method of proteomics was used to determine the potential of cellulase degradation of lignocellulose by Penicillium oxalate. On the basis of this, 4 main egg white, Cel5B, exoscester Cel7A-2, superoxide dismutase and cellulose expansion factor, were studied. The main contents and results of this paper are as follows: 1. in the process of biomass saccharification, the performance of Penicillium oxalate and Trichoderma rimycin is different in order to optimize the cellulase system of Penicillium oxalate, saccharification with different enzyme quantities, and the hydrolysis performance of the 3 kinds of commercial fibrinase preparation is compared and analyzed. The efficiency of saccharification delignification xylose residue (DCCR) with equal protein content is higher than that of xylose residue (CCR). When 20mg protein /g glucan is saccharification, the saccharification rate of DCCR can reach 90%, while the saccharification rate of CCR is only 40%., and the saccharification efficiency of Trichoderma Richter is 72% to CCR, which is obviously higher than that of Penicillium oxalate. The lignin with physical barrier and non productive adsorption affects the saccharification of lignocellulosic biomass, and has more significant effect on the cellulase of Penicillium oxalate. The two sugar hydrolase and xylanase activity of the Penicillium oxalate fiber are 2.3 and 2.17 times of Trichoderma ripenii, respectively, but have a lower activity of p- glucosidase. By adding p- glucosidase, the conversion rate of xylose residue was obviously improved. When the use of SP enzyme powder was 9 FPA/g glucan, the addition of BG was added to FPA:BG=1:4 by FPA:BG=1:4, and the conversion rate of dextran with DCCR as the substrate was reached, and the amount of enzyme needed for the saccharification of SP was reduced by 64%., which confirmed that the p- glucosidase is the cellulase of Penicillium oxalate. One of the main bottlenecks of saccharification is the addition of Cellulase of Trichoderma ripene, which promotes the efficiency of xylose degradation by Cellulase of Penicillium oxalate. By adding the cellulase component of Trichoderma ripene to cellulase of Penicillium oxalate, the protein component of Trichoderma arylate saccharification is preliminarily determined. The modified.2. proteomics of Cellulase of Penicillium gram showed that Penicillium oxalate and Trichoderma leicillium were different in the composition of enzyme system in order to explore the effect of cellulase composition on biomass saccharification. The proteomic tool was used to analyze the industrial strain of cellulase. The results showed that Penicillium oxalate could be used as an analytical tool. There are many kinds of cellulase and hemicellulase produced, including a variety of enzymes that act on the main chain and side chain of lignocellulosic materials, with complete lignocellulosic degrading enzyme system.SP cellulase containing 18 cellulase, including 3 fibrous two sugar hydrolase (2 CBH I and 1 CBH II) and 9 endonuclease.SP containing two B G, but low in quantity. The cellulase of Penicillium oxalate contains lysate polysaccharide monooxygenase (LPMO) and expansive protein (swollenin).CBH, EG, BG, LPMO, chitinase and amylase in ST, respectively, 37.15%, 15.03%, 0.66%, 2.11%, 0.02% and 0.01% respectively, and 39% in SP, respectively, 11. .27%, 0.25%, 1.37%, 0.19% and 0.44%. were compared with the cellulase SP of the Penicillium oxalate. In the ST proteome, several proteins were detected in SP. They were cellulose induced proteins (Cip1 and Cip2), xylanase, a-1,6- manna polyglucan, hydrophobin and cell wall egg white.SP containing more carbohydrate binding module first family (CBM). 1) of the protein, this difference may result in a larger substrate adsorption of SP, which leads to a lower enzymatic efficiency than the cellulase of Trichoderma RI. The effect of adsorption on the cellulase system is analyzed by the proteomics tool. The experiment shows that the content of LPMOs and expansive protein in the saccharifying liquid is very low in the process of saccharification and actively participates in the biomass water. In this study, the overall enzyme system composition and relative content of Penicillium oxalate and Trichoderma ripene were provided for the optimization and rational design of high efficiency and low cost fibrin enzyme system. The study provided a basic.3. for the exploration of Penicillium oxalate. Enzymatic properties of endonuclease Cel5B and exonuclease Cel7A-2 and saccharification effect, Penicillium oxalate endonuclease Cel5B and exonuclease Cel7A-2 are first and eighth protein in its cellulase system, and these two proteins have been expressed in A11 delta of Penicillium oxalate protein expression bacteria. In order to study the important protein of Penicillium oxalate and determine the saccharification process In the action of these two important proteins, the recombinant protein rCel5B and recombinant protein rCel7A-2.rCel5B were isolated and purified by molecular sieves. Compared with other internal cut beta -1,4- glucan enzymes of Penicillium oxalate, rCel5B had better thermal stability and pH tolerance. When the temperature was 30~50, the enzyme activity could remain at 90% for 6 hours. When the temperature rises to 60, and after 6 hours of heat preservation, the enzyme activity is reduced to 80%.Cel5B in the pH 4.2-6.0 range, and more than 95% of the enzyme activity can be preserved to a certain degree of hydrolysis, among which the ability to degrade sodium carboxymethyl cellulose is the strongest. The optimum temperature of the recombinant protein rCel7A-2 is 55 of the exoscase cellulase.Cel7A-2 is 55. When the temperature is 30~50, the enzyme activity can still remain above 90% for 6 hours. When the temperature rises to 60, the enzyme activity is reduced to 60%.Cel7A-2 in the pH3.6-4.8 range for more than 90% of the enzyme activity, the optimum pH is 4.2, the enzyme activity is 70% in pH 3, and when pH is raised to 5.4, the enzyme activity rapidly drops to 20%, when pH is 6.0,6.6 The enzyme activity almost disappeared. Adding Cel5B and Cel7A-2 could promote the degradation of lignocellulosic substrates by Penicillium oxalate cellulase. After adding Cel5B and Cel7A-2, the yield of glucose was improved. The conversion rate of glucan increased by 2% and 6%., respectively, and Cel5B and Cel7A-2 were designed to study Cel5B and Cel7A-2 for Penicillium oxalicum fiber. The effects of vitamin C saccharification on xylose residue showed that when the dosage of enzyme was 9 FPA/g cellulose, when 13%Cel7A-2,21% Cel5B and 29%BG were added, the conversion of glucose was increased by 33%.4., and the effect of Cu/Zn-SOD and expansin on protein secretion, cellulase synthesis and sugar residue of xylose residue were studied by 33%.4., and Cu/Zn-SOD and expansin were Penicillium oxalate. In order to study the effect of the two proteins on the lignocellulose substrate in the secretory group, in order to study the effect of the two proteins on the lignocellulose substrate, the strain 4-1 of the cellulase producing strain was used as the starting strain, and the missing strains were successfully constructed. The results showed that the absence of Cu/Zn-SOD affected the cellulase activity and the extracellular protein concentration of the Penicillium oxalicum fermentation broth. Cu/Zn-SOD itself has no direct effect on biomass saccharification. Through phenotypic and enzyme activity determination, it is found that knockout expandable protein also affects the p- glucosidase activity of the Penicillium oxalate fermentation broth, and there is no obvious change in the phenotype of the knockout plant. The swelling protein affects the saccharification efficiency of the xylose residue. The expansion protein expression strain is constructed with the protein expression plasmid preserved in the laboratory, and the extraneous saccharification experiment is carried out by the purified expansive protein. It is found that the expansion protein has a synergistic effect with the cellulase line of Penicillium oxalate. It has been successfully used in the wild type of Penicillium oxalate strain 114-2. An over expressing strain of expansive protein was constructed to optimize the cellulase system of Penicillium oxalicum.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q936
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本文编号：2033542