本文選題：草酸青霉 + 里氏木霉�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：目前石油等化石資源日趨枯竭,霧霾、氣候變化等環(huán)境問題日益嚴(yán)峻,急需尋找和開發(fā)可替代化石能源的新能源。可持續(xù)再生的木質(zhì)纖維素資源儲量非常豐富,而且這些資源并未得到充分的開發(fā)利用,有些還造成環(huán)境污染。利用微生物生產(chǎn)纖維素酶,進(jìn)而將可持續(xù)再生的生物質(zhì)轉(zhuǎn)變?yōu)橐后w燃料和化學(xué)品,是解決資源、能源和環(huán)境問題的有效途徑。絲狀真菌通常缺乏自然木質(zhì)纖維素降解酶系的某些組分,因此需要補(bǔ)充酶組分以完全水解復(fù)雜的木質(zhì)纖維原料。木質(zhì)纖維素降解酶系的組成應(yīng)該根據(jù)不同組成的生物質(zhì)材料而改變。優(yōu)化的纖維素酶混合物的成分可以提高生物質(zhì)的水解效率。然而,由于缺乏酶系組分的認(rèn)識,優(yōu)化設(shè)計(jì)纖維素酶系是有限的,應(yīng)在蛋白質(zhì)組學(xué)分析的基礎(chǔ)上對酶系進(jìn)行優(yōu)化,進(jìn)而開發(fā)出高效的生物轉(zhuǎn)化過程。草酸青霉具有完整的纖維素酶系,纖維素降解能力強(qiáng)、生長迅速,其高產(chǎn)纖維素酶菌株JU-A10自從1996年就已用于工業(yè)化生產(chǎn)纖維素酶制劑。本論文在草酸青霉基因組學(xué)、轉(zhuǎn)錄組學(xué)前期研究的基礎(chǔ)上,進(jìn)一步通過蛋白質(zhì)組學(xué)的方法,分析確定了草酸青霉纖維素酶系降解木質(zhì)纖維素的潛力。并以此為依據(jù),研究了草酸青霉內(nèi)切葡聚糖酶Cel5B、外切葡聚糖酶Cel7A-2、超氧化物歧化酶和纖維素膨脹因子4個(gè)主要蛋白,為草酸青霉纖維素酶系的優(yōu)化改良提供了思路和借鑒。本論文的主要研究內(nèi)容和結(jié)果如下：1.在生物質(zhì)糖化過程中草酸青霉與里氏木霉酶系的表現(xiàn)不同為了優(yōu)化草酸青霉纖維素酶系,使用不同酶量進(jìn)行糖化,比較分析了3種商業(yè)纖維素酶制劑的水解性能。結(jié)果表明,使用同等蛋白量的草酸青霉纖維素酶制劑糖化脫木素木糖渣(DCCR)比木糖渣(CCR)效率更高,20mg蛋白/g葡聚糖進(jìn)行糖化時(shí),DCCR的糖化率可以達(dá)到90%,而CCR的糖化率只有40%。另外,里氏木霉產(chǎn)纖維素酶對于CCR的糖化效率為72%,明顯高于草酸青霉。表明具有物理屏障和非生產(chǎn)性吸附作用的木素會影響木質(zhì)纖維素生物質(zhì)的糖化,而且對于草酸青霉纖維素酶系的的影響更為顯著。草酸青霉纖維二糖水解酶和木聚糖酶活較高,分別是里氏木霉的2.3和2.17倍,但具有較低的p-葡萄糖苷酶活性。通過添加p-葡萄糖苷酶,木糖渣轉(zhuǎn)化率明顯提高,當(dāng)SP酶粉使用量為9 FPA/g葡聚糖時(shí),按照FPA:BG=1:4額外添加BG,以DCCR為底物的葡聚糖轉(zhuǎn)化率達(dá)到了92%,與單獨(dú)使用SP進(jìn)行糖化所需的酶量減少了64%。這證實(shí)了p-葡萄糖苷酶是草酸青霉纖維素酶糖化的主要瓶頸之一。里氏木霉纖維素酶的添加促進(jìn)了草酸青霉纖維素酶降解木糖渣的效率,通過將分離的里氏木霉纖維素酶組分添加到草酸青霉纖維素酶中糖化的方法,初步確定了里氏木霉促進(jìn)草酸青霉糖化的蛋白組分。這一發(fā)現(xiàn)有望用于革酸青霉纖維素酶系的改良。2.蛋白質(zhì)組學(xué)結(jié)果表明草酸青霉與里氏木霉在酶系組成上不盡相同為了探究纖維素酶系組成對生物質(zhì)糖化的影響,使用蛋白質(zhì)組學(xué)工具解析了纖維素酶工業(yè)化生產(chǎn)菌株。研究結(jié)果表明草酸青霉能產(chǎn)生的纖維素酶和半纖維素酶的種類較多,包含多種作用于木質(zhì)纖維素材料主鏈和側(cè)鏈的酶,具有完全的木質(zhì)纖維素降解酶系。SP纖維素酶制劑包含18個(gè)纖維素酶,其中包括3個(gè)纖維二糖水解酶(2個(gè)CBH I和1個(gè)CBHⅡ)和9個(gè)內(nèi)切酶。SP包含兩個(gè)BG,但是量比較低。草酸青霉纖維素酶系含有對生物質(zhì)的糖化至關(guān)重要的裂解性多糖單加氧酶(LPMO)和膨脹蛋白(swollenin)。CBH、EG、BG、LPMO、幾丁質(zhì)酶和淀粉酶在ST中的蛋白比例分別為37.15%、15.03%、0.66%、2.11%、0.02%和0.01%,而在SP中的比例分別為39.00%、11.27%、0.25%、1.37%、0.19%和0.44%。與草酸青霉纖維素酶SP相比,在ST蛋白質(zhì)組中檢測到了SP所沒有的幾種蛋白,它們是纖維素誘導(dǎo)蛋白(Cip1和Cip2)、木葡聚糖酶、a-1,6-甘露聚糖酶、疏水蛋白和細(xì)胞壁蛋白。SP含有更多的碳水化合物結(jié)合模塊第一家族(CBM1)的蛋白,這種差異可能造成了SP更大的底物吸附,從而導(dǎo)致酶解效率低于里氏木霉纖維素酶。采用蛋白質(zhì)組學(xué)工具分析了吸附對纖維素酶系的影響,實(shí)驗(yàn)表明糖化過程中LPMOs和膨脹蛋白在糖化上清液中含量非常低,積極參與了生物質(zhì)水解。含CBM1過多的蛋白組分可能會產(chǎn)生較多非生產(chǎn)性吸附,阻礙其對含復(fù)雜成分纖維素底物的降解。本研究提供了草酸青霉和里氏木霉全面的酶系組成和相對含量,為優(yōu)化和合理設(shè)計(jì)高效低成本纖維素酶系奠定了基礎(chǔ)。3.探索了草酸青霉內(nèi)切酶Cel5B和外切酶Cel7A-2的酶學(xué)性質(zhì)和糖化添加效果草酸青霉內(nèi)切酶Cel5B和外切酶Cel7A-2是其纖維素酶系中含量第1和第8的蛋白,這兩種蛋白已經(jīng)在草酸青霉蛋白表達(dá)菌A11△中得到表達(dá)。為了研究草酸青霉的重要蛋白并確定糖化過程中這兩種重要蛋白的作用,使用分子篩分離純化了重組蛋白rCel5B和重組蛋白rCel7A-2。rCel5B為內(nèi)切纖維素酶,與草酸青霉其他內(nèi)切β-1,4-葡聚糖酶相比,rCel5B具有較好的熱穩(wěn)定性及pH耐受性。當(dāng)溫度為30～50℃時(shí),保溫6小時(shí),酶活仍可保持在90%以上。當(dāng)溫度升高到60℃,保溫6小時(shí)后,酶活降到80%。Cel5B在pH 4.2-6.0范圍內(nèi)可保存95%以上的酶活力。Cel5B對所用底物均有一定的水解能力,其中降解羧甲基纖維素鈉的能力最強(qiáng)。重組蛋白rCel7A-2為外切型纖維素酶。Cel7A-2的最適作用溫度為55℃。當(dāng)溫度為30～50℃時(shí),保溫6小時(shí),酶活仍可保持在90%以上,當(dāng)溫度升高到60℃時(shí),保溫6小時(shí)后,酶活降到60%。Cel7A-2在pH3.6-4.8范圍內(nèi)可保存90%以上的酶活力,最佳pH為4.2,在pH 3可保存70%的酶活力,當(dāng)pH升到5.4,酶活力迅速下降到20%,當(dāng)pH為6.0、6.6時(shí)酶活力幾乎消失。添加Cel5B和Cel7A-2對草酸青霉纖維素酶降解木質(zhì)纖維素底物有一定的促進(jìn)作用。單獨(dú)加入Cel5B和Cel7A-2后,葡萄糖產(chǎn)量均有提高,葡聚糖轉(zhuǎn)化率分別提高了2%和6%。使用中心組合實(shí)驗(yàn)設(shè)計(jì)研究Cel5B和Cel7A-2對于草酸青霉纖維素酶糖化木糖渣的影響,結(jié)果表明當(dāng)酶用量為9 FPA/g纖維素時(shí),添加13%Cel7A-2、21% Cel5B和29%BG時(shí),葡萄糖轉(zhuǎn)化率提高了33%。4.研究了草酸青霉Cu/Zn-SOD及膨脹蛋白對蛋白分泌、纖維素酶合成及木糖渣糖化的影響Cu/Zn-SOD和膨脹蛋白是草酸青霉分泌組中含量較高的纖維素酶輔助蛋白,為了研究這兩種蛋白對于木質(zhì)纖維素底物的作用,以纖維素酶高產(chǎn)菌株4-1為出發(fā)菌株,成功構(gòu)建了二者的缺失菌株。結(jié)果表明缺失Cu/Zn-SOD影響了草酸青霉發(fā)酵液的纖維素酶酶活力與胞外蛋白濃度。而Cu/Zn-SOD本身對生物質(zhì)糖化沒有直接作用。通過表型及酶活測定分析,發(fā)現(xiàn)敲除膨脹蛋白也影響了草酸青霉發(fā)酵液的p-葡萄糖苷酶活力,敲除株表型沒有發(fā)生明顯變化。敲除株粗酶液等蛋白量糖化和等濾紙酶活糖化結(jié)果顯示,敲除膨脹蛋白影響了木糖渣的糖化效率。以實(shí)驗(yàn)室保存的蛋白表達(dá)質(zhì)粒為載體構(gòu)建了膨脹蛋白表達(dá)菌株,用分離純化后的膨脹蛋白進(jìn)行外源添加糖化實(shí)驗(yàn),發(fā)現(xiàn)膨脹蛋白與草酸青霉纖維素酶系有協(xié)同作用。在草酸青霉野生型菌株114-2中成功構(gòu)建了膨脹蛋白過表達(dá)菌株,以優(yōu)化草酸青霉纖維素酶系。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q936
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本文編號：2033542