本文關(guān)鍵詞：纖維素酶補(bǔ)料發(fā)酵工藝及木糖渣生產(chǎn)葡萄糖酸鈉的研究　出處：《山東大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 草酸青霉 纖維素酶 補(bǔ)料發(fā)酵 葡萄糖酸鈉 酶固定化 統(tǒng)合生物加工

【摘要】：當(dāng)前石油、煤炭等化石日趨枯竭,能源短缺、環(huán)境惡化等問(wèn)題日益嚴(yán)峻,急需開(kāi)發(fā)化石資源的替代品�？沙掷m(xù)再生的木質(zhì)纖維素資源儲(chǔ)量豐富,并且未得到充分的開(kāi)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)化為液體燃料和化學(xué)品,是解決資源、能源和環(huán)境問(wèn)題的有效途徑。本論文對(duì)以木質(zhì)纖維素為原料生產(chǎn)纖維素酶和葡萄糖酸鈉的工藝進(jìn)行了研究,優(yōu)化了現(xiàn)場(chǎng)生產(chǎn)纖維素酶的發(fā)酵策略,以及雙酶法催化纖維素水解液生產(chǎn)葡萄糖酸鈉的工藝,并以草酸青霉為底盤(pán)構(gòu)建了能夠進(jìn)行葡萄糖酸鈉CBP生產(chǎn)的工程菌株,為未來(lái)木質(zhì)纖維素生產(chǎn)葡萄糖酸鈉實(shí)現(xiàn)產(chǎn)業(yè)化提供了一條完整的技術(shù)路線。本論文的主要研究?jī)?nèi)容和結(jié)果如下:1.用反復(fù)分批補(bǔ)料發(fā)酵策略提高了草酸青霉RE-10纖維素酶容積生產(chǎn)效率選擇廉價(jià)的天然原材料如麩皮,脫木素木糖渣和豆餅粉及少量無(wú)機(jī)鹽作為草酸青霉RE-10纖維素酶發(fā)酵培養(yǎng)基的主要成分,使用Plackett-Burman設(shè)計(jì)篩選發(fā)現(xiàn)麩皮和NaN03對(duì)纖維素酶的生產(chǎn)有顯著影響。經(jīng)過(guò)中心組合設(shè)計(jì)(Central composite design)優(yōu)化后得到最佳培養(yǎng)基配方,并在7.5 L發(fā)酵罐上驗(yàn)證,濾紙酶活(FPase)在120 h達(dá)到12.69 U/mL,較原始培養(yǎng)基配方提高1.76倍,CMCase,pNPCse,pNPGse活力同樣比原始培養(yǎng)基有顯著提高。采用反復(fù)分批補(bǔ)料發(fā)酵策略減少了發(fā)酵過(guò)程中的輔助時(shí)間,最終濾紙酶活的容積生產(chǎn)效率從66.85 U/L/h(分批發(fā)酵)提高到158.38 U/L/h。SDS-PAGE分析發(fā)現(xiàn)不同時(shí)間點(diǎn)的粗酶液之間沒(méi)有明顯的差別。用糖化脫木素木糖渣(DCCR)實(shí)驗(yàn)比較了不同時(shí)間點(diǎn)粗酶液、分批發(fā)酵粗酶液的纖維素降解能力,發(fā)現(xiàn)釋放出的糖量和葡聚糖轉(zhuǎn)化率沒(méi)有明顯差異。反復(fù)分批補(bǔ)料發(fā)酵的策略大幅提高了濾紙酶活的容積生產(chǎn)效率,在絲狀真菌纖維素酶工業(yè)生產(chǎn)方面具有較大的應(yīng)用潛力。2.亞銨黑液誘導(dǎo)草酸青霉分泌纖維素酶并可用于流加補(bǔ)料發(fā)酵工藝對(duì)亞銨黑液成分進(jìn)行分析,其組分中至少含有90-120 g/L低聚合度的半纖維素寡糖、30-50 g/L葡聚糖和20-40 g/L的單糖,此外以NH4+計(jì)殘銨含量為8-10 g/L。研究了添加亞銨黑液對(duì)草酸青霉RE-10在搖瓶中發(fā)酵產(chǎn)纖維素酶的影響,發(fā)現(xiàn)添加適量亞銨黑液可以提高纖維素酶的產(chǎn)量。但過(guò)量添加亞銨黑液時(shí),亞銨黑液含有的糠醛、S02、揮發(fā)性有機(jī)酸等物質(zhì)會(huì)抑制草酸青霉的生長(zhǎng)和產(chǎn)酶。以1 mL/L/h的速度連續(xù)流加亞銨黑液補(bǔ)料發(fā)酵生產(chǎn)纖維素酶,在144 h發(fā)酵液中濾紙酶活力達(dá)到16.12 U/mL,比分批發(fā)酵的最高酶活12.70 U/mL(120 h)提高了 26.93%。考慮到在發(fā)酵過(guò)程中纖維素酶在48 h才開(kāi)始大量合成,采用變速流加亞銨黑液的策略補(bǔ)料發(fā)酵生產(chǎn)纖維素酶,在144 h濾紙酶活達(dá)到17.66 U/mL,此時(shí)蛋白濃度、內(nèi)切纖維素酶、外切纖維素酶和β-糖苷酶活力分別為7.50 mg/mL、40.39 U/mL、1.77 U/mL和2.31 U/mL。變速流加策略沒(méi)有出現(xiàn)前期過(guò)量流加亞銨黑液時(shí)還原糖濃度一直較高的情況,說(shuō)明該策略減輕了亞銨黑液所含的抑制物在發(fā)酵前期對(duì)草酸青霉生長(zhǎng)的抑制。測(cè)定分批發(fā)酵和變速流加亞銨黑液補(bǔ)料發(fā)酵所生產(chǎn)的纖維素酶的各項(xiàng)酶活力和蛋白濃度,發(fā)現(xiàn)濾紙酶比活力從1.74U/mg提高到2.40U/mg。比較不同發(fā)酵策略生產(chǎn)的纖維素酶粗酶液在水解10%的DCCR時(shí)的效果,發(fā)現(xiàn)按等濾紙酶活添加纖維素酶(15 FPU/g葡聚糖)進(jìn)行水解的效果差異不大;而按等蛋白添加纖維素酶進(jìn)行水解時(shí),流加亞銨黑液補(bǔ)料發(fā)酵所產(chǎn)的粗酶液優(yōu)于分批發(fā)酵粗酶液。利用基于LC-MS/MS平臺(tái)的非標(biāo)定量蛋白組學(xué)手段,定性和相對(duì)定量地分析了變速流加亞銨黑液補(bǔ)料發(fā)酵和分批發(fā)酵兩者分泌組中蛋白的種類(lèi)和豐度,發(fā)現(xiàn)與分批發(fā)酵粗酶液相比,變速流加亞銨黑液補(bǔ)料發(fā)酵的粗酶液有21個(gè)蛋白豐度上調(diào),這些蛋白大部分都與生物質(zhì)的降解有關(guān),很好的解釋了比酶活提高的原因。用里氏木霉SN1驗(yàn)證了流加亞銨黑液補(bǔ)料發(fā)酵生產(chǎn)纖維素酶工藝,以與分批發(fā)酵相比,FPase、CMCase、pNPCase 和/pPGase 酶活分別提高了 44.98%、39.83%、72.17%、59.21%,表明流加亞銨黑液同樣對(duì)里氏木霉具有誘導(dǎo)分泌纖維素酶的作用。3.共固定化雙酶催化纖維素DCCR水解液生產(chǎn)葡萄糖酸鈉前期工作發(fā)現(xiàn),額外補(bǔ)加β-葡萄糖苷酶能夠提高纖維素糖化的效果。使用過(guò)表達(dá)β-葡萄糖苷酶的草酸青霉11-13菌株,以變速流加亞銨黑液補(bǔ)料發(fā)酵的策略生產(chǎn)纖維素酶,經(jīng)過(guò)168 h發(fā)酵,FPase、pNPGase分別達(dá)到13.63 U/mL、110.73 U/mL。對(duì)DCCR補(bǔ)料分批糖化工藝進(jìn)行了底物濃度、纖維素酶用量、補(bǔ)料策略等一系列優(yōu)化,確定最優(yōu)的水解工藝為初始底物濃度為15%,纖維素酶初始用量為26FPU/g葡聚糖,分別在24h,48h和72h補(bǔ)加3.33%的DCCR,補(bǔ)料同時(shí)按12 FPU/g葡聚糖補(bǔ)加與補(bǔ)料量相對(duì)應(yīng)的纖維素酶。在7.5 L攪拌式生物反應(yīng)器上驗(yàn)證了上述工藝,經(jīng)過(guò)120 h水解后水解液葡萄糖濃度達(dá)到145.80 g/L。優(yōu)化了 GOD-CAT共固定化酶顆粒制備工藝。在發(fā)酵罐中考察了反應(yīng)條件對(duì)使用GOD-CAT共固定化酶催化DCCR水解液生產(chǎn)葡萄糖酸鈉的影響,最佳反應(yīng)條件為GOD用量16U/g葡萄糖,pH5.4,溫度36℃,攪拌轉(zhuǎn)速300rpm,通風(fēng)量1.25 VVM,反應(yīng)48 h可以得到166.87 g/L的葡萄糖酸鈉,轉(zhuǎn)化率為98.24%。固定化酶反復(fù)使用6次以后仍然能夠保持60%以上的活力。4.以草酸青霉為底盤(pán)構(gòu)建了葡萄糖酸鈉CBP生產(chǎn)工程菌株利用草酸青霉A11△表達(dá)宿主表達(dá)了黑曲霉來(lái)源的GOD和CAT,SDS-PAGE顯示目的蛋白得到了表達(dá),對(duì)相應(yīng)酶活進(jìn)行了測(cè)定,結(jié)果表明黑曲霉來(lái)源的GOD和CAT可以在草酸青霉中正確表達(dá)。以草酸青霉114-2為出發(fā)菌株,以gpdA(p)為啟動(dòng)子成功構(gòu)建了同時(shí)過(guò)表達(dá)GOD和CAT的葡萄糖酸鈉CBP生產(chǎn)工程菌株z19,搖瓶發(fā)酵實(shí)驗(yàn)發(fā)現(xiàn)z19菌株的胞外纖維素酶活力與出發(fā)菌株114-2相比沒(méi)有明顯差別,但具有較高的GOD和CAT活力。采用兩階段控溫發(fā)酵策略,在發(fā)酵前120h以產(chǎn)酶為主,溫度控制在30℃;在120h后補(bǔ)加濾紙粉和Na2CO3,并將溫度提高到45℃進(jìn)行酶解轉(zhuǎn)化72 h后可以得到13.54 g/L的葡萄糖酸鈉,成功實(shí)現(xiàn)了以纖維素為原料"一鍋法"生產(chǎn)葡萄糖酸鈉。
[Abstract]:At present, fossil oil, coal and other fossils are increasingly exhausted, energy shortage, environmental degradation and other problems are becoming increasingly severe, and it is urgent to develop alternative materials for fossil resources. The resources of sustainable regenerated lignocellulose are abundant and have not been fully exploited and utilized, and some of them also cause environmental pollution. It is an effective way to solve the problems of resources, energy and environment by producing cellulase by microorganism, and then converting the renewable biomass into liquid fuel and chemicals. This paper studied the process of raw material for the production of cellulose and sodium gluconate with lignocellulose as the optimized fermentation strategy for cellulase production site, and the production process of sodium gluconate by double enzyme catalyzed hydrolysis liquid, and by p.oxalicum constructed the chassis to the engineering strain of sodium gluconate CBP production. Provide a complete technical route for the future production of sodium gluconate lignocellulose industrialization. The main research contents and results are as follows: 1. with repeated fed batch fermentation strategy to improve the natural raw materials such as wheat bran p.oxalicum RE-10 cellulase production efficiency volume of low-cost, the main component of delignification xylose residue and soybean powder and a small amount of inorganic salt as Penicillium oxalicum RE-10 cellulose enzyme fermentation medium, screening there is a significant effect of wheat bran and NaN03 on cellulase production using Plackett-Burman. The central composite design (Central composite design) after the optimization, the best medium, and validated in 7.5 L fermentor, the filter paper activity (FPase) in 120 h to 12.69 U/mL, compared with the original culture medium increased by 1.76 times, CMCase, pNPCse, pNPGse activity also increased significantly than that of the original medium. The fed batch fermentation strategy was used to reduce the auxiliary time in the fermentation process. Finally, the volume production efficiency of the filter paper enzyme activity increased from 66.85 U/L/h (batch fermentation) to 158.38 U/L/h. SDS-PAGE analysis showed that there was no significant difference between the crude enzyme solutions at different time points. The cellulose degradation ability of crude enzyme solution and batch fermentation of crude enzyme solution at different time points was compared with saccharification and Delignification xylose dregs (DCCR). It was found that the amount of sugar released and the conversion rate of dextran were not significantly different. The strategy of batch fed batch fermentation greatly improves the volume production efficiency of filter paper enzyme activity, and has great potential in filamentous cellulase industrial production. 2. ammonium sulfite liquor by Penicillium oxalicum cellulase and can be used to feed batch fermentation process were analyzed for ammonium sulfite liquor composition, the group of monosaccharide oligosaccharide, in hemicellulose containing at least 90-120 g/L 30-50 g/L low-polymirazed dextran and 20-40 g/L, in addition to NH4+ 8-10 g/L for residual ammonium content. The effect of adding ammonium sulfite black liquor on cellulase production from Penicillium oxalicum RE-10 in shaking flask was studied. It was found that adding appropriate amount of ammonium sulfite black liquor can increase the yield of cellulase. However, when ammonium subammonium black liquor was added, the furfural, S02 and volatile organic acids contained in the black liquor could inhibit the growth and enzyme production of Penicillium oxalate. With the continuous speed with 1 mL/L/h ammonium liquor feeding cellulase production fermentation in 144 h fermentation activity reached 16.12 U/mL, the highest enzyme in batch fermentation activity of 12.70 U/mL (120 h) increased by 26.93%. Taking into account the cellulase in the fermentation process began in large quantities at 48 h, with variable flow with ammonium liquor feeding strategy of cellulase production by fermentation, in the 144 h filter paper enzyme activity reached 17.66 U/mL, the concentration of protein, endo cellulase, cellobiohydrolase and beta glucosidase activity were 7.50 mg/mL, 40.39 U/mL, 1.77 U/mL and 2.31 U/mL. Variable speed feeding strategy does not appear excessive pre flow with ammonium liquor when reducing sugar concentration has been higher, indicating inhibition of the strategy to reduce the ammonium sulfite black liquor containing inhibitor on the growth of Penicillium oxalicum in prophase of fermentation. Determination of cellulase with variable flow batch fermentation and fed batch fermentation liquor of ammonium produced by the enzyme activity and protein concentration, found that the filter paper enzyme specific activity increased from 1.74U/mg to 2.40U/mg. To compare the effect of different strategies of fermentation production of crude enzyme liquid in cellulase hydrolysis of 10% DCCR, found by filter paper activity cellulase (15 FPU/g dextran) had little differences in the effect of hydrolysis; and by adding cellulase hydrolysis of protein, crude enzyme enzyme is better than that of black liquid flow with ammonium fed batch fermentation produced by batch fermentation. Based on non standard quantitative proteomics LC-MS/MS platform by means of qualitative and quantitative analysis of the species and abundance of variable flow with ammonium fed batch fermentation liquor fermentation and secretion of protein in both groups, compared with batch fermentation crude enzyme solution, crude enzyme solution with variable flow of ammonium fed batch fermentation with 21 black liquor protein abundance increases, most of these proteins and the degradation of biomass, very good explanation for the specific enzyme activity increased. With Trichoderma reesei SN1 was verified with ammonium liquor feeding flow production of cellulase fermentation, compared with batch fermentation, FPase, CMCase, pNPCase and /pPGase activity were increased by 44.98%, 39.83%, 72.17%, 59.21%, with black liquor also has indicated that ammonium induced secretion of cellulase by Trichoderma reesei. 3. co immobilized double enzyme catalyzed production of sodium gluconate from cellulose DCCR hydrolysate. Previous work found that supplementation of beta glucosidase could improve cellulose saccharification. Using Penicillium oxalicum expression p-glucosidase strain 11-13, with variable flow with ammonium liquor feeding fermentation strategy for cellulase production, after 168 h fermentation, FPase and pNPGase are respectively 13.63 U/mL and 110.73 U/mL. A series of optimization of substrate concentration, cellulase dosage and feeding strategy were carried out for DCCR fed batch saccharification process, and the optimal hydrolysis process was determined as the initial substrate concentration.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ929

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