纤维素酶补料发酵工艺及木糖渣生产葡萄糖酸钠的研究

发布时间：2017-12-28 16:29

本文关键词：纤维素酶补料发酵工艺及木糖渣生产葡萄糖酸钠的研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：当前石油、煤炭等化石日趋枯竭,能源短缺、环境恶化等问题日益严峻,急需开发化石资源的替代品。可持续再生的木质纤维素资源储量丰富,并且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化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。本论文对以木质纤维素为原料生产纤维素酶和葡萄糖酸钠的工艺进行了研究,优化了现场生产纤维素酶的发酵策略,以及双酶法催化纤维素水解液生产葡萄糖酸钠的工艺,并以草酸青霉为底盘构建了能够进行葡萄糖酸钠CBP生产的工程菌株,为未来木质纤维素生产葡萄糖酸钠实现产业化提供了一条完整的技术路线。本论文的主要研究内容和结果如下:1.用反复分批补料发酵策略提高了草酸青霉RE-10纤维素酶容积生产效率选择廉价的天然原材料如麸皮,脱木素木糖渣和豆饼粉及少量无机盐作为草酸青霉RE-10纤维素酶发酵培养基的主要成分,使用Plackett-Burman设计筛选发现麸皮和NaN03对纤维素酶的生产有显著影响。经过中心组合设计(Central composite design)优化后得到最佳培养基配方,并在7.5 L发酵罐上验证,滤纸酶活(FPase)在120 h达到12.69 U/mL,较原始培养基配方提高1.76倍,CMCase,pNPCse,pNPGse活力同样比原始培养基有显著提高。采用反复分批补料发酵策略减少了发酵过程中的辅助时间,最终滤纸酶活的容积生产效率从66.85 U/L/h(分批发酵)提高到158.38 U/L/h。SDS-PAGE分析发现不同时间点的粗酶液之间没有明显的差别。用糖化脱木素木糖渣(DCCR)实验比较了不同时间点粗酶液、分批发酵粗酶液的纤维素降解能力,发现释放出的糖量和葡聚糖转化率没有明显差异。反复分批补料发酵的策略大幅提高了滤纸酶活的容积生产效率,在丝状真菌纤维素酶工业生产方面具有较大的应用潜力。2.亚铵黑液诱导草酸青霉分泌纤维素酶并可用于流加补料发酵工艺对亚铵黑液成分进行分析,其组分中至少含有90-120 g/L低聚合度的半纤维素寡糖、30-50 g/L葡聚糖和20-40 g/L的单糖,此外以NH4+计残铵含量为8-10 g/L。研究了添加亚铵黑液对草酸青霉RE-10在摇瓶中发酵产纤维素酶的影响,发现添加适量亚铵黑液可以提高纤维素酶的产量。但过量添加亚铵黑液时,亚铵黑液含有的糠醛、S02、挥发性有机酸等物质会抑制草酸青霉的生长和产酶。以1 mL/L/h的速度连续流加亚铵黑液补料发酵生产纤维素酶,在144 h发酵液中滤纸酶活力达到16.12 U/mL,比分批发酵的最高酶活12.70 U/mL(120 h)提高了 26.93%。考虑到在发酵过程中纤维素酶在48 h才开始大量合成,采用变速流加亚铵黑液的策略补料发酵生产纤维素酶,在144 h滤纸酶活达到17.66 U/mL,此时蛋白浓度、内切纤维素酶、外切纤维素酶和β-糖苷酶活力分别为7.50 mg/mL、40.39 U/mL、1.77 U/mL和2.31 U/mL。变速流加策略没有出现前期过量流加亚铵黑液时还原糖浓度一直较高的情况,说明该策略减轻了亚铵黑液所含的抑制物在发酵前期对草酸青霉生长的抑制。测定分批发酵和变速流加亚铵黑液补料发酵所生产的纤维素酶的各项酶活力和蛋白浓度,发现滤纸酶比活力从1.74U/mg提高到2.40U/mg。比较不同发酵策略生产的纤维素酶粗酶液在水解10%的DCCR时的效果,发现按等滤纸酶活添加纤维素酶(15 FPU/g葡聚糖)进行水解的效果差异不大;而按等蛋白添加纤维素酶进行水解时,流加亚铵黑液补料发酵所产的粗酶液优于分批发酵粗酶液。利用基于LC-MS/MS平台的非标定量蛋白组学手段,定性和相对定量地分析了变速流加亚铵黑液补料发酵和分批发酵两者分泌组中蛋白的种类和丰度,发现与分批发酵粗酶液相比,变速流加亚铵黑液补料发酵的粗酶液有21个蛋白丰度上调,这些蛋白大部分都与生物质的降解有关,很好的解释了比酶活提高的原因。用里氏木霉SN1验证了流加亚铵黑液补料发酵生产纤维素酶工艺,以与分批发酵相比,FPase、CMCase、pNPCase 和/pPGase 酶活分别提高了 44.98%、39.83%、72.17%、59.21%,表明流加亚铵黑液同样对里氏木霉具有诱导分泌纤维素酶的作用。3.共固定化双酶催化纤维素DCCR水解液生产葡萄糖酸钠前期工作发现,额外补加β-葡萄糖苷酶能够提高纤维素糖化的效果。使用过表达β-葡萄糖苷酶的草酸青霉11-13菌株,以变速流加亚铵黑液补料发酵的策略生产纤维素酶,经过168 h发酵,FPase、pNPGase分别达到13.63 U/mL、110.73 U/mL。对DCCR补料分批糖化工艺进行了底物浓度、纤维素酶用量、补料策略等一系列优化,确定最优的水解工艺为初始底物浓度为15%,纤维素酶初始用量为26FPU/g葡聚糖,分别在24h,48h和72h补加3.33%的DCCR,补料同时按12 FPU/g葡聚糖补加与补料量相对应的纤维素酶。在7.5 L搅拌式生物反应器上验证了上述工艺,经过120 h水解后水解液葡萄糖浓度达到145.80 g/L。优化了 GOD-CAT共固定化酶颗粒制备工艺。在发酵罐中考察了反应条件对使用GOD-CAT共固定化酶催化DCCR水解液生产葡萄糖酸钠的影响,最佳反应条件为GOD用量16U/g葡萄糖,pH5.4,温度36℃,搅拌转速300rpm,通风量1.25 VVM,反应48 h可以得到166.87 g/L的葡萄糖酸钠,转化率为98.24%。固定化酶反复使用6次以后仍然能够保持60%以上的活力。4.以草酸青霉为底盘构建了葡萄糖酸钠CBP生产工程菌株利用草酸青霉A11△表达宿主表达了黑曲霉来源的GOD和CAT,SDS-PAGE显示目的蛋白得到了表达,对相应酶活进行了测定,结果表明黑曲霉来源的GOD和CAT可以在草酸青霉中正确表达。以草酸青霉114-2为出发菌株,以gpdA(p)为启动子成功构建了同时过表达GOD和CAT的葡萄糖酸钠CBP生产工程菌株z19,摇瓶发酵实验发现z19菌株的胞外纤维素酶活力与出发菌株114-2相比没有明显差别,但具有较高的GOD和CAT活力。采用两阶段控温发酵策略,在发酵前120h以产酶为主,温度控制在30℃;在120h后补加滤纸粉和Na2CO3,并将温度提高到45℃进行酶解转化72 h后可以得到13.54 g/L的葡萄糖酸钠,成功实现了以纤维素为原料"一锅法"生产葡萄糖酸钠。
[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.
【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】：TQ929

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