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固态发酵漆酶及其应用
Alternative TitleSolid state fermentation of laccase and its application
李冠华
Subtype博士
Thesis Advisor陈洪章
2014-04
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword固态发酵   固态基质   漆酶   漆酶应用
Abstract相比于液态发酵,固态发酵具有原料廉价,过程节能、节水、产品得率高等优点。固态基质是固态发酵的核心,但固态基质的研究相对薄弱,在一定程度上阻碍了固态发酵的发展。漆酶是一种含铜的多酚金属氧化酶,被称为绿色化学中的蓝色酶,以漆酶为核心的催化体系作用底物范围广,与人类关系密切,在工业生物催化中具有广泛的应用前景。但漆酶生产成本较高,成为制约漆酶开发应用的瓶颈。漆酶作为重要的木质素氧化酶,在生物质高值化的应用中有待加强。 针对上述问题,论文以固态基质为切入点解析固态发酵漆酶的过程,研究了固态基质与微生物的相互关系,固态基质水分布特点,汽爆预处理改性固态基质。在此基础上建立了固态发酵漆酶新工艺,研究了漆酶脱毒汽爆秸秆半纤维素水洗液,漆酶氧化提取汽爆秸秆木质素,漆酶纤维素酶协同酶解汽爆秸秆纤维素。 (1)从固态基质微观物理特性入手,分析固态基质多孔特征,以及汽爆预处理对固态基质微观多孔分布的影响。结果表明固态基质天然致密结构是影响微生物生长的重要因素,汽爆预处理可以改变固态基质微观多孔分布特征,促进微生物在固态基质中的生长。 (2)建立了铵盐预浸汽爆改性固态基质技术,研究不同汽爆预处理条件下固态基质水分布特征,固态基质含氮量、可降解性、发酵产酶量变化等。结果表明固态基质中水分为吸附水、毛管水和自由水,当含水量较低时固态基质中主要为结合水,随着含水量增加,固态基质中毛管水含量逐渐增多,水吸收峰逐渐向左移动且与固态基质结合力逐渐减弱;计算固态基质最大持水量,基质水分蒸发速率,低场核磁成像,结果表明汽爆预处理有助于提高固态基质持水能力和保水能力。铵盐预浸汽爆可以将外源铵离子引入固态基质,外源铵盐以可溶性氮源和不可溶性氮源两种形式存在。相对于未处理固态基质,铵盐预浸汽爆提高固态基质可降解性110%,提高固态发酵漆酶产量69%、提高木聚糖酶产量293%、提高纤维素酶产量274%,提高乙醇产量193%。 (3)筛选分离漆酶高产菌株,研究固态基质、氮源、诱导物对漆酶产量的影响。结果表明分离得到的硬毛粗盖孔菌可以利用花生壳为固态基质,以(NH4)2SO4为氮源,以CuSO4和皂苷为诱导物发酵15 d时,漆酶产量最大可以达到1169 U/g。在此基础上建立了气相双动态固态发酵漆酶新工艺,与传统的静态发酵方式相比,漆酶产量提高35%,不同料层间漆酶产量、含水量差异性缩小。该菌漆酶粗酶液最适催化温度50℃,pH值为3.5~4.5,在60℃保存3 h,酶活力仍可保持75%。 (4)以药用植物甘草为基质,固态发酵漆酶。结果表明硬毛粗盖孔菌固态发酵12 d时,漆酶产量可以达到162 U/g;固态发酵过程中该菌分泌的木质纤维素酶可以水解纤维素和半纤维素,氧化改性木质素,破坏甘草致密的物理化学结构;发酵甘草可以进一步提取黄酮活性成分,相比于未发酵甘草,提取率提高49%。 (5)建立了漆酶氧化脱毒汽爆秸秆水洗液工艺。结果表明漆酶脱毒处理可以除去香豆酸、香草醛等非挥发性抑制物,去除率达到40%以上。 (6)建立了汽爆耦合漆酶协同处理提取木质素工艺,结果表明汽爆漆酶协同处理增加了汽爆秸秆木质素碱提过程中反应起始作用位点,并提高了提取反应对温度的敏感性,秸秆木质素提取率提高20%。 (7)研究了漆酶-纤维素酶协同酶解汽爆秸秆纤维素,结果表明漆酶-纤维素酶协同酶解可以显著缩短汽爆秸秆纤维素酶解时间,减少酶解过程中纤维素酶用量。
Other AbstractCompared to submerged fermentation, solid state fermentation has many advantages such as water-saving, energy-saving, high-yield and clean production. The solid substrate is the core of solid state fermentation. However, the insufficient understanding of the essence of solid substrate and the lag in research of related process and engineering hinder the development of solid state fermentation. Laccase, blue enzymes for green chemistry, is one of the extracellular glycoprotein enzymes produced from white-rot fungi and other microorganism. Due to the feature of wide distribution and multifarious substrate, the laccase plays a crucial role in environmental pollution control, textile industry, biosensors, food industry and so on. However the cost of laccase is high because of the poor performance of laccase producing strains and the immaturity of fermentation technology, which results in the lag of laccase application. The high-value utilization of biomass is hotspot, as an important lignin oxidase, the application of laccase to this field should be strengthened. Hence, the solid substrate is used as the research point to analyse solid state fermentation process. The relationship between the solid substrate and microorganism, the distribution characteristics of water in solid substrate, and the pretreatment of solid substrate were studied. The novle laccase solid state fermentation technology was established. The application of laccase to the delignification of biomass, detoxification of steam exploded straw and the enzymatic hydrolysis of cellulose were studied. (1) The relationship between the physical structure and fungal growth was studied and the steam explosion was used to pretreat the solid subtrate. The effect of steam explosion (SE) on the solid substrate such as pore size distribution, crystallinity index, chemical compositions and enzymatic digestibility was evaluated. Results revealed that the rigid physical structure of solid substrate prevented the growth of fungi. What’s more, steam explosion destroyed the rigid structure of solid substrate and facilitated the following fungal growth. (2) Steam explosion associated with NH4Cl preimpregnation was carried out, and the effects of the pretreatment on characteristic of water distribution, nitrogen content, enzymatic digestibility, enzeme production and ethanol production were studied. The results showed that the water in solid substrate can be divided into three types: absorption water, capillary water and free water; when the water content was low, the majority of water was capillary water. The bonding connection between the substrate and the water was weakened and the water amplitude peak moved left gradually when the water content increased. The water holding ability and the water retaining capacity of solid substrate were improved by the pretreatment of steam explosion (SE) associated with NH4Cl preimpregnation. The ammonium can be introduced into the solid substrate and transformed into soluble nitrogen and insoluble nitrogen which can be used as nitrogen resource for fermentation. The enzymatic digestibility, laccase production, cellulase production, xylanase production, cellulase production, and ethanol yield from the pretreated sample were 1.1, 0.69, 2.93, 2.74, and 1.93 times higher than that of untreated sample, respectively. (3) A laccase producing strain was isolated from the rooten wood in nature, and the effect of solid substrate, nitrogen resource, and inducer on laccase production were studied. The results showed that the highest laccase production by Funalia trogii was 1169 U/g, when the solid state fermentation using peanut shell as substrate, (NH4)2SO4 as nitrogen resource, CuSO4 and saponin as inducer proceeded for 15 d. The novel gas double dynamic solid state fermentation was used to produce laccase. Compared to the static solid state fermentation, the laccase yield increased by 35%, and the nonuniformity of laccase yield and the water co
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/15547
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
李冠华. 固态发酵漆酶及其应用[D]. 中国科学院研究生院,2014.
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