【作者】 朱友双；

【导师】 黄峰；

【作者基本信息】 山东大学 ， 微生物学， 2011， 博士

【摘要】 生物质既是可再生能源,为人类提供能量；也是可再生资源,为人类提供物质性生产所需的原料。农作物秸秆是地球上最为丰富的生物质资源,这些生物质主要由三种成分组成,分别为纤维素、半纤维素、木质素,其中木质素是仅次于纤维素的第二大有机碳源,木质素难降解,并且木质素起到包被作用,因此木质素的存在阻碍了生物质的转化利用,木质素的降解构成了自然界碳素循环的限速步骤。白腐真菌是自然界中一类唯一能够彻底降解木质素的微生物,杂色云芝是其典型菌种。白腐真菌降解木质素主要依靠细胞分泌的木质素降解酶系统,其中漆酶(laccase)是木质素质降解酶系统中的代表性酶种。漆酶具有很好的底物广泛性(通过单电子传递机制能够氧化单酚、双酚、多酚、芳香族胺、对甲氧基酚以及维生素C等),漆酶氧化底物的过程中伴随着还原氧气生成水。漆酶以氧气为电子受体氧化酚类和部分非酚类化合物的特性以及漆酶具有非常宽的底物专一性,使得漆酶被广泛应用于纸浆的去木质化、染料污水处理、生物能源、生物传感器、食品饮料行业以及有机合成和药物合成中。本论文利用实验室筛选出的高产漆酶菌株Trametes versicolor sdu-4进行了液态和固态发酵培养,对所产漆酶进行了纯化,并研究了该漆酶的生物化学性质及其在染料脱色中的应用。各种生物质材料,如木材和农作物秸秆生产的第二代生物乙醇,有望成为一种有价值的汽油替代品或补足物。生物乙醇生产中关键的一步是将生物质中的纤维素水解为单糖,利用纤维素酶将纤维素水解为葡萄糖是最有发展前景的方法之一。但为了使原料更容易与酶接触,需要对木质纤维素原料进行各种类型的预处理。离子液体是近年来兴起的一类极具应用前景的绿色溶剂,以其良好溶解性、不挥发性、在水和空气中稳定等优点而被广泛应用。近年来的研究表明,离子液体可以用来溶解术质纤维素材料。本论文进行了离子液体对秸秆及木粉预处理的研究。本论文的主要研究内容及结果如下：1.菌株的鉴定。本实验室前期研究中利用愈创木酚培养基平板变色法筛选得到一株高效产漆酶的白腐真菌,并且该菌株生长迅速。通过子实体及菌丝体形态观察,并结合ITS1-5.8S rDNA-ITS2序列分析,该菌株被鉴定为杂色云芝菌,并被命名为Trametes versicolor sdu-4。2.T. versicolor sdu-4液态发酵产漆酶培养条件的优化通过部分因子试验、中心组合试验和响应面分析试验对影响T. versicolor sdu-4液态发酵产漆酶的因素进行了分析和优化,结果表明合成培养基中碳源(glucose)、氮源(yeast extract、CUSO4的浓度以及培养基初始pH值在漆酶液态发酵生产中是最为关键的影响因素。通过优化获得产漆酶的最佳培养条件为葡萄糖3.31g1-,酵母粉8.051 g1-,CuSO4 8.36 mg l-1,培养基初始pH为4.8,在此条件下漆酶最高产量为4146U1-。优化结果可以为发酵过程中碳源、氮源和CuSO4的合理添加提供指导,从而有效的提高漆酶产量,并降低发酵成本。3.T. versicolor sdu-4漆酶纯化和生物化学性质的研究对T. versicolor sdu-4漆酶进行了分离纯化,然后研究了该漆酶的生物化学性质,依据T. versicolor sdu-4漆酶的生物化学性质和光谱学特征,该漆酶为典型的真菌“蓝色”漆酶,其特征吸收光谱在600 nm和320 nm处都有光吸收。该漆酶的分子量为60 kDa；与已报道的其它漆酶的等电点接近,该漆酶等电点大约为3.0;金属离子含量测定表明,T. versicolor sdu-4漆酶中不含铁离子、锰离子和锌离子,每分子蛋白质中含有4个铜离子：该蛋白质的N末端氨基酸序列为AIGPAASLVVANA;氧化ABTS、DMP和丁香醛连氮的最适pH分别为2.2,3.7和7；该漆酶具有不同于其他典型真菌漆酶的特性,如热稳定性高,pH稳定范围广(3～10),对重金属有一定的耐受性,更宽的底物专一性,使其具有更好的理论研究价值和应用前景。该漆酶氧化ABTS的最适反应温度为75℃,当温度高于80℃,酶活在90 min时就完全丧失,但是70℃。C时的半衰期达到2.2h。漆酶能够在不存在介体的情况下直接氧化溴酚类物质(2-溴苯酚和4-溴苯酚)和非酚型化合物甲基红,具有广阔的应用前景。一般典型真菌“蓝色”漆酶不具备T. versicolor sdu-4漆酶的上述特性,这些特性仅在少量非典型性的“黄漆酶”或者“白漆酶”中有类似报道,表明T. versicolor sdu-4漆酶是一种新的真菌“蓝色”漆酶。本章还进行了T. versicolor sdu-4漆酶染料脱色的研究。选用八种不同类型的染料,分别研究了有无介体存在两种条件下T. versicolor sdu-4漆酶催化染料脱色的反应情况。研究表明不添加介体时T. versicolor sdu-4漆酶能够对偶氮染料甲基红和三苯甲烷染料溴酚蓝、亮绿、结晶紫和甲酚红很好地脱色,在有介体ABTS、TEMPO和HBT存在时漆酶也可以对三苯甲烷染料酸性品红、碱性品红和考马斯亮蓝R-250有效脱色。结果表明,T. versicolor sdu-4漆酶在染料脱色中有着比较好的应用前景。4.T. versicolor sdu-4固态发酵研究以稻草、麦草、玉米秸杆、甘蔗渣和木粉为固体基质,进行T. versicolor sdu-4固态发酵,发现以玉米秸秆(Corn stover, CS)为介质时漆酶产量最高。进而通过部分因子试验、中心组合试验和响应面分析试验对T. versicolor sdu-4在玉米秸秆中的固态发酵进行了详细的研究。结果表明T. versicolor sdu-4利用玉米秸秆进行固态发酵时产漆酶、纤维素酶(CMCase)和木聚糖酶(Xylanase),未检测到木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)。产漆酶的最优固态发酵条件为葡萄糖9mgg-1 CS, CuSO4 4.5μM g-1 CS,介质初始含水最80%,最高漆酶产量为45.1 Ug-1 CS。玉米秸秆中木质素降解的最优发酵条件为葡萄糖9.2 mg g-1 CS; CuSO4 4.3μMg-1 CS;介质初始含水量82%,在此条件下第21天时木质索最高可降解34.8%。5.漆酶在有机试剂、离子液体中的稳定性及其对木质素作用机制的研究由于离子液体几乎无蒸汽压,不具挥发性,因此被称为“绿色溶剂”,同时离子液体还具有液程宽、溶解能力强及可设计性等优点,另外据报道在离子液体中一些酶能保持较高的稳定性和活性,因此,离子液体作为酶的催化反应介质迅速成为研究热点。本文研究了不同浓度DMSO、1,4-二氧六环、离子液体([Bmim]Cl、[Emim][CH3COO]和[Pmim][CH3COO])对漆酶稳定性的影响。研究结果表明,在有机试剂与水、离子液体与水组成的均相溶液中漆酶仍具有较好的稳定性,但是漆酶稳定性随着溶液中有机试剂和离子液体浓度的升高而降低。当有机试剂或者离子液体的浓度在0～30%(w/w)范围内时,漆酶能够很好的保持活性,且在30%的[Bmim]Cl、[Emim][CH3COO]中,48 h后残余酶活仍在60%以上。但在高浓度的有机试剂和离子液体作用下,漆酶活性丧失很快。本论文研究了T. versicolor sdu-4漆酶在30%(W/W)的[Emim][CH3COO]中对酶分离木素(cellulolytic enzyme lignin, CEL)的作用,用红外光谱分析、1H-NMR、13C-NMR等方法对处理前后CEL的结构变化进行了表征。结果表明：T. versicolor sdu-4漆酶在[Emim][CH3COO]中对酶分离木素发生了氧化反应,使其结构中的羰基含量增多；漆酶破坏了酶分离木素中的部分甲氧基,使甲氧基的含量降低；破坏了酶分离木素中的愈创木基芳环和紫丁香基芳环结构。6.离子液体对木粉和秸秆的预处理利用离子液体[Pmim][CH3COO]溶解100目的杨术粉后,加入50%(Ⅴ：Ⅴ)丙酮-水溶液为提取剂,真空抽滤得到再生纤维素物质,将滤液中的丙酮旋蒸去除后真空抽滤可得到离子液体木质素(Ionic liquid-processed lignin, IL-processed lignin)。以酶分离木素为对照样,通过红外光谱和1H-NMR、13C-NMR等方法对离子液体木质素进行表征,分析表明制备的离子液体木质素物质即为木质素。利用[Bmim]Cl、[Emim][CH3COO]和[Pmim][CH3COO]分别对稻草、麦草和玉米秸秆进行了预处理,通过红外光谱和扫描电子显微镜(SEM)对处理前后的秸秆进行了表征,并对处理后的秸秆进行了纤维素酶解,以酶解后还原糖浓度和酶解得率为指标,发现三种离子液体中[Emim][CH3COO]对稻草的处理效果最好,[Pmim][CH3COO]对麦草和玉米秸秆的处理效果最好,说明不同的离子液体对不同木质纤维素材料处理效果也不相同。更多还原

【Abstract】 Biomass is not only the renewable energy source, provides energy to us, but also is the renewable resource, provides raw material which is needed in the production of compounds. Agricultural straw, which mainly consists of cellulose, hemicellulose, and lignin, is the most abundant renewable lignocellulosic biomass. After cellulose, lignin is the most abundant renewable carbon source on earth. The utilization of biomass is hindered by the presence of lignin which is quite resistant to degradation under natural conditions. Lignin degradation process is thought as a rate-determining step of the carbon cycle in the biosphere. White rot fungi are the most efficient lignocellulose de-graders and the only known organisms that can completely break down lignin to carbon dioxide and water. Among them, the most widely studied is Trametes versicolor. Lignin biodegradation by white rot fungi involves various enzymes and the most significant ones are laccases.Laccases can catalyze the oxidation of an array of substrates, such as mono-, di-, and polyphenols, aromatic amines, methoxyphenols, and ascorbate by one-electron transfer mechanism. Because of their high nonspecific oxidation capacity, and their use of readily available molecular oxygen as electron acceptor, laccases are useful as biocatalysts for a wide range of biotechnological applications. Laccases can be used in paper pulp bleaching, decolorization of synthetic dyes, wine clarification, fruit juice processing, bioremediation, ethanol production, biosensors, biofuel cells, organic synthesis, and drug synthesis. In this paper, I studied the submerged and solid-state fermentation by Trametes versicolor sdu-4 which could secret a high level of laccase. T. versicolor sdu-4 laccase was purified and characterized. I also studied its application in dye decolorization.The second generation bio-ethanols which are made form biomass materials, such as straw, wood residues, agricultural residues, are expected to be valuable alternatives of gasoline. A key step in production of bio-ethanol is hydrolysis of lignocellulosic biomass into monosackcharide. Enzymatic hydrolysis of cellulose by cellulase enzyme is the most promising methods. Due to the recalcitrant structure of lignocelluloses, a pretreatment step is needed prior to enzymatic hydrolysis in order to make the cellulose more accessible to cellulase enzyme. Over the past decades, ionic liquids (ILs), often referred to as ’green solvents’, have been the great focus of scientists in various fields due to their unusual physical and chemical properties like high thermal stability, lack of inflammability, low volatility, chemical stability and excellent solubility with many organic compounds. Some studies have shown that lignocellulosic materials can be dissolved in some hydrophilic ionic liquids, such as 1-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1). The pretreatment of the agricultural straw and wood powder by ILs have been studied in the present study.The main results of the present paper are as follows:1. Strain identificationDuring the previous study of our laboratory, we have screened a strain which could secret a high level of laccase by the mothed of guaiacol culture medium plate color deterioration, and the growth of this strain is rapid. The fungus was characterized as Trametes versicolor sdu-4 according to its morphology and ITS1-5.8S rrNA-ITS2 gene sequence.2. Optimization of laccase production by Trametes versicolor sdu-4 under submerged fermentationThe T. versicolor sdu-4 laccase production under submerged fermentation was optimized by the method of factorial design, central composite design, and response surface methodology. Results indicated that the concentration of carbon source (glucose), nitrogen source (yeast extract), copper sulfate (CuSO4), and nitial pH value of the culture medium play an important role in laccase production by Trametes versicolor sdu-4 under submerged fermentation. Maximum laccase production (4146 U l-1) was observed at glucose (3.31g l-1), yeast extract (8.051 g l-1), CuSO4 (8.36 mg l-1) and nitial pH (4.8). The optimization results led to reduction in culture medium cost for laccase production.3. Laccase purification and characterizationThe laccase was purified and then biochemical properties of the enzyme was studied. It has all the characteristics of a typical blue laccase:(1) its molecular mass was 60 kDa; (2) a peak at 600 nm due to type I blue copper atom; (3) a shoulder at 320 nm indicating the presence of the type 3 binuclear copper pair; (4) four copper atoms per enzyme molecule. Therefore, We classified the laccase from T. versicolor sdu-4 as a typical blue fungal laccase. The N-terminal amino acid sequence of the laccase was AIGPAASLWANA. The optimum pH values of T. versicolor sdu-4 laccase were 2.2,3.7, and 7 for the oxidations of ABTS, DMP, and syringaldazine, respectively. The enzyme was stable at pH 3.0～10.0. The optimum temperature for ABTS oxidation was 75℃. The laccase was relatively stable at 70℃. Moreover, the half-life of the purified laccase at 70℃was 2.2 h. When the temperature was at 80℃, laccase activity decreased rapidly and was completely inactivated after 1.5 h. The most noticeable characteristic of laccase was that it oxidized methyl red, 2-bromophenol, and 4-bromophenol without mediators. The laccase showed good decolorization of the triphenylmethane and azo dyes.4. Study of solid state fermentation by T. versicolor sdu-4Rice Straw, wheat straw, corn stover, sugarcane bagasse, and wood powder were used as the lignocellulosic substrates to study solid-state fermentation by T. versicolor sdu-4. Results showed that the laccase production was highest when corn stover was used as solid medium. The solid-state fermentation conditions of T. versicolor sdu-4 were optimized by the method of factorial design, central composite design, and response surface methodology. The result indicated that laccase, cellulase (CMCase) and xylanase were produced during solid state fermentation. While lignin peroxide enzyme (LiP) and manganese peroxide enzyme (MnP) were not detected. Maximum laccase production (45.1U g-1 CS) was observed at glucose (9 mg g-1 CS), CuSCO4 (4.5μM g-1 CS) and moisture level (80%). Maximal lignin (34.8%) was degraded at glucose (9.2 mg g-1 CS), CuSO4 (4.3μM g-1 CS) and moisture level (82%).5. The stability of laccace in organic reagents and ILs and the oxidation of CEL by laccase in ionic liquidRecently, ionic liquids (ILs) are considered as an alternative to organic solvents for biocatalysis and biotransformations in view of sustainable and ultimately "green" processes, not only because enzymes displayed high level of activity and stereoselectivity of many different chemical transformations, but mainly because of an overstabilization effect on biocatalysts. In this work the stability of T. versicolor sdu-4 laccace in different concentrations of organic solvents and ILs was studied. The results indicated that:laccase was stable in organic solvent and water, IL and water composed homogeneous solution. However, the laccase activity was decreased rapidly with increasing volume percent of the organic solvent and ILs. In 30% ILs ([Bmim]Cl and [Emim][CH3C00]) solution (w/w), the remaining activity of laccase was still above 60% after 48 h. When the concentration of IL was abave 70%, laccase activity decreased rapidly.In the present paper, oxidation of CEL by laccase in 30% [Emim][CH3C00] was also studied. Infrared spectrum,1H-NMR,13C-NMR methods were used to characterize the treated CEL. The results showed that:T. versicolor sdu-4 laccase break down methoxy group, the guaiacyl and syringyl structures of CEL in [Emim][CH3COO].6. ILs pretreatment of agricultural straw and wood powder100 mesh wood powder was dissolved in ionic liquid [Pmim][CH3C00], then acetone/water (1:1 v/v) was added and stirred at room temperature for 2 h. Cellulose material was regenerated by vacuum filtration. After evaporating the acetone, lignin was regenerated by vacuum filtration. Infrared spectrum analysis, 1H-NMR and 13C-NMR methods were used to characterize the Ionic liquid-processed lignin.Rice straw, wheat straw and corn stover were pretreated by [Bmim]CI, [Emim][CH3C00] and [Pmim][CH3COO], respectively. Infrared spectrum analysis and scanning electron microscopy (SEM) were used to characterize the treated straw. The results indicated that [Emim][CH3COO] is the best IL when pretreating rice straw and [Pmim][CH3COO] is the best IL when pretreating wheat straw and corn stover.更多还原