【作者】 张海波；

【导师】 黄峰；

【作者基本信息】 山东大学 ， 微生物学， 2010， 博士

【摘要】 能源、环境、粮食问题是人类二十一世纪所面临的三大主要问题。生物质能源的开发和利用让人们找到了化石能源的很好的替代品。生物质是地球上数量最丰富的可再生性资源,这些生物质主要由纤维素、半纤维素、木质素组成,它们能够为人类提供燃料、饲料、以及化工原料等,具有重要的开发价值。合理的利用自然界中的生物质发酵生产乙醇是目前最有应用前景的可持续发展的策略之一。而木质素的包被作用恰恰是阻碍生物质转化产乙醇的主要瓶颈之一。研究木质素的降解机理对于解决该问题有着重要的意义。造纸、印染等行业的污水中的有机污染物主要是木质素的小分子片断,以及木质素单体的衍生物及稠环类芳香族化合物。木质素是一种高聚芳香族化合物,木质素在自然界中的降解不产生毒性,而工业产生中的芳香族化合物有着在自然界中的难降解性和毒性,使得芳香族化合物的污染成为人们的主要研究对象。漆酶有着很好的底物广泛性(典型的单酚、双酚、多酚、苯胺、甲氧基酚以及细胞色素C等)。漆酶氧化的过程中伴随着还原氧气生成水。漆酶还能够直接氧化一些小分子物质生成自由基,自由基能够引发一系列的氧化反应。漆酶是木质素代谢过程中的一个主要酶,绝大多数漆酶不能够直接氧化非酚型芳香族化合物,这使得漆酶在木素代谢的研究中被人们所忽略。介体发现以后,人们对漆酶有了一个新的认识,漆酶的氧化机理和应用研究也越来越受到人们的重视。漆酶能够利用氧气为电子受体氧化芳香族化合物的特性以及具有很强的底物专一性和底物广泛性,使得漆酶被广泛应用于纸浆造纸、造纸污水处理、印染污水处理、食品饮料行业以及有机合成中。在本论文从分离菌株出发,并对菌株进行了鉴定和酶的纯化及酶学性质研究。主要工作如下：1.高效产漆酶的菌株的分离及鉴定。从本实验室菌株库、院菌种室以及选择性购买、筛选得到一株高效产漆酶的菌种,该菌株生长迅速,能够高效生产漆酶。经过形态学,及子实体形态观察,结合ITS序列分析,鉴定该菌株为Trametes hirsuta lg-9,并对该菌株进行了保藏,其保藏编号为：CGMCC No.2422。2.影响该菌株产酶的因素及C／N比值进行研究我们通过正交试验、全因子试验、最陡爬坡和响应面分析对影响T. hirsuta lg-9产漆酶的因素进行了分析(其策略如图1所示),结果表明C／N比值在漆酶发酵生产中有很大的影响。优化最佳碳源和氮源的值为C=10.31 g l-1和N=1.32 gl-1,而通常氮源的添加量是2.5 g l-1。优化结果可以指导性的在发酵过程中合理的少添加氮源,有效的降低发酵成本。优化结果表明T. hirsuta lg-9产漆酶发酵时需要较低的氮源,产漆酶发生在稳定期之后,这很好了支持了白腐菌在缺乏氮源时大量产生的观点。图1实验条件优化流程图3. Trametes hirsuta lg-9漆酶纯化和酶学性质的研究我们通过传统的方法对该漆酶进行了分离纯化和性质的研究工作(流程如图2),根据T. hirsuta lg-9漆酶的性质和光谱学特征,可以把它归类为白漆酶。图2漆酶的分离纯化步骤这类缺少600 nm处光吸收的漆酶也被叫做黄漆酶(特征吸收光谱如图3所示)。该漆酶的分子量大概为90 kDa；该漆酶等电点与已报道的其它漆酶的等电点接近,大约为4.3；金属离子测定表明,T. hirsuta lg-9漆酶中不含有铁离子和锌离子,每分子蛋白质中含有2.86±0.21分子铜离子和0.82±0.27分子锰离子；该蛋白质的N末端氨基酸序列为AIGPTADLTQSQA; ABTS作为底物的最适pH与DMP作为底物时基本一致,最适pH值在2.5左右；该漆酶的表现出许多比普通漆酶应用前景更好的特征,如热稳定性高,同时最适pH值低,有一定的理论研究价值。该漆酶的最适反应温度为85℃,虽然在最适反应温度的时候不太稳定,图3纯化漆酶的全波段扫描图谱但是75℃时的半衰期达到70 min。此外,该漆酶能够在不存在介体的情况下氧化非酚型化合物甲基红和稠环化合物茜素红,具有重要的应用前景和理论研究价值。T. hirsuta lg-9漆酶的氧化特性仅在少量黄漆酶中有类似报道。在研究过程中,发现不同浓度的草酸对漆酶氧化不同类型的底物的影响是不同的,并且该漆酶能够在没有介体的存在情况下氧化非酚型的芳香族底物,于是对它们进行了更加深入的研究。4.有机物对漆酶活性测定的影响我们以草酸和EDTA Na2作为影响因素,利用T. hirsuta lg-9和R. vernificera的不同漆酶以及ABTS和DMP的不同底物进行对照实验,可以得出,无论草酸还是EDTA Na2都能够通过影响漆酶与底物的结合以及反应体系的pH值来影响漆酶的酶活性测定,而不是传统的认为的能够螯合漆酶的T1铜离子来影响漆酶的活性。大多数腐生真菌的生境pH值在4.0左右,而大多数漆酶的抑制剂的测定也集中在4.0左右,因此EDTA Na2对漆酶测定缓冲体系的pH值的影响要小于其它酸性有机溶剂。而大多数漆酶有一个钟罩型的pH依赖曲线。这就是为什么草酸比EDTA Na2更能够有效地影响漆酶的酶活性测定。这个实验结果能够对已经发表的部分文献起一定的解释作用。同时也能够指导我们在考虑抑制物对酶活性测定的影响的时候应当考虑酶活性的pH依赖性和添加物的酸碱性以及缓冲体系的缓冲能力。5.漆酶氧化机制的研究及其在染料脱色中的应用在本章的研究中,我们通过紫外可见光谱对漆酶氧化甲基红进行了实时监测,并通过高压液相进行了产物的分离,后通过质谱对产物进行了鉴定,提出了可能的漆酶直接氧化甲基红的机制(图4)。由于甲基红不能够被氧化产生酚型自由基,因此非典型性漆酶氧化甲基红的机制与普通漆酶的氧化是不同的。紫外可见分光光度计的检测表明甲基红随着处理时间的延长是不断减少的。高压液相的结果也表明,至少有三种产物产生。根据高压液相质谱连用鉴定产物的结构,可以推测,非典型性漆酶的氧化机制跟普通漆酶类似,但是其氧化机制却是不同。两种氧化方式的断裂位点是一致的,但是产物却存在一定差别。反应过程中没有检测到醌型化合物。根据氧化产物和已经发表的文献推测,漆酶能够直接氧化甲基红,引起染料的脱甲基化,图4T. hirsuta lg-9漆酶氧化甲基红的可能机制类似反应在漆酶介体系统催化以及漆酶超声波处理的反应中报道过。氧化产生的苯胺基能够进一步被漆酶氧化。然后非典型性漆酶能够攻击叠氮连接,并引起一系列的反应。根据已经发表文献的观点,Leontievsky等人认为黄漆酶是普通漆酶被木质素代谢过程中的产物修饰,从而漆酶与降解产物构成漆酶及介体系统,而白漆酶是普通漆酶的T1铜离子位点被其它过渡态金属代替生成的。T1铜离子位点是漆酶氧化电子传递链的起始位点,它对漆酶的氧化还原电势起着关键作用,而T. hirsuta lg-9的漆酶应属于白漆酶。T. hirsuta lg-9漆酶的氧化特性是由于该漆酶中含有一个Mn离子造成的。通过部分因子实验、最陡爬坡和响应面分析对影响脱色的条件进行了优化,并通过二项式进行拟合。其拟合方程为：Y=+78.09+2.22X1-2.59X2+2.54X4+0.32X1X2-1.18X1X4-2.93X2X4-1.35X12-14.84X22-3.07X42经方差分析检验,该方程有效。根据方程和响应面分析的结果,可以求得对应的真实反应条件为：CEnzyme=1.8 U l-1,pH=4.93和Time=130.23 min。根据拟合得到的二项式,可以得知,最佳脱色值是79.41%。通过实验进行了验证,发现脱色率平均值约80%,与预测值的差别小于5%,表明模型是合适的,可以有效的指导实验。更多还原

【Abstract】 The people should seek solutions to the energy, environmental, and food challenges in the 21st century. The biofuel is a good candidate for fossil fuel. Biomass is the most abundant renewable resource in the world. The biomass is mainly composed of cellulose, hemicellulose and lignin, and it is of good use in fuel, silage, and chemical materials. Generation Liquid Biofuels from Biomass is one of the key strategies for sustainable development. Lignin is one of the key barriers to the utilization of biomass. The research on the degradation of lignin is signification to the application of biomass. The contaminations in pulp and dyes waste water are main small molecular lignin, the derivate of the monomer of lignin, and polyphenols. Lignin is mainly composed with three vinyl alcohols. The degradation of lignin does not make any pollution to the nature, while the most of the industry pollutions are aromatic substances.Laccases have a wide range of substrates (typically mono-, di-, and polyphenols, aromatic amines, methoxyphenols and ascorbate). This oxidation is coupled to the four-electron reduction of dioxygen to water molecules. Laccases also generate free radicals, which mediate a variety of subsequent reactions. Laccase is a main enzyme in the degradation of lignin. But it was ignored for a long time because it could not directly oxidize non phenol compounds. The discovery of the mediator attracts more attentions of the researchers. Laccases have great biotechnological potential because of their broad substrate specificity. They can be used in the paper industry, the food industry, in dye or stain bleaching, bioremediation, plant fibre modification, ethanol production, biosensors, biofuel cells, organic synthesis, and drug synthesisIn our study, we isolated and characterized a fungus. The laccase was purified and characterized. The main jobs are as follows:I. Isolation and characterization of laccase production fungusTrametes hirsuta lg-9 (CGMCC No.2422) was isolated from Mengshan Mountain (in Shandong Province, China) and characterized by our laboratory. This fungus can secrete more laccases than the other fungi in our lab, school and those were purchased from China General Microbiological Culture Collection Center (CGMCC). The fungus grows faster. The fungus was characterized as T. hirsuta lg-9 according to its’ morphology and ITS. The fungus was kept in CGMCC and the number was CGMCC No.2422.Ⅱ. The laccase production influence factors and the influence of C/NWe analyzed the laccase production influence factors of the fungus T. hirsuta lg-9 with the method of orthogonal experimental design, factorial design, steepest ascent, and response surface methodology (Fig.1). A quadratic model was obtained using the design expert, and the model was proved to be validated. According to the model, the maximum production of laccase was obtained at the concentration of 10.31 g l-1 glucose and 1.32 g l-1 ammonium dibasic phosphate, and the C/N ratioFig.1 the protocol of experiment condition optimization was 343.67 mM (glucose-C):19.99 mM (NH4+-N). The maximum laccase activity was about 16.56 U ml-1. We could reduce the nitrogen source in the laccase fermentation to reduce the cost according to the optimization. The result indicated that the secretion of laccase could be induced by the nitrogen starvation. Ⅲ. Purification and characterization of the laccaseThe purification of the laccase was carried according to the common method (Fig. 2). The characterization of the laccase was well studied. We classified the laccase from T. hirsuta lg-9 as a novel "white" laccase because of its atypical spectrum. White laccases lack absorption at 600 nm, and they also have been called "yellow"Fig.2 purification of laccase laccases (Fig.3). The laccase had a molecular weight of 90 kD. The isoelectric point of the enzyme was about 4.3. The metal content was determined by atomic absorption. The laccase from T. hirsuta lg-9 showed valued of 2.86±0.21 copper atoms and 0.82±0.27 manganese atoms per protein molecular. Zinc and iron was not detected. The N-terminal amino acid sequence of the purified protein was determined up to 13 amino acids as AIGPTADLTQSQA. A pH of 2.4 was optimal for the oxidation of ABTS and a pH of 2.5 was optimal for DMP. A temperature of 85℃was optimal for DMP oxidation. The half-life of this laccase was 70 min at 75℃, and 5 hours at 65℃.Fig.3 UV/visible spectrum of laccase from T. hirsuta lg-9In our study, we found that different concentration of oxalic acid can caused different inference to the laccase activity determination with different substrate, and the laccase from T. hirsuta lg-9 can directly oxidize some non-phenolic compounds. Both of them are worth further study. IV Comparative Study of the influences of organic compounds on laccase activity testsThe influence of oxalic acid and ethylenediaminetetraacetic acid disodium salt-2-hydrate (EDTA Na2) on laccase from T. hirsuta lg-9 and Rhus vernificera in different test systems utilizing 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol (DMP) as enzyme substrates were tested. Our study indicated that oxalic acid can influence the laccase activity determination mainly by changing the pH of the reaction system. The influences of both oxalic acid and EDTA on the laccase activity determination with different substrates were different. The results indicated that both oxalic acid and EDTA could influence the laccase activity determination by influencing and the binding of laccases substrates but not by chelating metal of the laccase. The organic compounds also can influence the laccase activity determination by changing the pH of the reaction system.V oxidation of non-phenol dye methyl red and conditions optimizationUV-Vis spectrophotometry was used for process monitoring of the oxidation products of methyl red. High performance liquid chromatography (HPLC) was used for detecting the products. Additionally, the structures of products were elucidated with electrospray injection mass spectroscopy. Probably degradation mechanism of the azo dye methyl red oxidized by the atypical laccase directly had been proposed (Fig.4).As methyl red can not be oxidized to generate a phenolic radical, the oxidation mechanism of the atypical laccase to oxidize non-phenolic methyl red may be also different from the oxidation mechanism of phenolic azo dyes with common laccase. UV-Vis spectrophotometry was used for process monitoring of the oxidation products of methyl red. The results of UV-Vis spectrophotometry indicated the productions of methyl red increased with the oxidation time running. HPLC was used for detecting the products. The results of HPLC indicate the quantities of the products are no less than three. Additionally, the structures of products were elucidated with electrospray injection mass spectroscopy. The structures of products indicate the oxidation mechanisms of the atypical laccase and common laccase are similar but different. The cleavage sites of them are the same, while the products are different. There were no quinones detected. We speculate the atypical laccase can directly attack the dye, which causes the N-demethylation of the dye. Similar reactions have been reported in decolorization by laccase-mediator system and laccase-ultrasound treatment. The formed amino-group can be further oxidized by laccase. Then, the atypical laccase attack the azo linkage, which induces a variety of subsequent reactions. Leontievsky et al. proposed that yellow laccases were formed by modification of blue laccases by mediators, so they can directly oxidize non-phenolic compound and hydroxy polyaromatic dye. The white laccases are formed by the replace of T1 Cu with other transition metals. The T1 site functions as the primary electron acceptor and is important to the redox potential of laccase. The laccase from T. hirsuta lg-9 may belong to white laccase which may be formed by the replace of T1 Cu with Mn. This is probably the main reason of direct oxidation of methyl red. Fig.4 The probably reaction scheme of methyl red directly oxidized by the atypical laccase from T. hirsuta lg-9Factorial design, steepest ascent design and central composite design were successfully applied to optimize the decolorization conditions of methyl red. The equations given below are based on the statistical analysis of the experimental data.The results of analysis of variance (ANOVA) imply the model is significant. We can calculate the value of Y was maximum when the algebraic solutions were X1= 0.66, X2=-0.11 and X4= 0.34. These values correspond to the uncoded value of CEnzyme= 1.8 U l-1, pH= 4.93 and Time= 130.23 min. The maximum predicated decolorization of methyl red was 79.41%. These optimum values were checked with experiments. The actual values of the methyl red decolorization were at an average of 80%. The error was less than 5%, and the model is significant.更多还原