【作者】 刘家扬；

【导师】 廖祥儒； Qingguo Huang；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【摘要】 漆酶（Laccase，EC1.10.3.2）是一种铜蓝氧化酶，能催化一大类酚类及非酚类化合物的聚合、分解及转化，期间伴随单电子的转移将分子氧还原成水。由于漆酶具有广泛的底物催化范围，因此在众多领域如食品、染织、有机合成、生物修复、传感器等方面都有应用的潜力。漆酶来源广泛，如植物、动物、细菌及真菌等，真菌是自然界中漆酶的主要生产者，相较其它来源的漆酶，真菌漆酶具有较高的氧化能力。目前，由于真菌漆酶产量低、不易放大生产等因素，制约了漆酶在工农业生产及环保等领域中的应用。筛选到优良的产漆酶菌株、实现高效生产、获得性能稳定的漆酶及开发漆酶的应用具有一定的理论意义和实践价值。本论文从中国亚热带地区筛选到一株白腐密孔菌属真菌，对其进行了分类学鉴定，然后针对该菌株发酵产漆酶的能力、酶学性质及其在预处理生物质促进糖化、对染织废水脱色及缓解土壤斥水性等方面进行了研究。论文的主要研究结果如下：从中国亚热带地区广东省筛选到一株产漆酶的白腐真菌，对该真菌进行了形态学观察及分子生物学鉴定，基于rRNA基因序列的聚类分析构建了进化树，将该菌命名为Pycnoporus sp. SYBC-L3。考察了不同营养元素及培养条件对该菌产漆酶的影响，得出了优化后的培养基为：麦芽糖14g/L，豆粕6g/L, KH₂HPO₄1g/L, MgSO₄·7H₂O0.5g/L,Na₂HPO₄·12H₂O0.2g/L, Cu²⁺1.5mmol/L,香兰素1mmol/L, Mn²⁺0.2mmol/L；较优的培养条件为：起始pH值5.0、种龄3天、接种量10%、摇床转速200r/min。在该培养基和培养条件下，在摇瓶中胞外漆酶的最高活力出现在第8天，为45U/mL，为优化前的57倍；生物量也较优化前提高了21倍（培养时间提前了约3天），约为3.5g/L。以摇瓶优化的培养基和培养条件为基础对Pycnoporus sp. SYBC-L3在不同规模发酵罐上的产酶水平进行了研究，在所测试的几个发酵罐水平上均取得了较好的酶活产量，其中在50L搅拌式发酵罐中取得了最高的酶产量，为110U/mL。对发酵罐中所产的粗酶进行了部分性质研究，发现粗酶在0-100℃的范围内均有较高的催化能力，最适温度为55℃，最适pH值为3.5，粗酶具有良好的热稳定性，在不同温度下的半衰期分别为4h （70℃）、6.5h （60℃）和10h （50℃），室温下粗酶以液体的形式储存一年后依然有80%的活力。对粗酶中包括漆酶在内的主要蛋白质成分进行了分析，发现发酵液中有至少存在13种不同的胞外蛋白质，其中主要组分是三中漆酶同工酶。采用喷雾干燥法将液体粗酶制成了粉剂漆酶，酶活的回收率达到80%左右。对漆酶进行了分离纯化及性质研究。通过硫酸铵沉淀、凝胶除盐及离子交换层析三步纯化，获得了漆酶纯酶lac-L，回收率为28%，比活提高了4.67倍。Lac-L的分子量约为58kDa，N端15个氨基酸序列为AIGPVADLTLTNAAV，lac-L活力的最适pH及最适温度有底物依赖性，pH在酸性范围内，温度在50-70℃之间具有较高的催化效率。Lac-L的最适底物为ABTS，其次为DMP、愈创木酚及丁香醛连氮。Lac-L具有较好的温度及pH稳定性，在70℃和60℃时的半衰期分别为3h和5h，在pH6-9的范围内于室温下保温24h后有80%以上的活力，lac-L对大部分金属离子具有一定的耐受性，但受到Ag⁺、Fe²⁺及Fe³⁺的抑制。用圆二色谱技术对lac-L进行了二级结构预测，发现lac-L含有2%的α-螺旋、51%的β-折叠及47%的无规则卷曲，三种抑制漆酶活性的金属离子(Ag⁺、Fe²⁺及Fe³⁺)在高浓度时均导致了漆酶二级结构的改变。能源作物switchgrass（柳枝稷）是生产燃料乙醇的重要原料之一，糖化过程是整个燃料乙醇生产中的限速步骤。用真菌Pycnoporus sp. SYBC-L3对switchgrass进行了预处理研究，旨在提高后续的糖化效率并同步实现产漆酶。真菌处理switchgrass后导致其化学组成发生了变化，其中真菌培养36天时发现选择性降解木质素的力度最大，相对残留的纤维素及半纤维素较高，对促进糖化最有利，但副产品漆酶的最高活力出现在第54天。预处理36天后的switchgrass比未处理的switchgrass在糖化效率方面提高了50%。糖化效率随纤维素酶用量的增加而增加，最高糖化效率为90%（葡聚糖）和40%（木聚糖）。在为期5天的糖化过程中，最高的单糖释放率出现在初始的12h内。FTIR光谱分析表明，真菌预处理导致生物质结构及化学组成上的变化，连接木质素与纤维素及半纤维素直接的化学键发生断裂。电镜观察证实了真菌的处理在switchgrass表面形成了不同程度的孔洞，破坏了生物质的致密性与坚韧度。对废弃物的处理及重新利用越来越受重视，染织废水的综合处理是当前的难题之一。尝试了用含有蒽醌类染料的染织废水作为液体培养基对真菌Pycnoporus sp.SYBC-L3进行了培养，达到了去除颜色及同步产漆酶的效果。在80%的染织废水中真菌能产出相对较高的酶活力，添加了木质纤维类废弃物芦苇后，产酶能力得到增强，在添加10g/L芦苇的条件下，最大漆酶活力为5.6U/mL，颜色的去除率为70%。研究结果可为废水处理及污染物的控制提供参考方法。高尔夫球场使用大量的沙质化土壤，土壤斥水现象的发生不可避免，由此引发草皮的大量死亡。将真菌Pycnoporus sp. SYBC-L3预处理switchgrass时产生的副产品（漆酶粗酶制剂）进行了缓解高尔夫球场土壤斥水性的应用研究。对不同球场采集的土壤样品分析发现，几乎所有的土壤都表现出了中度甚至严重斥水现象。在实验室水平上，该粗酶能缓解高尔夫球场表层土壤的斥水性，效果非常明显；酶活力越高，处理后的土壤亲水性越强。酶处理时间在第1天就可以基本消除土壤斥水现象。用酶处理土壤后，将废弃的酶液抽提出来，经活力测定发现，残余活力依然有一半以上，表明该漆酶在自然环境中具有较好的稳定性。因此，该漆酶有望用于高尔夫球场的草坪管理中，缓解由于土壤斥水引起的草皮死亡。更多还原

【Abstract】 Laccase （EC1.10.3.2） is a blue copper-containing oxidoreductase, capable ofpolymerizing, degrading and transforming a large variety of phenolic and non-phenoliccompounds. During one-electron transfer among copper atoms within active center, molecularoxygen is concomitantly reduced to water. In view of its wide spectrum of catalytic substrates,laccase is being increasingly found its applications in numerous fields such as food industry,textile dying and decolorization, biosynthesis and bioremediation as well as biosensor.Laccase is mostly produced by fungi in nature and fungal laccases represent the relativelyhigh catalytic capability among various lassase sources. Overview of published literaturereveals that in-depth applications of laccase can be restricted by low laccase yields with highproduction input. Screening potential wild-type fungus from nature with hyper-ability oflaccase excretion can hereof significantly contribute to full applications of this enzyme inindustrial and environmental aspects. The following research conducted a series of studiesbeginning with isolation of a white-rot fungus and ending with application of laccase by thisfungus, within which optimizing laccase production in flask level, conducting fermentationprocesses in various bioreactors, characterizing laccase properties and involving the fungusinto lignocellulosic biomass pretreatment and textile discharge treatment were also included.Below are the main findings from our research.1) A laccase-screting wild-type fungus isolated from a rotten wood was characterizedbased on its moyphological characteristics and molecular identification techniques anddesignated as Pycnoporus sp. SYBC-L3. Laccase production by this fungus was thenenhanced by optimizing some major medium components coupled with fermentationparameters and the favorable culture conditions are: initial pH value of5.0, seed age of3d,inoculum of10%and rotation rate of200rpm; the optimized medium consists of: maltose14g/L, soybean meal6g/L, KH₂HPO₄1g/L, MgSO₄·7H₂O0.5g/L, Na₂HPO₄·7H₂O0.2g/L, Cu²⁺1.5mmol/L, vanilla1mmol/L, Mn²⁺0.2mmol/L which finally yielded the highest laccaseactivity up to45U/mL,57-fold higher than the un-optimized one.2) Laccase excretion abilities of Pycnoporus sp. SYBC-L3were further tested invarious bioreactors in light of the flask-obtained investigation. The highest laccase activity,110U/mL, was obtianed in a50L stirred bioreactor. The crude laccase exhibited promisingproperties in terms of high catalytic activity under broad temperatures ranging from0-100oCwith optimum T of55oC within acidic pH spectra. It displayed inconceivable relative activityof as high as40%and80%at0oC and100oC, respectively. Long half-lives were obtained forlaccase under all varying temperatures assayed, especially at room temperature. Enzymecocktail of the culture broth was then subjected to nano-LC-MS/MS for predominantextracellular protein identification, which suggested that at least13differetn proteins existedin the fungal culture broth with three iso-laccases as the major ones. Crude laccase powderwas successfully prepared using spray-dryer with activity recovery of approximatedly80%.3) Pure laccase （designated as lac-L） was harvested by a three-step purification process,resulting in a purification fold of4.67and activity recovery of28%. Lac-L has an estimated molecular weight of58kDa and pI of4.7. N-terminal amino sequence of lac-L wasdetermined as AIGPVADLTLTNAAV. Optimal T and pH of lac-L were substrate-dependentbut generally in the range of T50-70oC and pH3-5. ABTS was proved to be the best substrate,followed by DMP, guaiacol and SGZ. Lac-L was tolerant to most metal ions but dramticallydepressed by Ag⁺, Fe²⁺and Fe³⁺at even low concentrations. Sr²⁺imparted weak stimuluseffect on lac-L activity. Stamina profiles of lac-L on elevated temperatures suggested thatlac-L was as excellent as or higher than other thermostable fungal laccases. The stabilities oflac-L towards high temperature and pH as well as deactivation by some metal ions wereelucidated using CD specturm technique.4) Energy crop switchgrass is one of the most promising lignocellulosic feedstocks forbioethanol production, in which cellulose conversion is a rate-limiting step. Fungalpretreatment by Pycnoporus sp. SYBC-L3was performed herein to enhance subsequentsaccharification and simultaneously produce useful enzymes as a co-product. The funguscaused dramatic changes in chemical composition in pretreated switchgrass over time. Thehighest lignin removal and saccharification efficiency was attained for cultivation time36dwhile the peak enzyme activity occurred at54d. SEM associated with FTIR techniquesdemonstrated that fungal pretreatmetn formed numorous pores or pits on the biomass surfaceand altered the chemical compositions of switchgrass, thereafter the recalcitrance coupledwith intensity of this biomass significantly reduced.5) Treatment and re-utilization of wastes have been foci worldwide in recent years.Fungal cultivation of Pycnoporus sp. SYBC-L3in textile effluent was for the first timeattempted to achieve both color removal and laccase production. The favorable concentrationof textile for maximum fungal growth and laccase activity was shown to be80%. Amendmentof80%textile effluent with12g/L lignocellulosic biomass Phragmites australis enhancedlaccase production, under which the peak activity reached5.6U/mL and70%color removaloccurred in this culture sytem. This novel scheme provides an alternative of management oftexteile effluent as well as other environmental pollutants control.6) Soil water repellency （SWR） is a re-occurring phenomenon on sandy soil on golfcourt and thus induce serious turfgrass death. The crude enzyme extract （CEE）, derived fromthe fungal pretreatment of switchgrass as above mentioned, was firstly employed to mitigateSWR seveity of sampled soils from variously located golf courts. All collected soils presentedwater repellency to varying extents and returned to hydrophilic status following with the CEEtreatment. Effective alleviation of hydrophobicity were achieved in1-d treatment with nofurther significant changes observed in the following4-d treatment. High residual enzymeactivities, around50%or higher, remained in the filtrate of enzymatic soil treatment for5days. The possible mechanisms for alleviation of SWR with enzymatic treatment were thusdiscussed. Potentially, laccase enzyme liquid might serve as a biological agent for preventionand amelioration of turfgrass soils in golf court management.更多还原