【作者】 陈敏；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2010， 博士

【摘要】 刺芹侧耳(Pleurotus eryngii)隶属于真菌门、真担子菌纲、伞菌目、侧耳科,侧耳属,是一种具有较强木质素降解能力的白腐真菌,极具工业应用价值。木质素降解酶是参与木质素生物降解过程中的酶类,具有高氧化降解能力和多底物性,能够降解木质素、多环芳烃类化合物、杂环类化合物和合成染料等多种难降解有机物。本研究以刺芹侧耳GIM5.280为研究对象,进行了能以2,2’-连氮基-双-(3-乙基苯并二氢噻唑啉-6-磺酸)二铵盐(ABTS)和愈创木酚为底物的高产胞外木质素降解酶的菌种选育和表达条件优化,并对发酵液中的木质素降解酶分离纯化路线、酶学性质、染料和木质素磺酸钠降解效果开展系统研究,为今后将其应用于环境保护、生物漂白与制浆、食品工业等领域提供理论和实践依据。为了选育高产木质素降解酶的刺芹侧耳菌株,系统研究了刺芹侧耳原生质体制备及再生体系。采用0.6mol/L(M)甘露醇为酶解渗透压稳定剂,在30℃、pH 5.5下,以4%蜗牛酶和2%溶壁酶的复合酶酶解3天菌龄的刺芹侧耳菌丝体3h,可获得最大原生质体得率,酶解液中原生质体数目可达1.82×108个/mL。所得原生质体经0.6M的蔗糖(再生渗透压稳定剂)配制的SYM培养基再生培养,上层琼脂浓度设定为0.7%,原生质体再生率最高,可达0.35%。通过紫外和60Coγ的二次复合诱变及继代培养筛选获得一株摇瓶酶活达110U/mL,较出发菌株酶活提高54.3%的稳定诱变株,命名为P. eryngiiCo007,用于后续发酵及分离纯化试验。通过单因素试验和中心组合响应面优化分析考察了影响P. eryngiiCo007菌株产木质素降解酶的因素,得到液态发酵产木质素降解酶的最适培养基组成和发酵参数。然后在发酵罐中进行了产酶扩大试验,在搅拌速度250rpm/min、通风量0.6vvm条件下,按最适培养基(3%葡萄糖,0.05%硫酸镁,0.4%磷酸二氢钾,0.2%酵母粉,0.2%胰蛋白胨,秸杆110%, Cu2+ 4.01mmol/L,吐温800.84g/L)和最适发酵参数(初始pH 5.63,发酵温度26℃,接种量15%)进行发酵,木质素降解酶酶活最高值出现在第10天,达到了324.72U/mL。建立了一条从P. eryngii Co007发酵液中快速分离纯化木质素降解酶的技术路线。通过35%硫酸铵饱和度初沉和75%硫酸铵饱和度二沉,可从P. eryngii Co007发酵液中去除掉大部分杂蛋白和获得绝大部分的目标蛋白沉淀。经DEAE-SepharoseTM FastFlow (DEAE FF)离子交换三步层析,可较好地纯化目标酶。该路线总纯化因子达10.3,回收率76.2%。经SDS-PAGE和Native-PAGE电泳分析,收集到的洗脱液中含有三种木质素降解酶,都具有活性。按其分子量大小,将其命名为Peco60-7-1dⅠ、Peco60-7-1dⅡ、Peco60-7-1dⅢ。采用肽指纹图谱分析方法和N末端测序法对分离纯化的三种单一组分进行鉴定,所得结果证实Peco60-7-1dⅠ是芳基乙醇酶,Peco60-7-1dⅡ是漆酶,Peco60-7-1dⅢ是多功能过氧化物酶,分别命名为AAO-Peco60-7 , Lac-peco60-7和VP-Peco60-7。AAO-Peco60-7酶是发酵液经硫酸铵盐沉、DEAE FF层析、SephacrylTM S-200 High Resolution层析和SourceTM 15Q层析后获得,该过程的纯化倍数为38.2,得率为25.5%。AAO-Peco60-7分子量约70kDa,等电点为4.2。以ABTS为底物,AAO-Peco60-7酶学性质如下：AAO-Peco60-7最适催化pH为3.0,pH为3.6时该酶稳定性最好,24h后残余酶活高达88%。AAO-Peco60-7最适催化温度为70℃,在4℃到60℃的温度范围内,酶较稳定,60℃保温50mmin后AAO-Peco60-7残余酶活还有80%左右,表现出较好的耐热性。氧化ABTS的动力学参数Vmax和Km分别为102.04U／mmg和214.96μmol。测试金属离子没有促进作用,其中的Fe3+、Fe2+、Ag+和Ca2+有明显抑制作用。Lac-Peco60-7酶是发酵液经过硫酸铵盐沉、DEAE FF层析和割胶纯化后获得,其分子量约62kDa,等电点为4.5。以ABTS为底物,Lac-Peco60-7酶学性质如下：Lac-Peco60-7最适催化pH为4.0,pH为4.6时该酶稳定性最好,24h后残余酶活约85%。最适催化温度为60℃,50℃以下稳定性较好,50℃保温40mmin后残余酶活还有40%左右,当温度超过50℃时,酶活损失加剧。氧化ABTS的动力学参数Vmax和Km分别为227.27U/mg和252.33μmol。金属离子中Zn2+和Cu2+对Lac-Peco60-7有明显促进作用,Ag+、Fe2+和Mn2+有明显的抑制作用。VP-Peco60-7酶是发酵液经硫酸铵盐沉、DEAE FF层析和SephacrylTM S-200 High Resolution凝胶层析后获得,其纯化倍数为27.0,得率为19.1%。VP-Peco60-7分子量约40kDa,等电点为4.1。以ABTS为底物,VP-Peco60-7酶学性质如下：VP-Peco60-7最适催化反应pH为3.0,pH为4.0时该酶稳定性最好,24h后残余酶活约50%。最适催化温度为50℃,VP-Peco60-7热稳定性差,30℃保温60min后其残余酶活力只有52%左右,当温度超过50℃时,酶活损失严重。氧化ABTS的动力学参数Vmax和Km分别为1 88.68U/mg和203.09μmol。金属离子中Zn2+和Cu2+对VP-Peco60-7有明显促进作用,而Fe3+、Fe2+和Mn2+有明显的抑制作用。采用经DEAE纯化的P. eryngiiCo60-7混合酶液对不同染料进行脱色试验,结果表明混合酶液对三苯甲烷类染料溴酚蓝降解效果最好,24h染液脱色率近99.3%。双偶氮类染料刚果红降解效果次之,24h染液脱色率达53.7%。杂环类染料亚甲基兰降解效果最差,24h染料脱色率只有11.5%。对脱色影响因素进行研究,以2h溴酚蓝脱色体系为例,在脱色温度30℃、pH4.0、H2O2 5μM、混合酶液浓度20U/mL、染料浓度25μM的条件下,脱色效率最高,达85.61%。小分子介体物质ABTS对溴酚蓝脱色体系脱色率无促进。采用经DEAE(?)屯化的P. eryngiiCo60-7(?)昆合酶液对木质素磺酸钠进行了降解试验,结果表明,降解体系中含有50mM pH 4.6乙酸钠-乙酸缓冲液,100mg/L木质素磺酸钠,1mM ABTS和20U/mL混合酶液,置40℃、150rpm下酶解反应8h,降解率可达82.13%。反应体系中添加小分子还原介体ABTS后,降解率明显提高。通过全波段光谱扫描对比分析发现降解后木质素磺酸钠278nm主吸收峰明显下降,显然ABTS可介导P. eryngiiCo60-7木质素降解酶氧化木质素磺酸钠,提高降解率。更多还原

【Abstract】 Pleurotus eryngii (P. eryngii) is a fungus belonging to Eumycophyta, Eubasidiomycetes, Agaricales, Pleurotaceae, pleurotus. As a kind of white rot fungi, it has been reported to have the capability to degrade lignin, and is therefore considered a model organism having extensive biotechnological applications. With remarkably oxidizing ability, degradation capacity and substrate non-specificity; ligninolytic enzymes involved in lignin biodegradation can oxidize various substrates including aromatic pollutants, heterocycle compounds, synthetic dyes and so on. In this work, using P. eryngii GIM 5.280 as starting strain, the mutagenesis of protoplast were performed for the screening of high-yield strains of ligninolytic enzymes with 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol as substrates. And then the production conditions in liquid submerged fermentation of ligninolytic enzymes of P. eryngii were optimized. Furthermore, the purification procedure of ligninolytic enzymes of P. eryngii was established, and purified enzymes were used to investigate their enzymatic properties, catalysis behaviors, dye decoloring ability, and lignin degradation ability. These studies will enhance our knowledge of the ligninolytic system of this edible mushroom. Furthermore, it will also clarify the potential applications of ligninolytic enzymes of P. eryngii in the environmental protection, paper pulp bleaching, processing of food and other industrial applications.In order to screen P. eryngii strains of high-yield ligninolytic enzymes, the preparation conditions of P. eryngii protoplasts were investigated in details. Results showed that the highest production rate of protoplast could be obtained by using 0.6 mol/L(M) mannitol,4% helicase,2% lywallzyme, and digesting for 3 hours at 30℃, pH 5.5. The protoplast numbers of P. eryngii reached to 1.82×108/mL. The optimal medium for protoplast regeneration of P. eryngii was SYM medium, using 0.6 mol/L sucrose as osmotic stabilizers, and 0.7% agar as upper-layer plate. The highest regeneration rate was 0.35%.The UV and 60Coγirradiation mutagenesis of P. eryngii protoplasts, and serial subcultivations were performed for screening high-yield strains of ligninolytic enzymes. The results indicated that the strain 007 (We named this strain as P. eryngiico007.) of P. eryngii exhibited outstanding genetic stability after four successive generations. Furthermore, the ligninolytic enzyme activity of this strain was up to 110U/mL, a 54.3% enhancement over that of the original strain. The optimum production conditions of ligninlytic enzymes of P. eryngiiCo007 in liquid culture was investigated through single factor experiment and response surface method, and the result were listed as follows: the compositions of optimal fermentation medium were glucose 30g/L, MgSO4·7H2O 0.5g/L, KH2PO4 4g/L, yeast extract 2g/L, tryptone 2g/L, straw 11g/L, CuSO4 4.01mmol/L, Tween 800.84g/L; The optimal cultivating conditions were 15%(v/v) inoculation volume,26℃and pH 5.63. The ligninolytic enzymes were prepared by cultivating P. eryngiiCo007 in a 3-liter fermentor under the conditions of 26℃,200 rpm, and aeration with 0.5 liter·min-1. Proteins with ligninolytic enzyme activity were purified from 10-day-old liquid cultures of P. eryngii, which exhibited maximal enzymatic activity (324.72 U/mL).The ligninolytic enzymes were separated and purified from the culture broth of Pleurotus eryngiiCo60-7 strain. An efficient purification procedure was establised, which was mainly composed of the precipitation of ammonium sulfate and ion exchange chromatography. In the precipitation of ammonium sulphate, two-stage precipitations were applied. In the first-stage precipitation,35% saturation of ammonium sulfate was used to remove the hybridproteins and in second-stage of precipitoation,75% saturation of ammonium sulfate was used to capture the ligninolytic enzymes. Next, the crude ligninllytic enzymes were purified by DEAE-SepharoseTM Fast Flow (DEAE FF) ion-exchange chromatography under three-step elution. Finally, a 76.2% activity yield was obtained with a purification factor of 10.3. The results of SDS PAGE and Native PAGE showed that three ligninolytic enzymes were present in the elution after DEAE FF ion-exchange chromatography, and these enzymes were named as Peco60-7-1dⅠ, Peco60-7-1dⅡ, and Peco60-7-1dⅢ, respectively. Using N-terminal sequence determination and peptide mapping analysis, Peco60-7-1dⅠ, Peco60-7-ldⅡ, and Peco60-7-1dⅢwere found to be similar to the aryl-alcohol oxidase, laccase and versatile peroxidase isoenzyme, respectively. Therefore, they were named as AAO-Peco60-7,Lac-Peco60-7 and VP-Peco60-7.AAO-Peco60-7 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, DEAE FF chromatography, SephacrylTM S-200 High Resolution chromatography, and SourceTM 15Q chromatography with a activity yield of 25.5% and a purification factor of 38.2. The molecular mass of AAO-Peco60-7 was estimated to be 70kDa by SDS-PAGE and its isoelectric point was 4.2. The optimal pH and temperature for oxidizing ABTS was 3.0 and 70℃respectively. The Vmax and Km values of the enzyme for ABTS were 102.04 U/mg and 214.96μmol. The enzyme was relatively stable at pH3.6, and 88% enzymatic activity was remained after incubating for 24 h. And the enzyme was also stable from 4℃to 60℃, and the survival enzymatic activity was over 80% after incubating for 50 min.0.1 mM various metal ions had no pronounced effects on enzyme activity of AAO-Peco60-7, However, the activity of the enzyme was strongly inhibited by Fe3+, Fe2+, Ag+ and Ca2+.Lac-Peco60-7 was purified to electrophoretic homogeneity by ammonium sulfate precipitation, DEAE FF chromatography and the gel slice of Native-PAGE. The molecular mass of Lac-Peco60-7 was estimated to be 62kDa by SDS-PAGE and its isoelectric point was 4.5. The optimal pH and temperature for ABTS were 4.0 and 60℃, respectively. The Vmax and Km values of the enzyme for ABTS were 227.27U/mg and 252.33μmol. The enzyme was relatively stable at pH 4.6, and 85% enzymatic activity was remained after incubating for 24 h. And the enzyme was also stable at 50℃, and the survival enzymatic activity was over 40% after incubating for 40 min.0.1mM various metal ions had different effects on enzyme activity, the enzymatic activity of Lac-Peco60-7 was strongly promoted by Zn2+ and Cu2+, however, its activity was inhibited by Ag+ and Fe2+VP-Peco60-7 was purified by ammonium sulfate precipitation, DEAE FF chromatography and SephacrylTM S-200 High Resolution chromatography with a activity yield of 19.1% and a purification factor of 27.0. The molecular mass of VP-Peco60-7 was estimated to be 40 kDa by SDS-PAGE and its isoelectric point was 4.1. The optimal pH and temperature for ABTS were 3.0 and 50℃, respectively. The Vmax and Km values of the enzyme toward substrate ABTS were 188.68U/mg and 203.09μmol. The enzyme was relatively stable following with 50% survival activity after incubating for 24 h at pH 4.0 and over 52% survival activity after incubating for 60 min at 30℃.0.1 mM various metal ions had different effects on enzyme activity, the enzymatic activity of Lac-Peco60-7 was promoted by Zn2+ and Cu2+, however, its activity was strongly inhibited by Fe3+, Fe2+ and Mn2+ Ligninolytic enzymes separated from DEAE FF were used to decolorize three dyes, including bromchlorphenol blue, methylene blue and Congo red, which could be classified as triphenylmethane, heterocyclic and disazo dyes. The results showed that the purified ligninolytic enzymes had the best decoloring effect for bromphenol blue, with a 99.3% decolorization rate after treating for 24 h. And the decolorization rate of Congo red and methylene blue were 53.7% and 11.5% after treated for 24 hours, respectively. The optimal decolorization conditions were as follows:decolorizing temperature was 30℃, decolorizing pH was 4.0, H2O2 concentration was 5μmol/L, concentration of ligninolytic enzyme was 20 U/mL, concentration of bromophenol blue is 25μmol/L. The decolorization rate of bromphenol blue reached up to 85.61% after treated for 2 hours. The adding of ABTS had no pronounced effect on decolorization rate of bromphenol blue.The study on sodium lignosulfonate biodegradation by ligninolytic enzymes purified from DEAE FF showed that about 82.13% of sodium lignosulfonate could be degraded in the system containing 100 mg/L sodium lignosulfonate,1 mmol/L ABTS,20 U/mL ligninolytic enzyme, and 50 mmol/L sodium acetate buffer (pH4.6) for 8 hours at 40℃. The absorption peak at the 273nm was obviously decreased by UV-VIS spectrogram (200-800nm), which was also demonstrated that the ABTS could clearly improve the biodegradation of sodium lignosulfonate.更多还原