【作者】 吴坤；

【导师】 闵航；

【作者基本信息】 浙江大学 ， 环境工程， 2002， 博士

【摘要】 本文以杂色云芝（Coriolus versicolor）和杂色云芝漆酶（laccase）为主线，从高产漆酶微生物的筛选、漆酶生产培养、漆酶的分离纯化、漆酶的酶学特性到漆酶和杂色云芝对部分环境化学污染物的降解等方面进行了较为系统的研究和分析并取得了国内目前相关研究中最为丰富、系统的研究成果，其中部分成果填补了国内甚至国内外相关研究的空白。 1．木质素和纤维素的生物降解是极其缓慢自然过程。本文对所收集到的60个担子菌和半知菌菌株进行了产漆酶和产纤维素酶能力检测，从中筛选到一株高产漆酶菌株——杂色云芝（Coriolus versicolor）、高产纤维素酶和半纤维素酶菌株——黑曲霉M15（Aspergillusniger M15）。其中杂色云芝在发酵条件下，产漆酶能力可以达到53U／ml；黑曲霉M15产纤维素酶和半纤维素酶最高能力分别为5007 U／ml和2639 U／ml。 2．研究了杂色云芝（C．versicolor）产漆酶的发酵条件。结果表明摇瓶实验产漆酶的最佳培养基成分为：可溶性淀粉2g/L，NH₄Cl 24mmol／L，微量元素混合液7ml／L，pH3.0柠檬酸—Na₂HPO₄缓冲溶液0.01mol／L，KH₂PO₄ 1.4×10^-2mol/L，MgSO₄ 1H₂O 2.03×1O^-3mol／L，CaCl₂ 2H₂O 6.8×10^-4 mol／L，VB₁ 2.97×10^-6mol／L，吐温80 4.0g/L，愈创木酚0.01mmol／L，CuSO₄．5H₂O 0.005mmol／L；最佳发酵条件为培养基初始pH3.0，菌体生长6d，培养基装量为250ml三角瓶中25ml培养液，25℃2条件下振荡培养（150r／min）9d。 3．利用杂色云芝发酵液，经超滤、DEAE-Sephadex A250柱层析两步分离纯化，发现杂色云芝发酵液中至少存在两种漆酶。在漆酶的分离、纯化中，漆酶酶谱以无色的底物愈创木酚与酶带在适当条件下反应呈特征性棕红色谱带而加以鉴定。分离纯化得到的两种漆酶分别命名为LacA和LacB，测定出两漆酶LacA和LacB的分子量分别为66.0kDa和30.7kDa；等电点pI分别为4.79和5.18。这是国内首次报道关于真菌漆酶分子量和等电点。 对杂色云芝漆酶LacA和LacB的酶学性质进行了研究。LacA的最适作用温度是40℃，LacB为50℃；保温2h，LacA的半失活温度(t_1/2)为60℃，LacB为90℃；LacA和LacB的最适作用pH值均为4.0；二者的稳定pH值也都在2.5～5.5之间。Al³⁺、Fe³⁺、Cu²⁺、Ag⁺和Hg²+)对两种漆酶均有抑制作用，其中Fe³⁺、Ag⁺的抑制作用最强，酶活完全损失。LacA催化氧化愈创木酚的Km和Vmax分别为0.2865mmol／L和292.40nmol／ml／min；LacB催化氧化愈创木酚的Km和Vmax分别为0.8130mmol／L和127.39nmol／ml／min。比较分析发现，LacB可能是一个非常特殊的漆酶类型。这是国内外首次发现杂色云芝中的低分子量漆酶。 4．测定和分析了杂色云芝漆酶和杂色云芝发酵液对对苯二酚的降解反应。通过对不同底物浓度下杂色云芝漆酶酶学反应动力学特征分析，求得漆酶LacA对对苯二酚的酶反应动力学方程V=1.523461Cs/（16.55047+Cs）；并发现在底物浓度达到一定高度时，存在底物抑制现象，求得LacA对对苯二酚的底物抑制反应速率方程rss=1.021／（1+12.86／Cs+Cs／137.17）。 杂色云芝发酵液对对苯二酚的降解反应进程方程符合一级反应动力学InC=-kt+b 摘 要 方程，并获得了不同底物浓度下的方程参数。发现杂色云芝发酵液对对苯二酚的降解反 应动力学基本符合酶学反应动力学，即符合Michelis－Me血en酶学反应动力学方程，并求得该反 应动力学方程为 Vpl．768347Csl（．626834＋Cs）。研究表明杂色云芝发酵液对对苯＝＄的降解 反应动力学与LacA对对苯二酚的酶学反应动力学具有较大的相似性，也存在着底物抑制现象， 其底物抑制的动力学方程为rts’－1．4011＋5尸5／CS＋CS／278．26)。 比较漆酶纯酶LacA和杂色云芝发酵液对对苯H酚反应的n和Vmax数值，发现杂色云 芝发酵液KJll较小而Vmax ＄值较大，显示出发酵液较漆酶纯酶具有更高的底物亲和力和催化 效率，这可能与发酵液中存在的某些mediators有关。这是国内外第一个相关的研究报告。 5．杂色云芝和杂色云芝漆酶对很多环境污染物具有降解能力。初步试验表明杂色 云芝在液体发酵条件下，3d内对添加水平50mg／L—500mg／L的苯胺降解率达到 10％以上；对 添加水平100mgh～300m叭的联苯肢降解率达到20％以上；对添加水平50mg／Lnd00m叭硝 基苯的降解率达到 50％，对添加水平 50mg／L－－si00mg／L 2，4二硝基氯苯的降解率达到 90％以 上。杂色云芝对黄曲霉毒素污染水平为320gb的阳性玉米样品，50d内的去毒率可达到100 ％，这是国内外第一个关于白腐菌对黄曲霉毒素降解的研究结果。本文建立了土壤中PCP的测 定方法，方法回收率和灵敏度分别为 68％和 0．ling／L；在无菌土壤基质中，杂色云芝对水平 50m日L一100m叭PCP的阵解率一个月内达50o以上，2个月左右可以达到完全降解。 6．分?更多还原

【Abstract】 Herein a systematic research of Coriolus versicolor and C. versicolor laccases has been made in the article, including election of high laccase-secreted strain, fermental condition for laccase production by C. versicolor, isolation, purification , characterization and enzymatic kinetic of laccases from C. versicolor, dynamics of m-cresol biodegradation as well. A plenty of active results have also been made as fellows:The biodegradation of lignin and cellulose is an extremely slow natural process. The production of laccase and cellulose was assayed in the 60 Deuteromycotina strain and Basidiomycetes strains collected. A highly productive laccase strain-Cono/us versicolor and a highly productive cellulase and hemicellulase strain - Aspergillus niger Ml 5 was screened, of which production of laccase by Coriolus versicolor in liquid fermentation reached 153U/ml, while the highest productivity of cellulase and hemicellulase by Ml 5 in solid fermentation were 5 007U/ml and 2 639U/ml, respectively.The optimal fermentation conditions for laccase production by Coriolus versicolor were studied and as the followings: The composition of fermentation medium was soluble starch 2g/L, NHjCl 24mmol/L, mixed liquid of trace metals 7ml/L, pH 3.0, citric acid-Na2HPO4 buffer O.Olmol/L, KH2PO4 1.4 X 10’2mol/L, MgSO4 ?7H2O 2.03X 10-3mol/L, CaCl2 ?H2O 6.8 X 10-4 mol/L, VB1 2.97 X 10 -6 mol/L, Tween 80 4.0g/L, guaiacol 0.01mmol/L and CuSO4 ?5H2O 0.005mmol/L, and the optimal conditions were the initial pH of medium 3.0, 25ml medium in 250ml-flask, and incubated at 25 癈 and 150r/minfor 9d.Two laccases were purified to electrophoretic homogeneity from Coriolus versicolor culture by the combination of superflltrate and DEAE-Sephadex A-250 ion exchange chromatography. The properties of the isoenzymes were determined by zymogram patterns from polyacrylamide gel and subsequently named Lac A and Lac B electrophoresis(PAGE). The purities were increased 9.3-fold and 3.41-fold with an overall yield of 18.2% for Lac A and 0.2% for Lac B, respectively. The molecular weights of Lac A and Lac B are 66.0 kDa and 30.7kDa as determined by SDS gel electrophoresis(SDS-PAGE) and their isoelectric points(pl) are 4.79 and 5.18 as assayed by polyacrylamide gel isoelectric focusing(PAGIF),82respectively. Both of these enzymes have same pH optimum (pH 4.0) and different temperature optimum (40 癈 for Lac A and 50 癈 for Lac B) for their optical activity. The temperature for half loss activity(t1/2) in 2 hours is 60 癈 for Lac A and 90 癈 for Lac B. The activities are constant at pH value in the range of 2.5~5.5. The Km and Vmax of Lac A for oxidizing guaiacol is 0.2865mmol/L and 292.40 mmoUL/min while those of Lac B is 0.8130 mmol/L and 127.39mmol/L/min, respectively. Their activities were inhibited by A13+, Fe3+, Cu2+, Ag+, Hg2+ and strongest by Ag+ and Fe3+.The degradation reaction process of hydroquinone by laccase and filtrate from C. versicolor was analyzed using the principle of M-M equation, leading to this enzyme catalytic reaction kinetics constant Km 16.55047mg/L and Vmax 1.52346 Img/L/min, However, there appeared the inhibition as the substrate concentration reached up to certain degree. The rs,max--1.021mg/min, Ks--12.86mg/L/min, KSI - 137.17mg/L, Cs.opt~40mg/L, and the reaction rate equation of substrate inhibition of rss=1.021/(l+12.86/Cs + Cs/137.17) was revealed after dealing with properties of the kinetics in different substrate density. The analysis and comparison of the parameters from theoretical equation and the practice assayed results suggested both Km and Vmax given by Lineweaver - Burke plots was quite different from the actual case, indicating it is possible to get kinetics parameter just from Lineweaver-Burke plots would lead to a great deviation.The degradation reaction process of hydroquinone by filtrate from C. versicolor was analyzed using the principle of M--M equation, leading to the kinetics equation of catalytic reaction Vf=1.768347Cs/(3.626834+ Cs). However, there appeared the inhibition as the substr更多还原