【作者】 王维乐；

【导师】 牟志美；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2011， 硕士

【摘要】 漆酶（Laccase,EC 1.10.3.2）是一种含铜的多酚氧化酶,具有广泛的底物,不仅能催化氧化多种芳香族化合物,还能降解木质素、去除许多有毒酚类物质如苯氧基类除草剂等的毒性,还可以使多种染料脱色及去除石油工业废水的毒性;因此在造纸、环保、食品、医药卫生、生物检测等领域具有较大的研究和应用价值。鉴于此,以本实验室筛选到的一株高产漆酶菌株Paraconiothyrium variabile GHJ-4 （简称:GHJ-4）为研究对象。采用统计学方法对GHJ-4菌株产漆酶的发酵培养基及发酵条件进行了优化,并进一步对菌株GHJ-4所产漆酶进行了分离纯化及其酶学性质的研究,主要内容如下:1.通过摇瓶培养,结合统计分析方法研究了发酵培养基和发酵条件对GHJ-4产漆酶活性的影响。通过单次因子试验和正交试验确定了发酵培养基的最佳碳源、氮源、碳氮比、无机盐。在此基础上,通过响应面法对发酵培养基进行优化,首先采用Plackett-Burman（PB）试验设计筛选出了影响该菌产漆酶的三个重要因素:可溶性淀粉、牛肉浸膏和CuSO₄;继而采用最陡爬坡试验逼近最大响应区域,并结合中心组合试验以及响应面分析,建立了以漆酶酶活为响应值的二次回归方程模型,从中获得了最佳发酵培养基为:麸皮30 g/L,可溶性淀粉58.08 g/L,牛肉浸膏3.39 g/L,NaNO₃ 1 g/L,KH₂PO₄ 3 g/L,CuSO₄ 0.554 g/L。在优化后发酵培养基的基础上,结合统计分析方法对发酵条件经了优化,首先用PB法筛选出四个影响较大的因素,即接种量、培养时间、装样量和转速;继而采用最陡爬坡试验逼近最大响应区域,并结合中心组合试验以及响应面分析,建立了以漆酶酶活为响应值的二次回归方程模型,从中获得了最佳发酵条件为:接种量6.43%（v/v）,温度28℃,初始PH 4,培养时间182 h,装液量72.5 mL/250 mL,转速157 r/min,种龄96 h。采用最佳发酵培养基和发酵条件,供试菌株的漆酶酶活达到710.44 U/mL,较优化前漆酶的产量提高了16.4倍,其试验值与预测值基本相符,说明预测模型可靠性高,可应用于漆酶发酵条件的优化。2. GHJ-4漆酶的分离纯化及其酶学性质菌株GHJ-4液态发酵所产的漆酶通过硫酸铵分级沉淀、DEAE-sepharose Fast Flow阴离子交换树脂、Sephadex G-75分子筛柱层析三步分离纯化后,得到电泳纯的漆酶,纯化倍数为8.1倍,回收率为24.2%,其分子量约53 kDa。纯化后的漆酶最适温度和最适pH分别为50℃和5.0,该酶在55℃以下pH3.5⁶.5范围内较为稳定;Cu²⁺、Mg²⁺、Zn²⁺对漆酶酶活有明显促进作用,而Hg²⁺、Fe³⁺、Ag⁺对漆酶酶活有显著抑制作用。以愈创木酚为底物时,测定该酶的米氏常数Km值为62.07μmol/L,最大反应速度Vmax为68.97 mmol/（L·min）。更多还原

【Abstract】 Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of polyphenols and other lignin-derived aromatic compounds. Laccases can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials; they can thus be widely used in paper pulp industry, food industry, medication and sanitation, biosensor and envir-onment protection. Hence, this study t a strain of high laccase producing microorganism was obtained from my laboratory. It was termed as Paraconiothyrium variabile GHJ-4 that was be called for GHJ-4 short. The fermentation medium and fermentantion conditions were statistical optimized. Lastly purification and characterization of laccase was operated from GHJ-4. The main contents were as follows.1. Through the cultivation in shake-flask fermentation, the effects of environmental conditions and nutrition on production of laccase by GHJ-4.Refering to the effects of nutrition on production of laccase, the optimum carbon, nitrogen, C/N and inorganic salt were confirmed by one-factor at a time and orthogonal test experiment. Firstly, we screened the important parameters by Plackett-Burman design. It showed that three factors playing the important roles in the medium, including soluble starch, beef extract and CuSO₄. After steepest ascent experiment approaching the optimal level of the three factors, the center composite design and response surface analysis were applied to establish the second-order equation model for the yield of laccase. On the last step, the optimal concentrations of components were determined by central composite design and response surface analysis.The optimal medium for GHJ-4 was composed of （in g/L）: wheat bran 30, soluble starch 58.08, beef extract 3.39, NaNO₃ 1, KH₂PO4 3, CuSO₄ 0.554.Based on the optimal medium, statistical analysis methods were applied to optimize the fermentation conditions. Firstly, Plackett-Burman design was used to investigate the main factors affecting laccase yield of GHJ-4. The results showed that four factors playing the important roles in the fermentation conditions, including inoculation volume, culture time, broth content and rotating speed. After steepest ascent experiment approaching the optimal level of the four factors, central composite design and response surface analysis were applied to establish the second-order equation model for the yield of laccase. On the last step, the optimal fermentation conditions were determined by central composite design and response surface analysis.The optimal fermentation conditions were obtained as following: 6.43% of inoculation, 28℃of culture temperature, initial pH value 4, 182 h of culture time, 72.5 mL of broth content, 157 r/min of rotating speed, and 96 h of seed age. Under this optimum conditions, the activity of laccase increased nearly 16.4 times to 710.44 U/mL and the experimental values argreed with the predicted values. These results suggested that the predicted model was reliable and available for the optimization of laccase fermentation conditions.2. Purification and characterization of laccase from GHJ-4.The laccase produced by strain GHJ-4 in liquid-state fermentation was isolated and purified by a three-step process: ammonium sulfate precipitation, DEAE-sepharose Fast Flow aion-exchange chromatography, Sephadex G-75 gel penetration chromatography. After the three-step purification, we obtained a electrophoresis pure laccase, its purity and yield were 8.1 folds and 24.2% of the crude extract respectively. The molecular mass of the laccase was about 53 kDa.The laccase was stable within the range of pH between 3.5 and 6.5 and at the temperature lower than 55℃with the maximal activity at 50℃, pH 5.0. Laccase could be markedly activated by Cu²⁺, Mg²⁺ and Zn²⁺ while be inhibited by Hg²⁺, Fe³⁺ and Ag⁺. The apparent Km of guajacolum was 62.07μmol/L, the Vmax was 68.97 mmol/（L·min）.更多还原