【作者】 周雯婧；

【导师】 曾光明； 刘剑潇；

【作者基本信息】 湖南大学 ， 环境科学， 2008， 硕士

【摘要】 堆肥一直被视为是处置工业和农业固体废物的一种非常有效的处理方式,在这个过程中的生物发挥着重要作用。电化学传感器是基于生物化学反应进行信号传导的电化学装置,具有方便、省时、精度高等优点,已成为环境监测领域的一种新型的检测技术。电化学生物传感器因为具有高效性和专一性的特点,成为电化学传感器的研究热点,其构建的关键技术在于基于多壁碳纳米管(CNTs)修饰的工作电极,创造了漆酶的电信号放大的微环境中。本研究致力于提高堆肥中分离的简青霉Penicillium simplicissimum的漆酶的产量,对于漆酶的电化学反应、表征及其在CNTs修饰后的放大效果进行了研究。主要完成了以下工作：对简青霉Penicillium simplicissimum产漆酶的液态发酵条件进行了研究,结果表明摇瓶培养产漆酶的最佳培养基组成为：麦芽糖2 g/L,蛋白胨1.2 g/L,大量元素体积分数为100 mL/L,微量元素体积分数为25 mL/L,VB1质量为100μg,吐温80浓度为0.05%,将含有107个分生孢子/mL的此培养液恒温30℃振荡(150r/min)培养8d,产漆酶量达12.44 U/L。培养液初始pH 3.5最有利于漆酶的产生,一定浓度的Cu2+有利于酶活提高,最适合浓度为60μmol/L。本实验研制成的一种电化学传感器检测分离于堆肥简青霉Penicillium simplicissimum所产生的漆酶的活性。该传感器是基于多壁碳纳米管(CNTs)修饰的玻碳电极。采用碳纳米管加入这一检测系统可以大大提高电化学分析法的信号,因此比常规的分光光度测定法具有更高的敏感度,选择性和速度。经研究发现,电解系统的最佳pH值为5.6。结果表明：电流和分光光度检测法测量出的漆酶酶活的浓度之间具有一个良好的线性关系,通过电流计时法检测出的电流的斜率所显示的相关系数为0.9835。因此,这个电化学传感器可用于快速检测堆肥中简青霉Penicillium simplicissimum所产生的漆酶的酶活。此外,它还在通过根据漆酶的酶活与生物量之间的相关性来来对Penicillium simplicissimum进行快速的定量分析方面具有潜在的可能性。更多还原

【Abstract】 Composting is considered as a useful process for the disposal of municipal and agricultural solid waste, in which enzymes play an important role. Electrochemical sensors are electrochemical devices that are based on the signal transmission of biochemical reactions. In comparison with the conventional chemical and biochemical analytical methods, the electrochemical sensors have the advantages of convenience, time-saving as well as high precision. Moreover, they are convenient for computers to collect and process data, and the sample won’t be damaged or polluted. Electrochemical sensor as a new kind of analytical tool is widely applicable in monitoring and controlling of composting.The sensors have been one of most concerned areas because of their specificity and high-efficiency. In the design and fabrication of electrochemical sensors, the key technology is to modify the working electrode with the multi-wall carbon nanotubes (CNTs) which can enhance the electrochemical signal of the laccase in the microenvironment. In this paper, the research was aimed to develop the production of the laccase from Penicillium simplicissimum isolated from the composting, and characterize the electrochemical activity of the laccase as well as the enhanced signal from the introduction of the CNTs. The detailed research contents are summarized as follows:The optimal fermentation conditions for laccase production by Penicillium simplicissimum in shake- flask cultivation were studied. The composition of fermentation medium was maltose 2g/L, peptone 1.2 g/L,100 mL/L Massive elements solution,25 mL/L Trace elements solution,100μg/L VB1,0.05% Tween 80. Shaking culture of the liquid containing 107conidia/mL for 8 day at the regime of 30℃and 150 r/min resulted in the enzyme activity of 12.44 U/L. The optimum pH of production Laccase was 4.0, the optimal concentration of Cu2+ was 60 u mol/L. An electrochemical sensor for detection of the activity of laccase from Penicillium simplicissimum isolated from the composting has been developed. The sensor is based on glassy carbon electrode modified with multi-wall carbon nanotubes (CNTs). The introduction of CNTs into this system can greatly enhance the electrochemical signal in this assay more sensitively, selectively and rapidly than that in conventional spectrophotometric assays. It was found that the optimal pH value of the electrolyte was 5.6. The results showed a good linear correlation between the current and the concentration of laccase activities measured by spectrophotometry, where the current slope was measured by chronoamperometry with a coefficient of 0.9835. Therefore, this electrochemical sensor can be used for rapid detection of laccase activity from Penicillium simplicissimum. Furthermore, it may be potentially used for rapid quantification of Penicillium simplicissimum according to the relationship between the laccase activities and the biomass.更多还原