灵芝漆酶的功能表达及其在工业废水脱色处理中的应用
发布时间：2018-09-13 14:16
【摘要】：漆酶(laccase,EC1.10.3.2)是一类含铜的多酚氧化酶,酚类、羧酸类、芳胺类、生物色素和甾体类激素等都是其作用底物。漆酶是木质素降解的关键酶,广泛应用于纸浆造纸,农林废弃物处理,而且在废水处理、环境污染物降解、食品加工等方面同样有着潜在的应用价值。本文合成了灵芝漆酶基因,整合于毕赤酵母进行异源表达,研究了灵芝漆酶的酶学特性并初步探索了漆酶脱色偶氮类染料氨基黑的能力,为灵芝漆酶工业化生产及应用提供依据。本研究根据中山大学程度博士的灵芝漆酶氨基酸序列,利用DNAworks 3.1软件优化设计引物。使用改良的重叠延伸PCR法合成具有毕赤酵母密码子偏好性的灵芝漆酶基因,通过电击转化法将带有目的基因的表达载体转入毕赤酵母GS115,用甲醇诱导,使其在毕赤醇母中分泌表达。对表达的灵芝漆酶使用Qiagen公司的Ni-NTA spin coloumn亲和层析柱纯化蛋白,通过SDS-PAGE电泳,检测纯化后灵芝漆酶蛋白大小和纯度,得到灵芝漆酶的分子量为55 KDa左右。以ABTS为底物,最适反应温度与p H值分别为30℃和4.6,在30℃时温度稳定性最高,在酸性范围内的p H具有良好的稳定性。针对漆酶对染料的降解作用,对灵芝漆酶作用于偶氮类染料脱色进行了研究。以氨基黑10B为例,采用响应面分析法,研究固定化灵芝漆酶(Lac)对氨基黑10B脱色反应体系进行优化。首先采用单因素实验分别获取反应体系中6个影响脱色率因素的最佳值;然后通过Plackett-Burman设计法对选取的6个影响因素进行筛选,确定主要影响因素为ABTS浓度和Lac酶量;再用最陡爬坡实验逼近ABTS浓度和Lac酶量2个主要因素的最大响应区域,并通过中心组合设计和响应面分析,确定2个主要影响因素的最佳值。优化后的氨基黑10B脱色200μL反应体系为:p H值4.6、温度30℃、硫酸铜浓度1.75 mmol/L、ABTS浓度0.07 mmol/L、Lac酶量6.23 mg、初始底物浓度100 mg/L。该反应体系氨基黑10B脱色率可达60.02%,比单因素法脱色率提高了44.46%。
[Abstract]:Laccase (laccase,EC1.10.3.2) is a kind of copper-containing polyphenol oxidase, phenols, carboxylic acids, aromatic amines, biological pigments and steroidal hormones. Laccase is a key enzyme in lignin degradation, which is widely used in pulp and paper, agricultural and forestry waste treatment, and also has potential application value in wastewater treatment, environmental pollutant degradation, food processing and so on. The laccase gene of Ganoderma lucidum was synthesized and expressed in Pichia pastoris. The enzymatic properties of laccase were studied and the ability of laccase to decolorize azo dye amino black was preliminarily explored. To provide the basis for the industrial production and application of laccase from Ganoderma lucidum. According to the amino acid sequence of Ganoderma lucidum laccase from Sun Yat-sen University, the primer was optimized by DNAworks 3.1 software. The laccase gene of Ganoderma lucidum with codon preference in Pichia pastoris was synthesized by a modified overlapping extension PCR method. The expression vector with the target gene was transferred into Pichia pastoris GS115, by electroporation and induced by methanol. It was secreted and expressed in bihematol mother. The purified Ganoderma lucidum laccase was purified by Qiagen Ni-NTA spin coloumn affinity chromatography. The size and purity of the purified lucidum laccase protein were detected by SDS-PAGE electrophoresis. The molecular weight of Ganoderma lucidum laccase was about 55 KDa. With ABTS as substrate, the optimum reaction temperature and pH were 30 鈩，
本文编号：2241407
[Abstract]:Laccase (laccase,EC1.10.3.2) is a kind of copper-containing polyphenol oxidase, phenols, carboxylic acids, aromatic amines, biological pigments and steroidal hormones. Laccase is a key enzyme in lignin degradation, which is widely used in pulp and paper, agricultural and forestry waste treatment, and also has potential application value in wastewater treatment, environmental pollutant degradation, food processing and so on. The laccase gene of Ganoderma lucidum was synthesized and expressed in Pichia pastoris. The enzymatic properties of laccase were studied and the ability of laccase to decolorize azo dye amino black was preliminarily explored. To provide the basis for the industrial production and application of laccase from Ganoderma lucidum. According to the amino acid sequence of Ganoderma lucidum laccase from Sun Yat-sen University, the primer was optimized by DNAworks 3.1 software. The laccase gene of Ganoderma lucidum with codon preference in Pichia pastoris was synthesized by a modified overlapping extension PCR method. The expression vector with the target gene was transferred into Pichia pastoris GS115, by electroporation and induced by methanol. It was secreted and expressed in bihematol mother. The purified Ganoderma lucidum laccase was purified by Qiagen Ni-NTA spin coloumn affinity chromatography. The size and purity of the purified lucidum laccase protein were detected by SDS-PAGE electrophoresis. The molecular weight of Ganoderma lucidum laccase was about 55 KDa. With ABTS as substrate, the optimum reaction temperature and pH were 30 鈩，
本文编号：2241407
本文链接：https://www.wllwen.com/kejilunwen/huanjinggongchenglunwen/2241407.html
最近更新
教材专著
