Hublik G, Schinner F
Institute of Microbiology, Technikerstrasse 24, University of Innsbruck, 6020, Innsbruck, Austria
Enzyme Microb Technol. 2000 Aug 1;27(3-5):330-336. doi: 10.1016/s0141-0229(00)00220-9.
A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.
漆酶是担子菌平菇菌株RK 36产生的唯一木质素分解酶，已被纯化至同质并进行了表征。该酶是一种单体蛋白，分子量为67000 Da，等电点为3.6。通过分光光度法鉴定出I型和III型Cu(2+)中心。以丁香醛连氮为底物时，漆酶在pH 5.8、50℃和40 mM磷酸盐缓冲液中显示出最高的氧化速率。在测试的稳定化参数中，漆酶分别在高离子强度缓冲液、pH 10、-20℃、存在10 mM苯甲酸和35%乙二醇的条件下保留了大部分活性。粗漆酶被共价固定到Eupergit((R))C上。发现苯甲酸盐在固定化过程中能稳定该酶。漆酶在25℃储存10天期间的活性损失平均为2%。固定化漆酶在填充床反应器中连续去除2,6-二甲氧基苯酚，然后过滤形成的沉淀。研究了氧化丁香醛连氮、邻联茴香胺和2,6-二甲氧基苯酚聚合物在温度、pH值和有机溶剂方面的溶解度。发现沉淀在非极端环境条件下不溶。
