Koschorreck Katja, Richter Sven M, Ene Augusta B, Roduner Emil, Schmid Rolf D, Urlacher Vlada B
Institute of Technical Biochemistry, University of Stuttgart, 70569 Stuttgart, Germany.
Appl Microbiol Biotechnol. 2008 May;79(2):217-24. doi: 10.1007/s00253-008-1417-2.
A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The enzyme has a molecular weight of approximately 65 kDa and demonstrates activity towards canonical laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants KM and kcat for ABTS were of 6.5+/-0.2 microM and 83 s(-1), for SGZ of 4.3+/-0.2 microM and 100 s(-1), and for 2,6-DMP of 56.7+/-1.0 microM and 28 s(-1). Highest oxidizing activity towards ABTS was obtained at 85 degrees C. However, after 1 h incubation of CotA at 70 degrees C and 80 degrees C, a residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid by CotA was observed, and highest activity of CotA was found towards sinapic acid.
从地衣芽孢杆菌中克隆出一个新的漆酶基因（cotA），并在大肠杆菌中进行表达。重组蛋白CotA经纯化后表现出蓝色多铜氧化酶特有的光谱特性。该酶分子量约为65 kDa，对典型漆酶底物2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）（ABTS）、丁香醛连氮（SGZ）和2,6-二甲氧基苯酚（2,6-DMP）具有活性。ABTS的动力学常数KM和kcat分别为6.5±0.2 μM和83 s-1，SGZ的分别为4.3±0.2 μM和100 s-1，2,6-DMP的分别为56.7±1.0 μM和28 s-1。在85℃时对ABTS的氧化活性最高。然而，将CotA在70℃和80℃孵育1小时后，测得的残余活性分别为43%和8%。此外，还研究了CotA对几种酚酸和一种非酚酸的氧化作用。CotA无法氧化香豆酸、肉桂酸和香草酸，而丁香酸被氧化为2,6-二甲氧基-1,4-苯醌。此外，观察到CotA使芥子酸、咖啡酸和阿魏酸发生二聚化，且CotA对芥子酸的活性最高。
