Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids
Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids
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DOI：
10.1007/s00253-008-1417-2
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发表时间：
2008-05-01
影响因子：
5
通讯作者：
Urlacher, Vlada B.
中科院分区：
文献类型：
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作者：
Koschorreck, Katja;Richter, Sven M.;Urlacher, Vlada B.
从地衣芽孢杆菌中克隆了一个新的漆酶基因(Cota)，并在大肠杆菌中表达。重组蛋白CotA经纯化后具有蓝色多铜氧化酶的典型光谱性质。该酶的相对分子质量约为65 kDa，对典型的漆酶底物2，2‘-azino-bis(3-ethylbenzothiazoline-6-sulphonic酸(ABTS)、丁香醛二氮(SGZ)和2，6-二甲氧基苯酚(2，6-DMP)具有活性。ABTS的K-M和k(CAT)分别为6.5+/-0.2亩M和83 S(-1)，SGZ为4.3+/-0.2亩M和10 0 S(-1)，2，6-DMP为5 6.7/-1.0亩M和2 8 S(-1)。在85℃时对ABTS的氧化活性最高，而CoTA在70℃和80℃孵育1h后，其剩余活性分别为43%和8%。此外，还考察了CoTA对几种酚酸和一种非酚酸的氧化作用。CoTA不能氧化香豆酸、肉桂酸和香草酸，而丁香酸被氧化成2，6-二甲氧基-1，4-苯二酚。此外，CoTA还对芥子酸、咖啡酸和阿魏酸进行了二聚反应，发现CoTA对芥子酸的催化活性最高。
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 similar to 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 K-M and k(cat) for ABTS were of 6.5 +/- 0.2 mu M and 83 s(-1), for SGZ of 4.3 +/- 0.2 mu M and 100 s(-1), and for 2,6-DMP of 56.7 +/- 1.0 mu M 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.