Xiao Y Z, Tu X M, Wang J, Zhang M, Cheng Q, Zeng W Y, Shi Y Y
Key Laboratory of Structural Biology, University of Science and Technology of China, Chinese Academy of Sciences, Hefei, Anhui, 230026, PR China.
Appl Microbiol Biotechnol. 2003 Feb;60(6):700-7. doi: 10.1007/s00253-002-1169-3. Epub 2002 Dec 18.
A recently isolated basidiomycete, Trametes sp. strain AH28-2, can be induced to produce a high level of laccases when grown on a cellobiose-asparagine liquid medium. After induction by kraft lignin, two major isozymes were detected in the fermentation supernatant of the fungus. The principal component laccase A, which accounts for about 85% of the total activity, can be purified to electrophoretic homogeneity by three chromatographic steps: DEAE-Sepharose FF, Superdex-200 and Mono-Q. The solution containing purified laccase is blue in color, and the ratio of absorbance at 280 nm to that at 600 nm is 22. The molecular mass of laccase A is estimated to be 62 kDa by SDS-PAGE, 57 kDa by FPLC, and measured as 58522 Da by MALDI mass spectrum. Laccase A is a monomeric glycoprotein with a carbohydrate content of 11-12% and an isoelectric point of 4.2. The optimum pH and temperature for oxidizing guaiacol are 4.5 and 50 degrees C, respectively. The half-life of the enzyme at 75 degrees C is 27 min. The enzyme shows a good stability from pH 4.2 to pH 8.0. The K(m) values of the enzyme toward substrates 2,2'-azino-bis (3-ethylbenzothazoline-6-sulfonate) (ABTS), guaiacol and 2,6-dimethoxyphenol are 25, 420 and 25.5 microM, respectively, and the corresponding V(max) values are 670, 66.8, and 79 microM min(-1) x mg(-1), respectively. Laccase A activity is strongly inhibited by 0.1 mM NaN(3) or 0.1 mM cyanide. Two units of laccase A alone is able to completely oxidize 100 micromol 2,6-chlorophenol in 6 h. In the presence of 1 mM ABTS and 1-hydroxybenzotriazole, 15.0 U laccase A is able to oxidize 45% and 70% of 50 micromol fluorene in 12 and 18 h, respectively. The laccase A gene was cloned by a PCR method, and preliminary analysis of its sequence indicates 87.0% similarity to the corresponding segment in the phenoloxidase gene from Coriolus hirsutus.
最近分离出的一种担子菌——栓菌属菌株AH28 - 2，在以纤维二糖 - 天冬酰胺液体培养基培养时，可被诱导产生高水平的漆酶。经硫酸盐木质素诱导后，在该真菌的发酵上清液中检测到两种主要的同工酶。主要成分漆酶A占总活性的约85%，可通过三步色谱法纯化至电泳纯：DEAE - 琼脂糖FF、Superdex - 200和Mono - Q。含有纯化漆酶的溶液呈蓝色，280 nm处的吸光度与600 nm处吸光度之比为22。通过SDS - PAGE估计漆酶A的分子量为62 kDa，通过FPLC为57 kDa，通过基质辅助激光解吸电离质谱测定为58522 Da。漆酶A是一种单体糖蛋白，碳水化合物含量为11 - 12%，等电点为4.2。氧化愈创木酚的最适pH和温度分别为4.5和50℃。该酶在75℃下的半衰期为27分钟。该酶在pH 4.2至pH 8.0范围内表现出良好的稳定性。该酶对底物2,2'-联氮 - 双（3 - 乙基苯并噻唑啉 - 6 - 磺酸）（ABTS）、愈创木酚和2,6 - 二甲氧基苯酚的K(m)值分别为25、420和25.5 μM，相应的V(max)值分别为670、66.8和79 μM min(-1) x mg(-1)。漆酶A的活性受到0.1 mM叠氮化钠或0.1 mM氰化物的强烈抑制。单独的两个漆酶A单位能够在6小时内完全氧化100 μmol 2,6 - 氯苯酚。在存在1 mM ABTS和1 - 羟基苯并三唑的情况下，15.0 U漆酶A能够在12小时和18小时内分别氧化50 μmol芴的45%和70%。通过PCR方法克隆了漆酶A基因，对其序列的初步分析表明，它与糙皮侧耳酚氧化酶基因中的相应片段有87.0%的相似性。
