Fernaud J R Hernández, Marina A, González K, Vázquez J, Falcón M A
Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna, Tenerife, Spain.
Appl Microbiol Biotechnol. 2006 Mar;70(2):212-21. doi: 10.1007/s00253-005-0221-5. Epub 2005 Dec 3.
Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures of Fusarium proliferatum (MUCL 31970). Initial pH value in the cultures was also an overriding factor for increasing its production. By gel permeation high-performance liquid chromatography, the enzyme eluted as an apparently homogeneous peak with a molecular mass of 54 kDa, but by isoelectrofocusing, two proteins with pI values of 5.17 and 5.07 were revealed. Two different phenoloxidase activities were also detected after nondenaturing polyacrylamide gel electrophoresis. By matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), both proteins showed unique fingerprints, so they were classifiable as isozymes, and were named laccase 1 (Lac1, pI 5.17) and laccase 2 (Lac2, pI 5.07). No clear matches were found when compared with other proteins. The tandem mass spectrometry of some peptides from both isozymes reanalyzed by nanoelectron ionization-ion trap-mass spectrometry (nESI-IT-MS) confirmed their unique character. The following interesting properties, particularly its stability at alkaline pH, make this laccase a promising industrial enzyme for biotechnological applications: maximum activity at 60 degrees C, thermal stability for 2 h at 40 degrees C, optimum pH 3.5 (km=62 microM) measured on 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate), and pH stability 4-8 (75% stability at pH levels 2.2 and 9) for 2 h at 25 degrees C.
苯甲醇和无淀粉的商用麦麸是串珠镰刀菌（MUCL 31970）培养物中漆酶活性的有效诱导剂。培养物中的初始pH值也是提高其产量的一个重要因素。通过凝胶渗透高效液相色谱法，该酶以一个表观均一的峰洗脱，分子量为54 kDa，但通过等电聚焦，发现了两种pI值分别为5.17和5.07的蛋白质。非变性聚丙烯酰胺凝胶电泳后也检测到两种不同的酚氧化酶活性。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS），这两种蛋白质都显示出独特的指纹图谱，因此它们可归类为同工酶，分别命名为漆酶1（Lac1，pI 5.17）和漆酶2（Lac2，pI 5.07）。与其他蛋白质相比，未发现明显匹配。通过纳米电喷雾电离-离子阱质谱（nESI-IT-MS）重新分析的两种同工酶的一些肽段的串联质谱证实了它们的独特性质。以下有趣的特性，特别是其在碱性pH下的稳定性，使这种漆酶成为一种有前途的用于生物技术应用的工业酶：在60℃时活性最高，在40℃下热稳定2小时，以2,2'-联氮双（3-乙基苯并噻唑啉-6-磺酸）为底物测得的最佳pH为3.5（km = 62 microM），在25℃下pH稳定性为4-8（在pH 2.2和9时75%的稳定性）2小时。
