Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum
Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum
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DOI：
10.1007/s00253-005-0221-5
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发表时间：
2006-03
影响因子：
5
通讯作者：
J. Fernaud;A. Marina;K. González;Jesús Vázquez;M. A. Falcón
中科院分区：
文献类型：
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作者：
J. Fernaud;A. Marina;K. González;Jesús Vázquez;M. A. Falcón
苯甲醇和不含淀粉的商品麦麸是增殖酵母（MUCL 31970）漆酶活性的有效诱导剂。培养物的初始pH值也是提高其产量的最重要因素。通过凝胶渗透高效液相色谱法，酶洗脱为明显均匀的峰，分子量为54 kDa，但通过等电聚焦，发现两个pI值分别为5.17和5.07的蛋白。非变性聚丙烯酰胺凝胶电泳还检测了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）分析，两种蛋白均具有独特的指纹图谱，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，没有发现明显的匹配。用纳米电子电离-离子阱-质谱（nESI-IT-MS）对这两种同工酶的部分肽进行串联质谱分析，证实了它们的独特性质。以下有趣的性质，特别是它在碱性pH下的稳定性，使这种漆酶成为生物技术应用的有前途的工业酶：60°C下的最大活性，40°C下2小时的热稳定性，2,2 ' -azino-bis（3-乙基苯-噻唑啉-6-磺酸盐）的最佳pH值为3.5 (km=62 μM)，在25°C下2小时的pH稳定性为4-8（在pH水平2.2和9下稳定性为75%）。
Benzyl alcohol and starch-free commercial wheat bran were effective inducers of the laccase activity in cultures ofFusarium 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°C, thermal stability for 2 h at 40°C, optimum pH 3.5 (km=62 μM) 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°C.