Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum

• Published in: Applied microbiology and biotechnology Vol. 70; no. 2; pp. 212 - 221
• Main Authors: Hernández Fernaud, J.R, Marina, A, González, K, Vázquez, J, Falcón, M. A
• Format: Journal Article
• Language: English
• Published: Berlin Berlin/Heidelberg : Springer-Verlag 01.03.2006; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Benzyl Alcohol; Biological and medical sciences; Biotechnology; Biotechnology - methods; Culture Media - chemistry; Dietary Fiber; Enzyme Activation; Enzymes; Fundamental and applied biological sciences. Psychology; Fusarium - enzymology; Fusarium - genetics; Fusarium - growth & development; Fusarium proliferatum; Hydrogen-Ion Concentration; Ionization; Ions; Laccase - chemistry; Laccase - genetics; Laccase - metabolism; Liquid chromatography; Mass Spectrometry; Molecular Sequence Data; Peptides; Proteins; Scientific imaging; Temperature; Triticum aestivum; Wheat bran; Fungi; Characterization; Enzyme; Production; Fusarium; Fungi Imperfecti; Oxidoreductases; Extracellular; Mass spectrometry; Thallophyta; Laccase
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-005-0221-5

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°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.