Production, partial characterization and mass spectrometric studies of the extracellular laccase activity from Fusarium proliferatum.
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.
Fernaud
J R Hernández
JR
Departamento de Microbiología y Biología Celular, Facultad de Farmacia, Universidad de La Laguna, Tenerife, Spain.
Marina
A
A
González
K
K
Vázquez
J
J
Falcón
M A
MA
D000595:Amino Acid Sequence; D019905:Benzyl Alcohol; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media / Q000737:chemistry; D004043:Dietary Fiber; D004789:Enzyme Activation; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D013696:Temperature
D003470:Culture Media; D004043:Dietary Fiber; D042845:Laccase; D019905:Benzyl Alcohol