Study On Laccase Produced By Pleurotus Ferulae

Lignin is an aromatic polymer compound, intertwined with cellulose and hemicellulose.The presence of lignin is not only restricted the use of the cellulose, but also caused seriousenvironmental pollution. The ligninase mainly including three enzymes: lignin peroxidase(LiP), manganese peroxidase (MnP) and laccase (Lac). Laccase (EC1.10.3.2), also knowncouplet phenol oxidase, is a copper polyphenol oxidase. Laccase are widely distributed inplants, insects, and fungi, the most widely used is the white-rot fungus laccase. Laccase, asbiocatalysts, is attracting much attention in terms of environmental protection and industrialproduction. Especially in the food industry, pulp bio-bleaching, industrial wastewatertreatment, dye decolorization，detoxification and degradation of environmental pollutants.Therefore, laccase has the necessity of research value, and has a very large applicationpotential to become a hotspot for protecting the environment by enzyme.In this study, the effect of culture medium on laccase production by Pleurotus ferulaeJM30X was investigated by single-factor experiment in submerged fermentation. Theoptimized medium for laccase production by Pleurotus ferulae JM30X was: glucose20g/L,corn flour10g/L, wheat bran10g/L,1mmol/L K2SO4, initial pH9.0.150mL medium in500mL flask, incubate at25oC,120r/min. Under this condition, the highet laccase production was6832.86U/L, which was signicantly enhances by6.09folds compared with control.The relationship between morphology and laccase production was studied. The resultsindicated that the morphology of P. ferulae pellets was changed when glass beads were added.Laccase production showed higher with spherical mycelium than with filamentous orflocculent mycelium. In addition, the spherical mycelium with a diameter of0.2-0.4mmhighly affected laccase production. Effect of the composition of culture medium on pelletswas investigated and results indicated that various concentrations of glucose, corn meal andwheat bran were important to the formation of pellets in diameter of0.2-0.4mm. Besidesnutrients, the addition of non-nutritional substrates influenced the distribution of P. ferulaepellets. However, the production of laccase was not promoted by non-nutritional substrates.Laccase in fermentation broth was purified by ammonium sulfate precipitation, Mono Q5/50GL strong anion exchange chromatography and SuperdexTM7510/300GL gel filtrationchromatography. The obtained laccase was purified4.85folds compared with crude broth,with a26.91%yield. The molecular weight of purified laccase was66kDa, analyzed bySDS-PAGE and Native-PAGE.The optimum catalytic pH and temperature were3.0and50-70oC for ABTS, respectively.P. ferulae JM30X laccase exhibited high thermostability with t1/2of7.59h under50oC. The P.ferulae JM30X laccase is relatively stable in the acidic pH conditions, while inactivatedrapidly under alkaline pH conditions. In four inhibitors, laccase activity was inhibited50%by1.0mmol/L ethylenediamine tetraacetic acid (EDTA), while completely inhibited by1.0mmol/L sodium azide (NaN3), dithiothreitol (DTT) and phenylmethylsulfonyl fluoride(PMSF). However, with the increase of the concentration of the metal ions, the enzymeactivity is reduced and inhibited. However, laccase was completely inhibited by Fe2+and Fe3+at any concentration. The best substrate for laccase was ABTS, in which the Michaelis-Menten constant (km) and catalytic efficiency (kcat/km) value for P. ferulae JM30Xlaccase was0.193mmol/L and2.73×106(mmol/L S)-1, respectively.In this study, a novel Pleurotus genus laccase was obtained from P. ferulae JM30X. Toour knowledge, the laccase production by P. ferulae JM30X submerged fermentation has notbeen reported. Laccase produced by P. ferulae JM30X exhibit excellent thermal property andhave potential for industrial and biotechnology applications.