Process Optimization Of Laccase Production From Corious Versicolor And Application Of Laccase

Laccase is an enzyme which could be well used in the industry offood, papermaking and environmental protection etc, has an cheerfulprospect. Unfortunatly, its production is always poor, which refrain itswild range of application.So that, the condition of pure culture to produce laccase had beentested firstly, the optimal medium composition （/L） was200g potato,10g glucose and10g wheat bran as mixed carbon source,7g soybeanpowder as nitrogen source,0.085g （0.5mmol） gallic acid as introducer,1.873g（0.75mmol）CuSO₄·5H₂O,1.5g MgSO₄·7H₂O,3g KH₂PO₄, seedvolume2.8%（V:V）, in the optimum condition, the highest activity oflaccase reached10.511U/mL, which was about9.32times higher thanthat in original fermental condition.Co-culture of two kinds of fungus was investigated. Added1.4%（V:V） yeast after fermention of C.versicolor for3days, the laccase avtivity could be38.9%more than the contro（l10.511U/mL）in the tenthday. So co-culture is an effective process to enhance laccase producingand its application is valuable.Some enzymatic properties of the laccase from pure culture ofC.versicolor and co-culture of C.versicolor and Candida rugosa werestudied with2,2’-azino-bis（3-ethylbenzothiazoline-6-sulfonate）（ABTS）as substrate. The optimal temperature for them was55℃, the laccaseproduced merely by C.versicolor was stable when the temperature was20-45℃or the pH values were4.0,5.5. Laccase activity of both theculture was higher as the pH value was lower. Laccase from pure cultureof C.versicolor got different activity in different buffers, aceticacid-sodium acetate buffer solution had the most significant influence onrelative activity. Metal ions of Co²⁺, Fe²⁺, Mn²⁺decreased the laccaseactivity apparently while Zn²⁺, Cu²⁺, Mg²⁺obviously increased thelaccase activity in certain concentrations. Negative ion of I-had anextreme inhibition in certain concentration, SO₄^2-had an positive impacton laccase activity.Dosage of （ABTS, as a mediator）, indigo blue, laccase, reaction pHvalue and temperature on decolorization of indigo blue by laccase weretested. The results showed that the optimal condition of the reactionsystem （70mL） is6.0μmol ABTS,50mg/L（50mL） indigo,20Ulaccase, pH5.0,45℃; in that condition, vanillin（7.0μmol）was more effective than ABTS（6.0μmol）, vanillin （2.0μmol） mixed withABTS（1.2μmol） as mediator is preferable for decolorization of indigoblue. In other systems which could hardly decolorize dyes, mediatorsshould be added to improve. Under this condition there was almost nodifference in decolorization of indigo blue by laccase produced by pureculture of C.versicolor and mixed culture of C.versicolor and C. rugosa.Laccase, cellulose and the synergism of both could be used in denimwashing, the results showed that merely cellulose be used, thedecolorization was obvious, but high indigo backstaining of indigo blueemerged; laccase only, the decolorization was not very apparent and nobackstaining of indigo blue was found; the synergism of laccase andcellulase could decolorise the indigo blue well with no backstaining.