Purification, Characterization And Functional Analysis Of Laccase From Vitis Vinifera

Laccase (ECI.10.3.2) called for-two phenol (double):oxygen oxidoreductase, Researches to date indicate that laccase is widely present in the fungi, bacteria, plants, and insects. The researches related to plant laccases are relatively fewer, but the functions of laccases in lignin and pigment biosynthesis, disease and stress resistance are studied and some results have been obtained. Grape is deciduous vine plants, fruit yield occupy almost1/4of the world. The nutritional value of grape is very high, which contains large amounts of protein, glucose, fructose, tartaric acid, oxalic acid and many trace elements. So far no research has been reported about the laccase from Vitis vinifera. In this thesis, we chemically synthesized the laccase gene, named VvLCCI, the VvLCCI gene was secretory expressed in Pichia pastoris, and the function of the recombinant laccase was characterized. This study shows that VvLCCI can play an important role in the treatment of industrial wastewater containing dyes or phenolic compound. At the same time, it provides the theoretical foundation for the function of laccase in Grape.In this paper, a new laccase gene from Vitis vinifera was synthesized chemically according to the yeast bias codon and integrated into Pichia pastoris GS115genome by electroporation. the yeast strains showed the highest laccase activity were selected by screening using G418and PCR, then the strains was stored at-70℃in20%glycerol. A large-scale production of the recombinant laccase was performed with100mL of BMGY growth medium and subsequently with BMMY methanol induction medium, The supernatant containing crude recombinant laccase was purification by an affinity Ni column specific to His-tailed proteins at4℃. SDS-PAGE analysis showed that the recombinant laccase has a molecular mass of approximately64.0kDa. Its biochemical properties was carriedout using substrate2-2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS). It was showed that the optimum pH and temperature of the laccase is3.0and40℃, respectively. Cu2+has a strong promoting effect on the activity of VvLCCI, while Fe2+, L-cysteine and sodium thiosulfate inhibit the enzyme’s catalytic function strongly. The Km values of the ABTS and guaiacol are0.0923and16.8mM, respectively, and the Vmax values are39.55and10.79mM*mg-1*min-1, respectively. The recombinant laccase degraded87.2%2,4,6-TCP after7.5h under the optimal conditions. At the same time, VvLCCI has very strong decolorization ability on azo dyes and aromatic methane dyes.