Construction Of Bifunctional Enzyme With Endoglucanse And Laccase From Melanocarpus Albomyces And Analysis Of Their Enzymatic Properties

Waste paper is a very important renewable resource, recycling of waste paper is aneffective way to solve the problem that the paper industry is facing now. However, thereare still many problems need to be solved. Enzymatic deinking has been widely used inthe paper industry. Cellulase, hemicellulase has been used for deinking of old newspaper, which already realized industrialization. To discover new enzyme is of vital significance for the development of enzymatic deinking. In industrial production, compared to culturing two single enzyme, cultivating difunctional enzyme can save materials and inputs, so it’s more economic and effective to use difunctional enzyme in waste paper deinking. In this paper, endoglucanase gene Maeg and laccase gene Malac were derived from Melanocarpus albomyces, due to endoglucanase and laccase of similar character, they are appropriate for constructing difunctional enzyme.In this study, endoglucanase gene Maeg and laccase gene Malac from Melanocarpusalbomyces were synthesized by the company. The gene Maeg encoding235amino acidresidues and the gene Malac encoding623amino acid residues were inserted into the plasmid pPICZαA and expressed in Pichia pastoris KM71H(Muts,Arg+). Than, an artificialfusion protein MaEG-(GS4)2-MaLac of Melanocarpus albomyces endoglucanase(MaEG) andMelanocarpus albomyces laccase (MaLac) was constructed by (G4S)3linker. Although endoglucanase and laccase activities can be both exhibited in yeast culture supernatant, there weretwo bands on SDS-PAGE. Since the α-factor signal peptide only connected with N-terminal of Maeg. In the preminary analysis, fusion protein expressed correctly, but degraded when secreting the outside of cell.So we thought that the fusion enzyme was degraded at the linker peptide.Different types of fusion enzymes were constructed in order to solve the problem ofdegradation of the fusion protein. However, the fusion proteins were degraded as well. Than, anartificial fusion protein MaLac-(GS4)3-MaEG was constructed and expressed in Pichia pastorisKM71H. The fusion protein was partially purified by the ion exchange column and Ni-NTAAgarose and the purification yield was8.28%. The specific activities of endoglucanase ofMaLac-(GS4)3-MaEG is2214.8±48.95IU/μmol. Compared with the parent enzymes the specificactivities of MaLac-(GS4)3-MaEG were reduced by25.4%, for2%CMC. While the specificactivities of laccase of MaLac-(GS4)3-MaEG is1048.28IU/μmol. Compared with the parentenzymes, the specific activities of MaLac-(GS4)3-MaEG were reduced by16.4%, for ABTS. Thechimeras and parent enzymes showed the similar temperature and pH properties.MaLac gene expressed in Pichia pastoris has not been reported. The Expression was severaltimes higher than reported by shake flask culturing. Based on this, LacMa in Pichia pastorisKM71H was used for high-density fermentation. The activity of the laccase reached39.15IU/mL in first six days. The laccase activity in first seven days was increased by5times.Finally, we do some deinking experiments on waste paper with MaLac-(GS4)3-MaEG. Bycomparison, when MaLac-(GS4)3-MaEG,MaEG,MaLac were in the same number of molecules,MaLac-(GS4)3-MaEG work better on waste paper. All in all, the bifunctional enzymeMaLac-(GS4)3-MaEG was more effectively in deinking than the natural substrates.