Study On Manganese Peroxidase Gene Expression In Pichia Pastoris And Lignin Degradation In Corn Straw

Due to economic development driving the rapid development of animalhusbandry, feed shortage becomes more and more serious. China is an agriculturalcountry, and the annual production of straw reaches700million tons. Because of thelack of technical conditions, straw can not be used well, and most of them are decayedor burned, which cause resource loss and serious environmental pollution. Therefore,how to convert straw into feedstuffs become more and more important.It is difficult to degrade straw under routine conditions due to the lignincomponent types and diverse chemical bond types, and the biodegradation process ofstraw is very complex. In addition, the combination of lignin and hemicellulose inplant cell walls is through covalent bond forms to embed the cellulose molecules forforming more complex polymer, so that cellulase is not easy to contact with thecellulose molecules. It is necessary to degrade lignin through microbial degradation orother biotechnology for completely degradation of straw.Lignin is a type of the material formed by polymerization of aromatic alcohol,existing in the wood organization, the main effect of lignin is to harden the cell wallby weaving nets. The degradation enzymes of lignin include three kinds: laccase,lignin peroxidase, manganese peroxidase. The purpose of this study is to improve theenzyme activity and fully degrade lignin by manganese peroxidase genesheterologous expression.Manganese peroxidase gene was isolated from phanerochaete chrysosporium byPCR. The gene was connected with PMD19-T and transferred to competent E. coli（DH5α）. After verification, the gene was connected with expression vector ofpGAPZαA and transferred to competent E. coli. After the vector ofpGAPZαA-manganese peroxidase gene was obtained, it was then transferred into thecompetent Pichia pastoris (X-33) by electrophoresis. The enzyme activity, optimumtemperature and pH value, and lignin degradation ability from recombinant yeast werestudied.Sequence alignment of manganese peroxidase gene showed that the DNAsequence length was1480bp, base homology was98.43%, protein homology was99%, compared with manganese peroxidase gene (L29039.1) in GeneBank. Theprotein was composed of382amino acids, and the molecular weight of the expressedprotein was40.01kDa by SDS-PAGE analysis. Under the same conditions of liquidfermentation, manganese peroxidase activity was0.197U/L after72h incubation for Phanerochaete chrysosporium, and0.3178U/mL after48h incubation for therecombinant yeast（P<0.05）. The enzyme activity has been increased by1.56timeswith gene transformation. The enzyme activity was reduced to0.1862U/mL after72h incubation for the recombinant yeast. The optimal temperature and pH of theexpressed enzyme were40℃and4.5（P<0.05）. Lignin degradation rate in corn strawwas24.09%for the recombinant yeast, and16.53%for Phanerochaete chrysosporium（P<0.05）.