Study Of Expression Of LigD, LigF, And LigG To Selectively Cleave ?-O-4 Bond Of Lignin By Saccharomyces Cerevisiae

Conditions of physicochemical methods for degradation of lignin are harsh and un-environmental.Meanwhile,the degradation of lignin by traditional fungal can cause the cracking of benzene ring,and degradation products are in a dynamic equilibrium between depolymerization and polymerization.The most abundant linkage bond in lignin is?-O-4 bond.Studying the selective degradation of the?-O-4 bond of lignin is crucial to the effective lignin monomer extraction.In this study,cleavage of?-O-4 bond in lignin was achieved by beta-etherase of Sphingobiumsp.SYK-6 expressed by Saccharomyces cerevisiae.Through expression the beta-etherase in cell to study the effect of co-factors on the dimer degradation and optimization action conditions of?-etherase,the results showed that co-factors had great influence on the dimer degradation and the optimized conditions of beta-etherases was best under 50?,pH 9.0,Tris-HCl buffer,and Cu²⁺metal ions.By using MF?and SED1 signal peptide to secrete the target protein,the substrate dimer Guaiacylglycerol-beta-guaiacyl ether was degraded in the culture supernatant.It was also found that MF?signal peptide mediated degradation of 34%was stronger than SED1 signal peptide mediated degradation of 28%dimers.By secretion expressing the genes of the three enzymes in one cell and in three cells,the results showed that the degradation percentage in three cells was 34%,and the effect was better than that in one cell of 24%degradation percentage.The ratio of the three enzyme of secretion expression was optimized.When the optimal ratio of ligD:ligF:ligG was 2:1:5,the synergistic effect of the three enzymes was best,and achieved 27%degradation percentage.Through the optimization of the promoter to enhance the secretion amount of protein,different strength promoters such as PGK1p,TDH3p and Gal1p were selected,and the final degradation of the dimer by the Gallp promoter was 0.376?mol,increase of 34%.Optimization culture temperatures and culture of S.cerevisiae to enhance the yield and activity of the target protein.The results showed that 30? and the culture medium YPD were best.