Synergistic Effect And Mechanism Of Novel Lignocellulosic Enzyme System

Lignocellulosic materials such as agricultural waste and energy crops are abundant renewable resources. Make full use of lignocellulosic materials for fermentative production of bioenergy (ethanol, biogas, etc.) is the research focus for directional bioconversion of lignocellulose. The pretreatrnent effect of lignocellulose and the high cost of enzymes are the two essential points in the cellulosic ethanol production process. The lignin component cannot be completely removed by existing pretreatment procedures, and the residual lignin has a strong inhibitory effect on cellulase and xylanase enzyme activity. Therefore, the role of ligninase in degradation and its synergistic effect with cellulase or hemicellulase should be studied in order to reduce the costs of lignocellulolytic enzymes while achieving a high yield of fermentable sugars. These are the main challenges of cellulosic ethanol bio-processing.In this study, ligninase derived from the low temperature-resistant strain C. cladosporioides Ch2-2 screened by our team with the good ability of selective lignin degradation was employed. After optimization of the preparation method of the ligninase, including applied amount of commercial cellulase and xylanase, and the tempreture of glycation, an efficient lignocellulose complex enzyme system containing ligninase Ch2-2, commercial cellulase and xylanase was established to replace the expensive commercial cellulases requiring strict temperature conditions. The synergystic coefficient of this system was 4.7 while the concentration of reducing sugar reached 10.1 mg/mL. The results indicated that the synergistic effect between ligninase and xylanase was remarkably.The synergystic degradation mode of the xylanase production microbial community EMSD5 was investigated through metagenome sequencing and information analysis. The results showed that the proportion of glycoside hydrolases was 52.35% in the lignocellulolytic enzyme family, indicating the production of glycoside hydrolases was the main procedure of the degradation process of natural lignocellulosic substrates by EMSD5. In the glycoside hydrolase family, acetyl xylan esterases and xylanases represented a secondary proportion, indicating acetyl xylan esterases were the main auxiliary enzymes through hydrolyzing the ester bond of the hemicellulose side chains, synergystic interaction with xylanase and promoting the degradation of hemicellulose in the degradation process of lignocellulosic substrates. Endoglucanases and cellobiohydrolases represented the least proportion, indicating cellulases were not accounted for the major proportion in the degrading enzyme system of EMSD5. The functional domains of new lignocellulolytic enzyme genes from EMSD5 indicated that there is synergistic interaction among cellulases, hemicellulases and hydrolytic enzymes. Moreover, there were plenty of lignocellulosic catalytic domains with a carbohydrate-binding module (CBM, 11.92%), these catalytic domains were derived from esterases, glycosidase transferases, glycoside hydrolases, oxidases and polysaccharide lyases, indicating the catalytic mechanism of secretion enzymes with CBM played an important role in the degradation process of lignocellulosic substrates by EMSD5. The catalytic mode of the connection of outer membrane protein domain coding SusE and hemicellulases catalytic domain was also found. There were multiple degradation modes of natural lignocellulosic substrates in EMSD5. This novel bacterial based lignocellulosic degradation mode may provide a new reference for synthetic of lignocellulose metabolic pathways.According to the information of metagenome sequencing, a new xylanase gene exyl-his derived from GH11 family was cloned. The manganese peroxidase gene pcmnp-his of Phanerochaete chrysosporium was also cloned. The two genes were expressed in Pichia pastoris respectively. The purified xylanase Exyl was employed to degrade Jerusalem artichoke stems with recombinant manganese peroxidase PcMnP. The synergistic interaction between xylanase and manganese peroxidase was first proposed and demonstrated in this study, laying the foundation for further study of mechanism of complex enzymes synergystic interaction.