Cloning Of Lytic Polysaccharide Monooxygenases Genes From Asperillus Niger And Research On Its Synergism Activity

Lignocellulose is the most abundant and promising renewable resource on theEarth,but it has a complex network structure that hinders its degradation and utilization.Increasing the efficient of enzymatic hydrolysis is the primary task of reducing the cost of degradation of lignocellulose.AA9 family of LPMO,which is the most promising accessory protein,can effectively promote the degradation of lignocellulose.Aspergillus niger,which is widely used in cellulase secretion,containing seven genes encoding AA9 protein,study them can expand the range of AA9 and has a very important significance on effective use of lignocelluloseIn this study,two AA9 family proteins from Aspergillus niger,AnLPM014g and AnLPMO15g,were successfully expressed in Pichia pastoris.Substrate specificity analysis showed that they could act on avicel,CMC,lignocellulose(straw,corncob,filter paper)and xylan.MALDI-TOF/TOF analysis of their hydrolysis products of avicel showed that there were sodium adduct of fiber oligosaccharide,1,5-?-lactone,aldonic acid and their sodium adduct in the products,inferring that the two proteins act on the Cl position of the glucoside chain.When acting on avicel and straw combined with cellulase,the adding of AnLPM014g could increase the yield of reducing sugar by 92.66%and 131.42%,and the adding of AnLPM015g could increase the yield by 96.75%and 131.55%.On corncob,the two proteins increased the yield of reducing sugar by 46.61%and 44.51%.The yield of reducing sugar increased by 37.92%and 24.69%on filter paper.When acting on CMC,the synergistic activity was the lowes,7.51%and 14.75%.When the two proteins act on beech xylan combined with xylanase,the yield of reducing sugar increased by 14.37%and 8.85%,respectively.Experiments show that pH,metal ions,enzyme loading and the concentration of reductive electron donors have effects on the synergistic activity,in which ascorbic acid is the most suitable type of reduced type electron donor and the promotion degree increase with concentration.AnLPMO15g consists of a binding domain(CBM),a catalytic domain and a linker.By cloning and expressing AnLPM015g lacking of CBM or CBM and Linker and investigating their synergism activities with cellulase,it was found that the absence of CBM and Linker could improve the synergetic activity with cellulase in avicel degradation inhibit that in lignocellulose degradation.It was predicted thatAnLPM015g contains three glycosylation sites(151,334 and 385).AnLPM015g with mutants on different postions were cloned and expressed successfully.By investigating their synergism acticities with cellulase in avicel degradation,it was found that the N-glycosylations at 385 position were not favorable for synergism activities with cellulase,while that at 334 position favored the synergistic activity,and that at 151 position had little influence on the synergistic activity.When acting on lignocellulose substrate,N-glycosylation at 151 position favored the synergism activity with cellulase,whereas those at 334 and 385 position were not conducive to the synergism activity with cellulase.