Mechanism Of Enhancing Lignocellulose Hydrolysis By Hemicellulases From Penicillium Chrysogenum

Low hydrolysis efficiency and high enzyme cost are still the main bottlenecks that limit the efficient utilization of lignocellulose.Developing high-efficiency auxiliary enzymes and investigating the mechanism have attracted widespread attention at home and abroad.Hemicellulose is an important factor limiting the accessibility and hydrolysis efficiency of cellulase to cellulose.Therefore,exploring hemicellulases with complete composition and high enzyme activity as auxiliary enzymes to commercial cellulase is of great significance for improving the overall conversion of lignocellulose.Penicillium chrysogenum P33 with good ability of lignocellulose degradation screened in the previous laboratory work was selected to study.The optimal carbon source for inducing P33 to secrete lignocellulose-degrading enzymes was studied,and the effect and mechanism of extracellular enzyme cocktail on promoting commercial cellulase were investigated.The results showed that wheat bran plus microcrystalline cellulose was the best carbon source for inducing P33 to produce enzymes.P33 enzyme cocktail had a good promoting effect on the hydrolytic performance of commercial cellulase.Without increasing the total enzyme dosage,50%of commercial cellulase was replaced by P33 enzyme cocktail for the hydrolysis of delignified corn stover;the release of reducing sugars increased by 78.6%,and the conversion of glucan and xylan increased by 37%and 106%,respectively.P3 3 enzyme cocktail also promoted the commercial cellulase hydrolysis against various biomass.LC-MS/MS analysis of P33 enzyme cocktail revealed that P33 secreted complete and high-abundance hemicellulases,including xylanases,?-xylosidases,xyloglucanases,esterases and a-L-arabinofuranosidases,accounting for 10.1%of the total protein.In addition,P33 produced abundant amylases,pectinases,oxidoreductases and some non-hydrolytic proteins.The abundant auxiliary enzymes in P33 enzyme cocktail were responsible for its significant promotion of hydrolysis of commercial cellulase.Based on the results of secretome,a novel bifunctional glycoside hydrolase rPcAxe from P33 was cloned and expressed for the first time.rPcAxe had acetyl xylan esterase and a-L-arabinofuranosidase activities.The optimum pH and temperature of rPcAxe against p-nitrophenyl acetate were 7.0 and 40?,respectively.It had good pH stability and could maintain 100%enzyme activity after incubation at pH 6.0-9.0 for 1 h.It also tolerated the presence of various metal ions.The optimum pH and temperature of rPcAxe against p-nitrophenyl-a-L-arabinofuranoside were 7.0 and 50?,respectively.rPcAxe showed a significant synergistic effect with xylanase rScXYL from Schizophyllum commune,with a maximum degree of synergy of 1.35.Release of cellobiose was decreased by 45%and release of glucose was increased by 51%when 20%of commercial cellulase was replaced by equivalent amount of rPcAxe,indicating that rPcAxe could promote the hydrolysis of cellulase against cellooligosaccharide.All three xylanases Xyl1,Xyl2 and Xyl3 secreted by P33 under induction of wheat bran plus microstalline cellulose were cloned and expressed.Xyll and Xyl3 belong to GH10 family and Xyl3 contain a CBM1 at its C-termini,and Xyl2 belong to GH11 family.The synergistic hydrolysis experiments showed that Xyl2 and Xyl3 had a obvious synergistic effect and could significantly enhance the hydrolysis of commercial cellulase.Analysis of the hydrolytic product from mode substrate by TLC revealed that the synergy among the three xylanases was mainly the cooperation of the hydrolysis mode.It was proved for the first time that the hydrolysates of GH11 xylanase could be further hydrolyzed by GH10 xylanase,but not vice versa.CBM1 in Xyl3 played an important role in the hydrolysis of natural substrates.To investigate the effects of multiple CBMs on xylanase,a GH11 xylanase 46506 containing CBM13 and CBM2 from bacterial consortium EMSD5 was cloned and expressed,and three different truncated CBM xylanase mutants were constructed.Hydrolysis of mode and natural substrates showed that both CBM 13 and CBM2 did not affect the hydrolysis mode of xylanase.CBM2 mainly participated in the hydrolysis of soluble substrates,while CBM13 mainly played a role in the hydrolysis of natural lignocellulosic substrates.