Inhibitory Effect Of Mannan On Cellulose Enzymatic Hydrolysis And Strategies For Minimizing Its Effect

The development and commercialization of bioethanol are currently limited by the high cost of cellulases.How to improve the hydrolysis efficiency of lignocellulosic biomass and reduce the dosage of cellulases have become the key problem.In lignocellulosic biomass,the access of cellulases to cellulose surface is limited by hemicelluloses,which are found in close association with cellulose and to some extent cover the fiber surfaces.To overcome this barrier,original-bound hemicelluloses should be converted into free form or hydrolyzed to soluble sugars.It was found that free xylan(the major composition of hemicelluloses in hardwood and gramineae)and its derivatives showed strong inhibitory effect on cellulose hydrolysis.However,effect of free mannan(the major hemicelluloses in softwood)and its derivatives on the hydrolysis of cellulose is still not fully clear.Therefore,in this work,the effects of free mannan and its derivatives on cellulose hydrolysis were investigated.The mechanism of the inhibition of cellulose hydrolysis by mannan and its derivatives and the methods to reduce the inhibition were also studied.The aim of this work is to provide theoretical guidance to efficient hydrolysis of lignocellulosic biomass with high hydrolysis yield and low enzyme dosage.The major research contents and results are shown as follows:Effect of different mannans(linear mannan,low molecular weight carob galactomannan,and high molecular weight carob galactomannan)on the hydrolysis efficiency of cellulose was investigated.The results indicated that all the three mannans showed inhibitory effect on cellulose hydrolysis.The hydrolysis yield of Avicel clearly decreased by 13.75%-39.10%after the addition of 2.5 mg/mL linear mannan,low molecular weight carob galactomannan,or high molecular weight carob galactomannan.Additionally,it was observed that the inhibitory effect of mannan on cellulose hydrolysis was strongly associated with its structural characteristics.High molecular weight galactomannan showed a stronger inhibitory effect on cellulose hydrolysis as compared with low molecular weight galactomannan.Galactomannan with a low degree of galactose substitution showed a stronger inhibitory effect on cellulose hydrolysis as compared with that with a high degree of galactose substitution.The effects of mannan on the hydrolytic action of individual components of cellulases(endoglucanase Cel5A,cellobiohydrolases Cel6A and Cel7A,and?-glucosidase Cel3A)were determined and the kinetics of inhibition of Cel7A by mannan were analyzed to elucidate the inhibition mechanism of mannan on cellulose hydrolysis.The results showed that mannan clearly inhibited the hydrolytic action of Cel5A,Cel6A and Cel7A,but had no inhibition effect on the hydrolytic action of Cel3A.Kinetic experiments revealed that mannan could occupy the active site of Cel7A and thereby competitively inhibit Cel7A activity.Among the tested mannans,high molecular weight galactomannan showed the strongest inhibitory effect on Cel7A activity(inhibition constant K_i is 0.43 mg/mL),followed by low molecular weight galactomannan(inhibition constant K_i is 0.47 mg/mL)and linear mannan(inhibition constant K_i is 0.50 mg/mL).Effects of mannan derivatives(manno-oligosaccharides,mannose,and galactose)on the hydrolysis efficiency of cellulose were further investigated.The results showed that all the mannan derivatives could inhibit the enzymatic hydrolysis of cellulose,the order of the inhibitory potency was manno-oligosaccharides>galactose>mannose.During the hydrolysis of Avicel by cellulases,the addition of 5 mg/mL manno-oligosaccharides,galactose,and mannose lowered the hydrolysis yield by 12.70%,4.23%,and 3.97%,respectively.Inhibition by manno-oligosaccharides with different chain length and degree of galactose substitution was further compared.It was observed that manno-oligosaccharides with low chain length showed a stronger inhibitory effect on cellulose hydrolysis as compared with that with high chain length.Additionally,manno-oligosaccharides with a low degree of galactose substitution showed a stronger inhibitory effect on cellulose hydrolysis as compared with that with a high degree of galactose substitution.To elucidate the inhibition mechanism of mannan derivatives on cellulose hydrolysis,the effects of manno-oligosaccharides on the hydrolytic action of individual components of cellulases were determined.The results indicated that manno-oligosaccharides showed inhibitory effect on the hydrolytic action of Cel6A and Cel7A,but not on Cel5A and Cel3A,while mannose and galactose could clearly inhibit hydrolytic action of Cel5A,Cel6A,and Cel7A.Kinetic experiments further revealed that manno-oligosaccharides preparation was a competitive inhibitor of the Cel7A(inhibition constant K_i is 1.57 mg/mL).The role of mannanase in reducing the inhibition of mannan on cellulose hydrolysis was investigated.The results showed that mannanase was capable of efficiently suppressing the mannan-induced inhibition effect.In the hydrolysis of Avicel by cellulases,the addition of 5mg/ml galactomannan reduced the glucose yield from 38.75 to 18.68%,after further supplementation of endo-mannanase,?-mannosidase,and?-galactosidase,the glucose yield clearly increased to 35.33%.In the hydrolysis of spruce by cellulases,the addition of mannanase did not only increase the glucose yield by 14.29%,but also enhanced activities of Cel5A and Cel7A by 14.30%and 10.91%,respectively.The results thus indicated that the addition of mannanase could relieve the inhibition of mannan on Cel5A and Cel7A,recover activities of inactivated cellulases,and hence boost the hydrolysis of cellulose in spruce.Based on the results presented,an underlying mechanism behind the promotive effect of mannanase on lignocellulosic biomass hydrolysis was proposed.The role of Tween 80 in reducing the inhibition of mannan and its derivatives on cellulose hydrolysis was studied.In the hydrolysis of Avicel by cellulases,addition of galactomannan(5 mg/ml),manno-oligosaccharides(5 mg/mL),mannose(10 mg/mL),and galactose(10 mg/mL)reduced the glucose yield from 48.60%to 26.64%,39.96%,39.15%,and 36.90%,respectively,after further addition of Tween 80,the glucose yield clearly increased to 36.00%,46.58%,46.35%,and 45.00%,respectively.The results thus indicated that Tween 80 could suppress the inhibition of mannan and its derivatives on cellulose hydrolysis.Kinetic experiments further revealed that Tween 80 was an activator of the Cel7A,but not of Cel5A and Cel3A.The activation effect of Tween 80 showed a specific positive role in suppressing inhibition of Cel7A by mannan and its derivatives and maintained the enzyme in high activity during the enzymatic hydrolysis process,thus boosting the enzymatic hydrolysis of lignocellulosic biomass.