Effect And Mechanism Of Xylo-oligosaccharides On Enzymatic Hydrolysis Of Cellulosic Materials

Lignocellulosic biomass,as one of the most abundant and available resources in the world,is the promising raw material for producing biofuel.However,the natural biomass recalcitrance caused by the complex structure and characteristics of lignocellulose,limits its efficient degradation by cellulase in industrial production of biofuel.Therefore,pretreatment of lignocellulose is a necessary procedure before enzyme hydrolysis,which renders the substrates more digestible to cellulase by enlarging the access of cellulase to plant cell wall and reducing the crystallinity of cellulose.Moreover,in order to improve the sugar yield and final concentration of ethanol during the lignocellulose conversion processes,the enzymatic hydrolysis of lignocellulose is always carried on at higher substrate concentration to reduce the cost of cellulosic ethanol production.However,hemicellulose-drived sugars,such as xylan and xylo-oligosaccharides(XOS),were widely existed in the prehydrolysate and solid materials from pretreatment process of lignocellulose,especially in acid pretreatment.Moreover,XOS were the important intermediate during hemicellulose degradation,and the concentrations of its components(DP 2-6)in hydrolysis system were changed during enzymatic hydrolysis of lignocellulose for their continuous formation and degradation.It has been reported that XOS had strong inhibitory effect on the enzymatic hydrolysis of cellulose.However,currently,the change rules of XOS during enzymatic hydrolysis and its inhibition mechanism on enzymatic hydrolysis are still unclear.And the efficient approaches for reliving the negative influence of the XOS on enzymatic hydrolysis are still needed to be further explored as well.Based on the above-mentioned background,this study mainly focused on the influence of XOS on enzymatic hydrolysis of cellulose,variation pattern of each XOS components during enzymatic hydrolysis and its effect on enzymatic hydrolysis of cellulose and the influencing mechanism.The major contents and conclusions of this study were as follows:(1)Studying the effect of XOS on enzymatic hydrolysis with Avicel as substrate and the enzymatic activities of main cellulase components.The results showed that the inhibition level of XOS on Avicel conversion was growing with its concentration.Compared with those without adding XOS,when XOS concentration reached 10 mg/mL,the Avicel conversion dropped 78%and 28%at hydrolysis time of 1 h and 72 h,respectively.In addition,the influence level of XOS on cellulase components was that cellobiohydrolases(CBH)>β-glucosidase(BGL)>endoglucanases(EG)through determining the decrease degree of enzymatic activities.And kinetic analysis showed the inhibitory effect of XOS on CBH and BGL were a kind of mixed type of inhibition between competitive inhibition and non-competitive inhibition(increased the value of K.and decreased the value of Vmax).(2)Effect of the substrate concentration,cellulase dosage and addition of different pure enzyme components had been studied during the hydrolysis of Avicel.It had been found that increasing the substrate concentration or cellulase dosage could partly relieve the inhibitory effect of XOS on Avicel hydrolysis.Furthermore,supplying pure cellulase components,such as CBH or BGL,could improve glucose yields and Avicel conversion,and reduce the apparent inhibition degree.However,the addition of β-xylosidase couldn’t obviously affect the glucose yields and Avicel conversion,and only relieved the apparent inhibition degree at a small extent.The apparent inhibition degree could be significantly reduced by CBHI addition,which meant that CBHI might be one of the key factors of the inhibitory of XOS on Avicel hydrolysis.(3)Studying the effect of supplying hemicellulase on hydrolysis of ammonia steam pretreated corn stover.The results showed that adding extra hemicellulase could efficiently improve cellulose hydrolysis through the synergistic effect of hemicellulase with cellulase,but the addition methods of hemicellulase could affect the final effects of enzymatic hydrolysis.Based on the 40 mg of cellulase,compared with those without adding hemicellulase,adding 100mg/g of hemicellulase at 12 h of hydrolysis could raise the cellulose conversion by 27%with ammonia steam pretreated corn stover as substrate.Moreover,supplying pure acetylxylan esterase and EG could also enhance the lignocellulose hydrolysis and increase the conversion of cellulose and xylan.(4)The change patterns of XOS components during enzymatic hydrolysis had been investigated.It was founded that TLC assay could efficiently separate the oligosaccharides in the enzymatic liquid.Thus,this assay method could be used for analyzing the concentration variation of each sugar components in the XOS during enzymatic hydrolysis.In the process of enzymatic hydrolysis,the concentrations of different components of XOS varied differently.In general,the concentration of xylose was continuously increased while the content of sugars which DP>3 were rapidly reduced.And the relative content of xylotriose were gradually decreased during enzymatic hydrolysis but still held a certain concentration at the hydrolysis time of 24 h.Combined with the change rules of the apparent inhibition degree(gradual reduction),it was indicated that xylotriose should be a major inhibitor during enzymatic hydrolysis.(5)The analysis of binding mode of xylotriose and CBHI by molecular docking.It was indicated that xylotriose preferred to bind at the tunnel of catalysis domain of CBHI,which could hinder the combination of cellulose substrates and cellulase molecules.Thus,this kind of non-productive combination of xylotriose and CBHI might reduce the enzymatic activity of cellulase,and affect the enzymatic hydrolysis of cellulose in the end.