| The processivity of cellulase means that the enzyme can keep the connection with the substrate and act on the substrate repeatedly before leaving the substrate.It is usually expressed by the ratio of soluble reducing sugar to insoluble or total reducing sugar produced by enzymatic hydrolysis of crystalline cellulose.Cellobiohydrolase is a typical processive cellulase,while endoglucanase is a typical non-processive cellulase.With the extensive understanding of cellulase,a small amount of endoglucanase also has the processivity,and the processivity of endoglucanase comes from the contribution of endo-action and exo-action.At present,most of the processive endoglucanases are from bacteria,a few from fungi,and there are few reports about endoglucanases which come from fungi.Previous studies on endoglucanase EG1 from Volvariella volvacea showed that it was a kind of processive endoglucanase,but the mechanism of its processive hydrolysis was not clear.The processive endoglucanase EG1 from Volvariella volvacea is the main research object.From the perspective of cellulose substrate properties,the effects of degree of polymerization(DP),crystallinity(Cr I),crystal form and cellulose reducing end concentration(CREs)of insoluble cellulose substrate on the processivity of EG1 were analyzed,and the mode of processive action of EG1 was revealed.At the same time,the adsorption capacity of EG1 on insoluble cellulose substrate was compared,and the influence of the above factors on the adsorption capacity of EG1 was studied.Compared with EG1(expressed as EG1-CD)without carbohydrate binding domain(CBM),the role of CBM in the processive hydrolysis of EG1 was analyzed.On this basis,QCM(quartz crystal microbalance)technology was used to investigate the adsorption and hydrolysis of EG1 at the nano level,and further explain the processive hydrolysis mode of EG1.The main results are as follows:1.The effects of crystallinity and polymerization degree of cellulose on the processivity of EG1Three cotton linter pulps(CLPs)with degree of polymerization(DP)of 659,1985,2873 were used as substrates to study the effect of DP on the processivity of endoglucanase EG1.The results showed that the processivity of EG1 was positively correlated with the polymerization degree of CLP.Cotton linter pulp with DP of 1985 was treated by ball milling to obtain cellulose samples with different crystallinity(CLP2Ms).The adsorption and hydrolysis of EG1 on CLP2 Ms were studied,and the correlation between crystallinity,DP and reducing end concentration of cellulose and the processivity of EG1 was discussed by Pearson multivariate analysis and multivariate least square regression model.The results showed that the crystallinity of cellulose had the greatest effect on the processivity of EG1,and the lower the crystallinity was,the stronger processivity of EG1 was.The higher the concentration of cellulose reducing end,the stronger the processivity of EG1.The effect of degree of polymerization of cellulose on the processivity of EG1 is reflected by the concentration of reducing end.2.The effect of cellulose crystal form on the processivity of EG1In order to study the effect of cellulose crystal form on the sustained action ability of EG1,Avicel(DP = 210)with similar crystallinity and lower degree of polymerization,CLP(DP = 1985)with higher degree of polymerization and its corresponding cellulose-II were used as substrates respectively,and the effect of cellulose crystal form on the processivity of EG1 was discussed.The results showed that after hydrolysis for 24 hours,the processivity of EG1 to hydrolyze Avicel-II and CLP-II was increased by 52% and 33% respectively compared with the corresponding cellulose I.The X-ray diffraction(XRD)analysis of cellulose and the adsorption of EG1 showed that,compared with cellulose I,the spacing of crystal planes on the hydrophobic surface of cellulose-II increased,and the interaction between cellulose chains became weaker,which led to the increase of cellulase hydrolysis ability.At this time,the increase of processivity of EG1 was largely due to the contribution of endo-action.3.The effect of cellulose reducing end concentration on the processivity of EG1In order to study the effect of reducing end concentration of cellulose on the processivity of EG1,the ball milled cellulose(CLP2M45)and Avicel with higher initial reducing end concentration were used as raw materials to obtain cellulose samples with different crystallinity and reducing end concentration by phosphoric acid swelling and sodium borohydride reduction,respectively,the effect of cellulose reducing end concentration on the processivity of EG1.The results showed that the processivity of EG1 increased with the increase of cellulose reducing end concentration,regardless of the crystallinity of cellulose.Even for cellulose after phosphoric acid swelling,when the reducing end concentration of cellulose was low,the total reducing sugar concentration produced by EG1 hydrolysis was very high,but the processivity of enzyme was relatively low.Therefore,it is suggested that the contribution of EG1 to the processive exo-action is mainly due to its processive exo-action starting from the reductive end of cellulose chain.Combined with the study of EG1 adsorption on cellulose with different crystallinity and different cellulose reducing end concentration,it is concluded that EG1 adsorption on crystalline cellulose substrate is more inclined to the cellulose reducing end,and thus began to continue to circumscribe.When the crystallinity of cellulose was high,EG1 was mainly adsorbed on the reducing end of cellulose chain,and the processivity of EG1 was mainly contributed by its processive exo-action mode;When the crystallinity of cellulose decreased,the overall adsorption capacity of EG1 on cellulose increased,but still tended to preferentially adsorb on the reducing end of cellulose chain(including amorphous and crystalline regions),and partially adsorb on the non chain end.At this time,the mode of action of EG1 depends on the site of its adsorption.The hydrolysis of EG1 adsorbed on the reducing end of cellulose is exo-action,while the hydrolysis of EG1 adsorbed on the non chain end is endo mode.The ability of sustained action is still mainly contributed by exo-action,and endo-action also contributes to sustained action,it depends on the degree of polymerization of cellulose substrate and hydrolysis time of enzyme.4.The effect of carbohydrate binding domain of enzyme on the processivity of EG1The effects of cellulose degree of polymerization,crystallinity and reduction end concentration on the adsorption and processivity of EG1-CD were studied by ball milling samples(CLP2Ms)and reductive Avicel respectively,and the effects of CBM on the adsorption and processivity of EG1-CD were studied.The results showed that the adsorption of EG1-CD on the crystalline cellulose substrate was no longer affected by the concentration of cellulose reduction end after CBM removal.The hydrolysis and processivity of EG1-CD on the crystalline cellulose substrate were significantly reduced.It indicated that the main role of CBM was to help EG1 combine with cellulose reductive end,and then start the processive exo-action;The EG1-CD without CBM can also adsorb on the insoluble cellulose substrate,but it is more inclined to amorphous zone of crystalline cellulose or non crystalline cellulose.The hydrolysis of cellulose is mainly endo-action,so the processivity is far lower than that of EG1,especially when the crystallinity of cellulose is high.5.The interaction between EG1 and cellulose at nanometer scaleAvicel and CLP,and their nanocellulose crystals(CNC-Avicel and CNC-CLP)and nanocellulose microfibril(CNF)are used as the cellulose substrates for EG1.The interaction between EG1 and CNC and CNF is monitored by QCM-D method,and the processivity of EG1 is further examined and verified.The results of EG1 adsorption on nano cellulose showed that the total adsorbed protein of EG1 on CNC-CLP was about 1.3 times that of CNC-Avicel at the same protein concentration,which was close to the results of micron scale,indicating that the adsorption of EG1 on crystalline cellulose was not affected by the degree of cellulose aggregation(cell wall layer);Although the adsorption capacity of EG1 on CNC-Avicel was significantly lower than that on CNC-CLP,the hydrolysis capacity of EG1 was significantly higher than that on CNC-CLP,which further confirmed the research conclusion in micron scale,that is,when the crystal form of cellulose was transformed from cellulose-I to cellulose-II,the adsorption capacity of EG1 decreased,but the hydrolysis capacity increased;In addition,the adsorption and hydrolysis ability of EG1 on CNC was stronger than that of EG1-CD on CNF,which further confirmed that the role of CBM was to help EG1 adsorb on the reducing end of crystalline cellulose,and then to assist its processive exo-action. |
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