Ligninophilic Modification Of β-glucosidase And Its Effect And Mechanism On Enzymatic Hydrolysis Of Lignocellulose

The cellulose in the lignocellulosic substrate can be converted into fermentable sugars by cellulase enzymatic hydrolysis,and then fermented to produce cellulosic ethanol.The high cost of enzymatic hydrolysis restricts the development of cellulosic ethanol.The synergy of cellulases is an important method to reduce the cost of enzymatic hydrolysis.The cationized modification of β-glucosidase was designed to make it adsorb on the lignin preferentially,constituting a heterogeneous enzymatic hydrolysis system of β-glucosidase/lignin.The β-glucosidase/lignin heterogeneous enzymatic hydrolysis system retains high catalytic activity and reduces the non-productive adsorption of endo-and exo-glucosidases on lignin,achieving the enhancement of the lignocellulose enzymatic hydrolysis.First,ethylenediamine is used to amidate the carboxyl group of β-glucosidase to increase the adsorption of β-glucosidase on lignin and improve the enzymatic hydrolysis efficiency of lignocellulose.The influence of the amount of activator(EDC/NHS)and ethylenediamine on the activity and molecular weight of modifiedβ-glucosidase was studied.It is found that when the molar ratio of β-glucosidase,EDC and NHS is 1:20:30,the ethylenediamine grafting reaction can be better activated.When the molar ratio of β-glucosidase to ethylenediamine is less than 1:20,there were few crosslinking reactions between enzymes,and the activity retention rate of β-glucosidase is greater than 74%.When β-glucosidase adsorbed on lignin,a heterogeneous system is formed,which increase the activity of ethylenediamine modified β-glucosidase by 12.1%.The effect of ethylenediamine modified enzyme on the enzymatic hydrolysis performance of lignocellulose was further studied.The results showed that as the grafting amount of ethylenediamine increased,the enzymatic hydrolysis performance of lignocellulose first increased and then decreased.As the p H of the enzymatic hydrolysis system decreases,the enzymatic hydrolysis performance of the ethylenediamine-modified β-glucosidase is enhanced.When p H=4.0,the 72 h enzymatic hydrolysis efficiency of corncob can be increased by15.5%,while it can only increase by 10% at p H=4.8.The QCM test showed that ethylenediamine modified β-glucosidase can reduce the non-productive adsorption of endo-and exo-glucanases on lignin through competitive adsorption,and therefore promote the enzymatic hydrolysis of lignocellulose.This result preliminarily verified the effectiveness of β-glucosidase modified with amino compound to enhance enzymatic hydrolysis.In order to further increase the grafting amount of cationizable groups and reduce its influence on enzyme activity,a modified β-glucosidase with acrylamide-dimethylaminoethyl methacrylate block polymer was synthesized by imitating the "catalytic module-linker peptide-binding module" structure of the exo-glucanases.The block polymer is compounded with cellulase and used for enzymatic hydrolysis of lignocellulosic substrates.As the proportion of cationic blocks in the polymer increases,the enzymatic hydrolysis efficiency first increases and then decreases under the same concentration and same molecular weight.When the molar ratio of acrylamide to dimethylaminoethyl methacrylate in the polymer is80:30,the enzymatic hydrolysis enhancement effect is the best,and the 72 hour enzymatic hydrolysis efficiency of corncob can be increased by 45.8%.After the block polymer was grafted to β-glucosidase,the p H stability of the enzyme was improved.The enzyme activity at p H=3.0 and 6.0 increased by 9.1% and 27.7%,respectively.The β-glucosidase activity retention rates after 168 h in the buffer system were all greater than 84.5% at p H4.8.Adding 15 CBU/g glucan block polymer modified β-glucosidase can increase the enzymatic hydrolysis efficiency of corncob72 h by 7.5%.QCM tests show that block polymers can reduce the non-productive adsorption of endo-and exo-glucosidases on lignin by 21%,thereby increasing the efficiency of enzymatic hydrolysis of lignocellulose.However,due to the large molecular weight of β-glucosidase,the adsorption driving force of the block polymer modified β-glucosidase is weakened and makes it difficult to reduce the non-productive adsorption of lignin to endo-and exo-glucosidases,and has little effect on the enzymatic hydrolysis of lignocellulose.In this paper,the ligninophilic modification of β-glucosidase was achieved by grafting ethylenediamine which reduce the non-productive adsorption of cellulase and thereby improve the enzymatic hydrolysis efficiency of lignocelluloses.The stability of β-glucosidase was improved by grafting polyamine-based block polymers.These result can provide new ideas and guidance for the improvement of enzymatic hydrolysis technology and the transformation of cellulase strains.