Mechanism Of Effect Of Sulfonated Lignin On Enzymatic Hydrolysis And Enzymatic Recovery Of Cellulose Substrates

With the increase of energy crisis and environmental problems,using lignocellulose as the raw material to produce clean and renewable energy instead of fossil energy through the three main steps of pretreatment,enzymatic hydrolysis and fermentation has attracted widespread attention.It not only can alleviate the energy crisis,but also is environmentally friendly.However,due to the low efficiency of enzymatic hydrolysis and the high price of cellulase,the cost of lignocellulosic biorefining is high.Therefore,increasing the enzymatic hydrolysis efficiency of cellulose and the recycling rate of cellulase is an important prerequisite for the large-scale production and use of lignocellulose energy.The adsorption characteristics of lignocellulosic substrates and cellulase are not only the key influencing factors for enzymatic hydrolysis of cellulose substrates,but also have an important impact on the recycling of cellulase.As the main component of lignocellulose,lignin usually bonds cellulase by nonproductive adsorption,thereby it hinders the cellulase adsorption on cellulose and inhibits the enzymatic hydrolysis of cellulose.However,our previous studies have shown that after lignin sulfonation,the amount of cellulase adsorption is reduced,which promotes the enzymatic hydrolysis of cellulose.But in fact,the effect mechanism of sulfonated lignin on enzymatic hydrolysis is still unclear.Therefore,this study discussed the adsorption characteristics of sulfonated lignin and cellulase from the perspective of lignin-cellulase interaction and the physical and chemical properties of lignin,and studied the effect of lignin-cellulase adsorption characteristics on the enzymatic hydrolysis of cellulose substrate and the recycling of cellulase.The specific research contents are as follows:(1)The effect of lignosulfonate on cellulase adsorption and enzymatic hydrolysis of cellulose substratesBefore and after adding lignosulfonate(LS),by analyzing the enzymatic hydrolysis efficiency of dilute acid pretreated bagasse(DA-SCB)and Avicel,the productive adsorption and non-productive adsorption of cellulase,zeta potential and particle size distribution of the substrate,and the effect of LS on enzymatic hydrolysis of cellulosic substrates was discussed.The result showed that for DA-SCB,the addition of LS promoted the formation of LS-lignin complexes and LS-cellulase complexes,LS occupied the adsorption site of the cellulase,and the complexes had more negative charges.As a result,the non-productive adsorption of cellulase decreased and the productive adsorption increased,thus LS promoted the enzymatic hydrolysis of DASCB.On the contrary,for Avicel,adding LS to Avicel suspension could form the AvicelLS complexes and LS-cellulase complexes.LS occupied the adsorption site of the cellulase and increased the electrostatic repulsion between Avicel and cellulase.Therefore,LS decreased the productive cellulase adsorption of Avicel and inhibited the enzymatic hydrolysis of Avicel.(2)The effect of lignin sulfonation degree on cellulase adsorption and enzymatic hydrolysis of cellulose substrateDilute acid pretreatment(DA)and acid sulfite pretreatment(SPORL)lignocellulose will produce lignin with different degrees of sulfonation.Therefore,in order to explore the adsorption properties of lignin residues and cellulase at different sulfonation degrees,the physicochemical properties of the dilute acid pretreatment for enzymatic hydrolysis lignin residue(DA-EHLR)and the sulfite pretreatment for enzymatic hydrolysis of lignin residue(SPORL-EHLR),and the types of adsorbed enzymes were analyzed.The result showed that SPORL-EHLR adsorbed a large amount of ?-glucosidase(?-GL)through hydrogen bonds and electrostatic interaction force,resulting in the adsorption amount of SPORL-EHLR and cellulase was significantly greater than that of DAEHLR and cellulase.The binding site of SPORL-EHLR and ?-GL was located on the front side of ?-GL,and the binding catalytic site of cellobiose and ?-GL was on the opposite side of ?-GL,and thus,SPORL-EHLR would not inhibit the catalytic activity of ?-GL.Furthermore,recycled SPORL-EHLR into the new SPORL-SCB hydrolysis system saved 40% cellulase and achieved the purpose of saving cellulase.(3)Enzymatic hydrolysis lignin residues immobilized cellulaseThe enzymatic hydrolysis lignin residues have many active functional groups,easy modification and low price,and thus,they have good application potential to recycle cellulase as the carriers for enzyme immobilization.In this study,SPORL-EHLR and sodium periodate oxidized modified SPORL-EHLR(O-SPORL-EHLR)were used as carriers to immobilize ?-GL.The immobilization conditions were optimized through single-factor experiments,and the storage stability and enzymatic hydrolysis efficiency of the immobilized ?-GL were measured.The results found that optimal immobilization conditions for ?-GL by SPOEL-EHLR and O-SPORL-EHLR were p H 4.8 and 12 h.?-GL activity of SPORL-EHLR immobilized enzyme and O-SPORL-EHLR immobilized was the highest at p H 5.3.?-GL activity of SPORL-EHLR immobilized enzyme was the highest at 70 ?,but ?-GL activity of O-SPORL-EHLR immobilized enzyme was the highest at 50 ?.After 35 days of storage,the ?-GL activity of SPORL-EHLR immobilized enzyme was not significantly changed,and thus the storage stability of SPORL-EHLR immobilized enzyme was better.SPORL-EHLR immobilized enzyme replaced 40% of cellulase,and O-SPORL-EHLR immobilized enzyme replaced 50%of cellulase.In this study,mainly explored the effect of sulfonated lignin on the enzymatic hydrolysis and enzymatic recovery of cellulose substrates in a systematic and in-depth exploration way.It provided a theoretical basis for increasing the enzymatic hydrolysis efficiency of lignocellulosic and the recycling rate of cellulase,and reducing the cost of enzymatic hydrolysis.