The Effect Of Lignin Structure On Cellulase Adsorption And Enzymatic Hydrolysis By Using QCM

Lignin has an inhibitory effect on enzymatic hydrolysis of lignocellulosic materials,thus reducing the hydrolysis efficiency.The effect of lignin on enzymatic hydrolysis is not only expressed in space hindrance,but the chemical structure of lignin molecule also plays an important role in the enzymatic hydrolysis.In this study,masson pine,wheat straw and poplar with different lignin structure units were used as raw materials.MWLs were separated and purified from these three materials by the method of modified Bj?rkman.Chemical and spectral methods were used to study the differences of lignin structure from different raw materials,and with the help of quartz crystal microbalance?QCM?,the effect of lignin structure on enzyme adsorption and the substrate enzyme hydrolysis was detected online.The primary main research contents and results are as follows:1?The isolation and characterization of masson pine,wheat straw and poplar MWLs:The purity of isolated MWLs by the method of modified Bj?rkmanis can reach more than 80%.Analysis methods like alkaline nitrobenzene oxidation,infrared spectroscopy,the 2-dimensional nuclear magnetic resonance?NMR?and ³¹P NMR were used for the structural characterization of three kinds of MWLs.The results indicate that the structural units of lignin are different from each other.The structural unit of masson pine is relatively simple,mainly composed of guaiacyl?G?and S/G=0;The structure of poplar lignin is composed of the syringyl?S?and guaiacyl and S/G=1.47;In the lignin of wheat straw,there are three structural units of guaiacyl,syringyls and p-hydroxyphenyl?H?,S/G=0.50.It can be seen from the analysis of ¹H-¹³C HSQC NMR that the lignin structural unit is mainly connected with the?-O-4,and there are less other linkages like the?-5,?-?and 5-5.In particular,there is 65.7%?-O-4 linkages in poplar MWL,higher than that in masson pine and wheat straw.This is because there are more syringyl structures in the poplar MWL,which also makes poplar lignin less condensed than wheat straw and masson pine.Masson pine MWL contains more amount of G units,so the linkage of?-5 is more than that of wheat straw and poplar MWL.Masson pine lignin contains a small amount of ferulic acid?FA?,poplar lignin contains 15.8%of PCA,and both of these phenolic acids are present in the wheat straw lignin.The data of ³¹P shows that the content of total hydroxyl especially phenolic hydroxyl in masson pine and wheat straw MWL is higher than that in poplar MWL.2?The preparation and characterization of biosensor films:The preparation of a smooth and overall coating on the sensor surface is the premise and key of using QCM.Three kinds of MWLs were dissolved with 90%dioxane?v/v?and 8%LiCl/DMSO?w/w?,then the dissolved lignin solution was directly spin coated on the gold sensors.The results of the atomic force microscopy?AFM?show that lignin was adsorbed on the surface of the gold sensors,thus making the ideal lignin films.Compared with lignin films made by dioxane,lignin films made of LiCl/DMSO solvents are more uniform and smooth.The surface roughness of cellulose film prepared by LiCl/DMSO is higher than that of lignin films,and the roughness of Cel/MWL bicomponent films is between that of single cellulose and lignin films.There is no obvious difference of surface roughness in bicomponent films with different origin and proportion of lignin prepared by LiCl/DMSO.3?The effect of lignin structure on the absorption of cellulase EG1:QCM was used to detected the enzyme adsorption behaviors on different cellulose film,lignin films and bicomponent films at 15°C.The QCM results indicate that all the adsorption process can be divided into two phases:the rapid adsorption and the slow adsorption phrase.After washing with the sodium acetate buffer,the part of reversible adsorption was washed away.In this study,QCM data was fitted to establish an enzyme adsorption kinetic equation model and the relevant information about the maximum adsorption and adsorption rate was achieved.According to the adsorption of enzyme on the lignin films from different raw materials,the maximum adsorption of masson pine MWL was the largest and the adsorption rate was the fastest,followed by wheat straw MWL.When the cellulose and lignin was mixtured with the proportion of 4:1 and 1:1,the adsorption amount increases and the adsorption rate slows.The QCM data shows higher adsorption extent and lower adsorption speed with higher lignin proportion.The films after enzyme injection were characterized by AFM,the raised and round particles on the surface of films confirm the adsorption of enzyme on the substrate.4?The effect of green liquid?GL?pretreatment on lignin structure and substrate enzyme hydrolysis:To simulate the enzymatic hydrolysis of lignocellulose,using common poplar wood as raw material and then pretreating on it.The purity of GL-MWL isolated from GL-poplar is higher than that of untreated poplar.Alkaline nitrobenzene oxidation,2D NMR and ³¹P NMR were used to characterize lignin structure.After the GL-pretreatment,the?-O-4 ether bond is broken,the condensation degree increases.According to the ³¹P NMR data,the total hydroxyl content in lignin increases after the pretreatment,especially the content of phenolic hydroxyl.Commercial composite enzyme CTec 2 was used to research the effect of lignin on enzyme adsorption and the substrate enzymatic hydrolysis at 15°C.The results of QCM show that the adsorption capacity?adsorption extent and rate?of the enzyme on GL-MWL films is stronger than that of MWL.Bicomponent films were prepared by mixing up with cellulsoe and MWLs in the proportion of Cel/MWL=4:1 and 1:1.The results show that the higher lignin content in the substrate,the extent of enzymatic hydrolysis is lower.In addition,Cel/GL-MWL bicomponent films have a lower degree of hydrolysis than that of Cel/MWL bicomponent films.