Effect And Mechanism Of Sulfomethylated Tannic Acid On Enzymatic Hydrolysis

Lignocellulosic biomasses,such as agricultural and forestry residues,pulp mill wastes,and energy crops,are viewed as a prospective alternative for bioethanol production,due to their high carbohydrate storage.Therefore,tremendous efforts have recently been made on producing sugars from lignocellulose over an enzymatic hydrolysis strategy.Cellulase can bind to lignin nonproductively during enzymatic hydrolysis,negatively impacting the digestibility of enzymes.The pretreatment can significantly promote lignocellulosic biomass enzymatic digestibility by removing lignin from lignocellulosic biomasses.However,the cost of completely removing lignin is expensive,and the harsh treatment process will result in high carbohydrate loss.Additives can be employed to decrease nonproductive adsorption of cellulase on substrate lignin to enhance enzymatic saccharification.Sulfonated lignin can significantly enhance the enzymatic hydrolysis of lignocellulose substrates.Lignin is a type of polyphenol,therefore,sulfonated polyphenols are likely to have similar effects.In this study,five kinds of sulfomethylated tannic acid(STA)with different sulfonation degrees were prepared by sulfomethylation of tannic acid(TA).STA was used as an additive for enzymatic hydrolysis of 8% Na OH pretreated wheat straw,and the influence of STA on enzymatic hydrolysis and cellulase adsorption was explored.The effect of STA on the p H value of hydrolysis system and the effect of different p H values on the sugar conversion rate of enzymatic hydrolysis with STA were explored.(1)Preparation of sulfomethylated tannic acid(STA)with different sulfonation degrees.The sulfomethylated tannic acid(STA)with different sulfonation degrees(i.e.,different contents of sulfonic acid content)was prepared by controlling the dosage of sodium sulfite.STA containing0.9,1.2,1.8,2.4 and 3.0 mmol/L of sulfonic acid are called STA-0.9,STA-1.2,STA-1.8,STA-2.4and STA-3.0,respectively.Within the sodium sulfite dosage range in this work,the sulfonic acid content of STA increased linearly with the dosage of sodium sulfite.(2)Effect of STA on enzymatic hydrolysis of 8% Na OH pretreated wheat straw.The effects of TA and STA on enzymatic hydrolysis efficiency of pretreated wheat straw were studied.The results showed that the hydrolysis efficiency was closely related to the sulfonation degree of STA.The enzymatic hydrolysis was strongly inhibited by TA,whereas was significantly promoted by STA.The hydrolysis yield of total sugars continues to decrease with the addition of TA.The addition of 1.6g /L TA almost completely inhibited the enzymatic hydrolysis.The promotion effect of STA with different sulfonation degrees have different effects on enzymatic hydrolysis.The promotion effect on enzymatic hydrolysis increased with the addition(0-1.6 g/L)of low sulfomethylated STA(<2.4 mmol/g).However,excessive STA with high sulfonation degrees(≥ 2.4 mmol/g)was not conducive to further improving the sugar conversion.The addition of0.8 g/L STA-2.4 has the most obvious promoting effect on enzymatic hydrolysis.The glucan yield increased from 60.6% to 97.9% at at a low cellulase dosage(5 FPU/g-glucan).(3)The effect of TA and STA on the cellulase adsorption during enzymolysis.During enzymolysis,most cellulases are rapidly adsorbed by the substrate.The addition of TA inhibited the content of soluble free protein in the hydrolysate.While the addition of STA increased the free protein content,and the non-productive adsorption of cellulase on substrate lignin was significantly reduced.The adsorbed cellulase protein on substrate and enzyme protein of hydrolysate were close to reaching equilibrium within 24 hours.After hydrolysis for 96 h,the free protein in supernatant with 0.8 g/L STA-2.4 increased from 0.0174 mg/m L to 0.0734 mg/m L,i.e.,the cellulase amount adsorbed onto substrate dropped from 87% to 44%.Within the range of STA addition(0-1.6 g/L)in this research,the content of free protein raised almost linearly with the increase of STA with lower sulfonation degree(< 2.4 mmol/g).The content of free protein first increased and then decreased with the addition of STA(≥ 2.4mmol /g)with high degree of sulfonation.STA wtih high sulfnation degrees can bind more cellulases,however,they may precipitate at high concentrations.Compared to STA-2.4,the binding between STA-Cel aggregates formed by STA-3.0 and cellulose was relatively unstable,resulting in lower glucan conversion efficiency per unit of free protein.In summary,STA with higher sulfonation degree effectively inhibited the nonproductive cellulase adsorption on substrate lignin,thereby significantly promoting substrate enzymatic hydrolysis.Fluorescence spectra can be used to study the binding mechanism between chemical molecules and enzyme proteins,because it reflects the quenching constant,binding constant and other information.The fluorescence emission spectra showed that TA,STA did interact with cellulase,and the fluorescence quenching mechanism was static quenching.(4)The effect of different p H buffer on the enzymatic hydrolysis and cellulase adsorption.For the hydrolysis system without STA and with the addition of STA-0.9 and STA-2.4,as the buffer p H increased from 4.4 to 4.8,the sugar conversion increased first.When p H value exceeded 4.8,the sugar conversion rate decreased slightly.The effect of p H on enzymatic hydrolysis was related to the effect of p H on the electronegativity of cellulase,substrate electronegativity,substrate lignin hydrophilicity and cellulase activity.The free protein content in supernatant increased first and then decreased slightly with the increase of buffer solution p H.The free protein content in hydrolysate did not change obviously in buffer with different p H.p H=4.8 was the most favorable for enzymatic saccharification of Na OH-pretreated wheat straw.