| The utilization of biomass is a promising approach to achieve sustainable development.Cellulose is one of the most abundant biomass resources in the world.Ion liquid(IL)pretreatment with in situ enzymatic hydrolysis has attracted wide attention as a novel method for efficient utilization of cellulose.However,bacterial cellulase has not been fully developed and utilized in this field,despite the common use of commercial fungus cellulase.Moreover,disadvantages of in situ enzymatic hydrolysis including high IL cost and inhibition of enzyme activity still exist.In order to overcome these problems and improve the application of in situ enzymatic hydrolysis,intensive studies were carried out as follows:A total of 110 bacterial strains were isolated from the soil samples of Shangfang Mountain,Suzhou.After preliminary screening with carboxymethylcellulose sodium plates and subsequent assay of cellulase production capability,an efficient cellulase producing strain was selected and named as LLZ1.With 1.00 g/L of microcrystalline cellulose as substrate,0.13 g/L of glucose was produced after 24 h incubation by LLZ1 cellulase.Then,LLZ1 was identified as Paenibacillus sp.via morphological characterizations and 16 s rRNA sequence alignment.It showed highest homology with a P.cellulosilyticus(99%).Dimethyl sulfoxide(DMSO)/IL,an advanced binary pretreatment solvent,was then introduced into the in situ enzymatic hydrolysis system.After investigating the efficiency of five kinds of DMSO/IL combination,a novel aqueous-DMSO/[EMIM]DEP-cellulase system was constructed and subsequently optimized.Under the optimum condition,the conversion of microcrystalline cellulose and bagasse cellulose increased by 39.3% and 37.6%,respectively.The application of binary pretreatment solvent resulted in a 70.0% decrease in the usage of IL,which reduced the cost of this process.In the following enzymatic saccharification process,the optimum pH and temperature of the LLZ1 cellulase were identified as 6.0 and40 °C,respectively.Compared to current in situ enzymatic hydrolysis,this new system has advantages of lower operating temperature and milder pH,indicating energy-saving and environmentally friendly features of the LLZ1 cellulase.Based on the problem that cellulase activity is generally inhibited by IL,the inactivation mechanism of LLZ1 cellulase was investigated.In addition,the approach to enhance the activity of LLZ1 cellulase in IL solutions was also studied.Firstly,the relative activity of LLZ1 cellulases was analyzed in the presence of [EMIM]DEP withrespect to different substrates.With increasing [EMIM]DEP concentration,LLZ1 cellulases were differentially inhibited depending on the substrate.Subsequently,the kinetic parameters Km and Vmax were analyzed.A significant increase in the Km indicated that [EMIM]DEP exerted a negative impact on the affinity of the enzyme on the substrate.Comparatively,the Vmax was influenced only slightly.Further study on the relative activity with different substrate content showed that LLZ1 cellulases were less inhibited at higher substrate concentration.Moreover,results of SDS-PAGE showed that the inactivation of endo-1,4-?-glucanase was reversible at low[EMIM]DEP concentration(<30.0%),and the irreversibility was onset at high[EMIM]DEP concentration(>40.0%).The varied effects of [EMIM]DEP were observed in different endo-1,4-?-glucanase.Finally,addition of metal ions and surfactants improved the cellulase activity in [EMIM]DEP solution,where 0.7 mM of Ca2+ and 0.5‰ of Tween 80 led to 31.2% and 48.4% increase in cellulase activity,respectively.This study is a support for the cellulose hydrolysis in aqueous-DMSO/IL-cellulase system.Particularly,this investigation provides the knowledgebase related to the acitivity alteration of cellulase in IL. |
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