| Currently,the in situ enzymatic hydrolysis process of ionic liquid,cellulase and lignocellulose in one-pot treatment has become a research hotspot.?-Glucosidase?BG?is the key enzyme in the process of cellulose hydrolysis.However,BG tends to be inactivated in ionic liquids.Therefore,in this study,Paenibacillus sp.LLZ1 was selected as the BG-producing strain,focusing on the inactivation of enzyme in ionic liquids and the improvement of enzyme activity.The researches of this study are as follows:Firstly,the effects of five carbon sources,such as microcrystalline cellulose?MCC?and starch,on BG production were studied.The results showed that the activity of BG induced by MCC was relatively high?2.52 U/mL?.Furthermore,the effects of BG induced by MCC was explored in various concentrations of ionic liquids,which is composed of 1-ethyl-3-methylimidazolium?[EMIM]+?cation and different anions?[DEP]-,[OAc]-,[Br]-,[Cl]-,and[BF4]-?.Circular dichroism?CD?indicated that there was almost no change in the secondary structure of BG after 50%1-ethyl-3-methylimidazolium diethyl phosphate?[EMIM]DEP?treatment,it indicated that BG has better tolerance to[EMIM]DEP.However,the?-helix content of BG treated with 50%1-ethyl-3-methylimidazole tetrafluoroborate?[EMIM]BF4?was reduce to 0.7%,[EMIM]BF4 led to the irreversible inactivation of BG.Finally,0.4 mM sodium bis?2-ethylhexyl?succinate sulfonate?AOT?was added to the 25%?v/v?[EMIM]DEP system to treat BG for one hour,and the addition of cellobiose for in situ enzymatic hydrolysis was continued.As a result,the BG activity increased by 20.05%,and the glucose yield increased by 23.87%.Secondly,in reaction system with 5%?v/v?[EMIM]DEP,the effects of six surfactants,such as rhambolipid or PEG4000,on BG activity were studied,it was found that the relative activity of BG increased by 21.85%with the addition of 1‰?v/v?rhamnolipid.The kinetic study of BG enzymatic reaction showed that the addition of 5%[EMIM]DEP and 1‰rhamnolipid reduced the Km value of BG by 2.11 mM,indicating that the affinity of BG to substrate was increased.Fluorescence spectroscopy indicated that the maximum emission wavelength of BG treated with 5%[EMIM]DEP and 1‰rhambolipid was appeared red-shifted to 363 nm,and the results of differential scanning calorimetry?DSC?showed that 1‰rhamnolipid and 5%[EMIM]DEP caused the midpoint temperature Tm of BG to change from60.7°C to 61.4°C,and the average unfolded enthalpy?H was increased to 395.5 J/g;In situ enzymatic hydrolysis of cellobiose was carried out in reaction system with 1‰rhamnolipid and 5%[EMIM]DEP,and the conversion rate of cellobiose was increased by 21.93%.Finally,Synergistic effects of rhamnolipid 1‰?v/v?and three ionic liquids?1-ethyl-3-methylimidazolium acetate?[EMIM]OAc?,[EMIM]DEP and[EMIM]BF4?on BG activity were investigated,respectively.It was found that[EMIM]OAc increased the relative activity of BG by 38.55%.Fluorescence spectroscopy indicated that the maximum emission wavelength of BG treated with 1‰rhambolipid and[EMIM]OAc,or treated with 1‰rhambolipid and[EMIM]DEP,were appeared red-shifted,while[EMIM]BF4 caused blue shift.BG was treated in 2.5%[EMIM]OAC and 1‰rhamnolipid system for an hour before adding cellobiose for in situ enzymatic hydrolysis,it was found that the relative activity of BG increased by 47.68%,and the conversion rate was increased by 21.60%.Moreover,the near-UV CD spectrum showed that the absorption peak and disulfide bond of phenylalanine in the BG protein were changed after treatment. |
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