| Cellulase,as a green and efficient biocatalyst,existing widely in organism.However,the high cost and inactivation of enzyme seriously restrict its wide application in industry.At present,the main problems of water as the catalytic medium for enzyme are low catalytic efficiency and short enzyme activity maintenance time.Meanwhile,the chromatographic techniques in separation and purification of enzyme are complex,time-consuming and costly.Therefore,it is a significant work to find a solvent that can not only serve as a good catalytic medium,but also achieve the separation and purification of enzyme.Ionic liquids(ILs)and deep eutectic solvents(DESs)provide a new idea as green solvents.Based on above researches,on the one hand,this study discussed the factors and mechanism of cellulase stability in ILs/DESs,and proved the feasibility of using ILs/DESs as enzyme catalytic medium.On the other hand,the appropriate choline-based ILs/DESs+salt aqueous two-phase system(ATPS)were constructed to achieve the separation and purification of the?-glucosidase with choline-binding protein LytA(Glyt A)from broth.This study had carried out the following contents:(1)The stability of cellulase in ILs/DESs was studied.Firstly,four DESs(Ch Cl-Gly,Ch Cl-EG,Bet-Gly and Bet-EG)were prepared by using choline chloride(Ch Cl)and betaine(Bet)as hydrogen bond acceptors,ethylene glycol(EG)and glycerol(Gly)as hydrogen bond donors at a molar ratio of 1:2,and their structures were characterized by FT-IR.The viscosity of two purchased ILs(choline lactate[Ch][Lac]and choline acetate([Ch][Ac])and four prepared DESs were also discussed.It can be seen that the viscosity of ILs/DESs is proportional to the concentration and inversely proportional to the temperature.By discussing the effects of ILs/DESs types,concentration,temperature and time on stability of cellulase and related kinetic studies,the sequence of stability for cellulase at 50?,30%ILs/DESs was determined as follows:[Ch][Lac]>[Ch][Ac]>Bet-EG>Ch Cl-EG>Bet-Gly>Ch Cl-Gly.In addition,fluorescence spectra showed that DESs could provide a more hydrophobic environment,which was conducive to the stability of cellulase.The results of circular dichroism showed that ILs/DESs had no negative effect on the structure of cellulase.(2)In this study,a choline-based ILs/DESs+salt ATPS was constructed,and used to separate and purify the recombinant?-glucosidase(GLytA).Firstly,the recombinant plasmid p ET-Glu-Linker-GLytA that contained the choline binding protein(LytA)tag was designed and constructed.Through double digestion and induction expression in E.coli,the optimal induction conditions were determined as follows:0.2m M IPTG,induction temperature 25?and induction time 12 h.Then,choline based ILs/DESs+K2HPO4 ATPS were constructed,and the data of binodal and tie lines of ATPS were obtained.The effects of temperature and ILs/DESs types on the phase separation ability of ATPS were further analyzed.The results showed that Bet-Gly was the most sensitive to temperature,its phase separation ability decreased significantly with the increase of temperature,followed by Ch Cl-Gly and Bet-EG,while the two ILs and Ch Cl-EG were not sensitive to temperature.Finally,based on the affinity of the choline binding tag on the GLytA,the appropriate ATPSs were determined by analyzing the effects of DESs type,mass fraction of top or bottom phases,temperature and extraction time on the extraction efficiency and partition coefficient of GLytA,the separation and purification of the recombinant enzyme GLytA was achieved.Under the optimum conditions of 23.11%Bet-EG+40.50%K2HPO4ATPS,the recovery of enzyme activity and purification factor reached 89.07%and 8.3359,respectively.When the ratio of GLytA to cellulase in DES rich phase is 0.5,the saccharification rate of microcrystalline cellulose can reach 81.20%after 48 h,which is 22.4%higher than that of pure cellulase.The residual activity of mixture was 64.22%after 35 days stored at 4?,showing good storage stability. |
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