Study On The Deactivation Mechanism And Kinetics Of Paenibacillus Sp. LLZ1 Cellulase In Ionic Liquids

Recently,in situ enzymatic hydrolysis of lignocellulose with ionic liquids(ILs),cellulase and cellulose in one-pot process has been hot in research.The inactivation mechanismofPaenibacillussp.LLZ1cellulasesinthepresenceof1-ethyl-3-methylimidazolium diethylphosphate([Emim]DEP)has been preliminarily investigated.To improve the in situ enzymatic hydrolysis efficiency of biomass in reaction system with ILs,the primary studies are as follows:Firstly,attempts were made to investigate the influence of various carbon sources including microcrystalline cellulose(MCC),sucrose,carboxymethylcellulose(CMC),cellobiose,starch,and glycerol on the production of cellulase by Paenibacillus sp.LLZ1.The results showed that both FPA and CMCA induced via MCC were clearly higher than the other examined carbon sources.In addition,zymography analysis revealed the existence of seven protein bands(CMC3,CMC4 and CMC6 to CMC9)with endoglucanase activity when MCC was used as the carbon source for the culture of Paenibacillus sp.LLZ1.The endoglucanase of Paenibacillus sp.LLZ1 that was induced by MCC still retained 67%of the initial enzyme activity in 25%[Emim]DEP solution,and ionic liquid resistance is higher than that of recently published endoglucanase.Secondly,effects of the nonionic surfactants(Tween 80,AEO-9 and PEG 4000),anionic surfactants(SDS),cationic surfactants(CTAB)and amphoteric surfactant dodecyl dimethyl betaine(BS 12)on the stability of cellulase in reaction system with[Emim]DEP were investigated detailedly.The analysis of fluorescence spectra indicated that the maximum emission wavelength of cellulase was appeared red shifted with 1‰(v/v)Tween80 addition.Simultaneously,cellulase activity was increased by 19%.When 1‰Tween 80was added into reaction mixture,Circular dichroism(CD)spectroscopy showed that the contents of?-helix and?-sheet were increased by 0.58%and 0.70%respectively.Analysis of enzymatic kinetic parameters revealed that K_m value reduced from 22 to 17.42 with 1‰Tween 80 addition,the results also indicated the increasement of between enzyme and substrate.Furthermore,the in situ enzymatic hydrolysis system included 5%[Emim]DEP(v/v),1‰Tween 80 and cellulase was constructed,in which the enzymatic hydrolysis conversion of bagasse cellulose(BC)was reached by 83%.Finally,three ILs including[Emim]DEP,1-ethyl-3-methylimidazolium Chloride([Emim]Cl),and 1-ethyl-3-methylimidazolium acetate([Emim]OAc),were used to evaluate the cellulase biocompatibility,respectively.The results of enzyme activity showed that the cellulase retained 87.4%of the initial enzyme activity in 5%(v/v)[Emim]OAc solution.The analysis of Fluorescence spectroscopy suggested that the maximum emission wavelength of cellulase was appeared blue shift in[Emim]DEP or[Emim]Cl solution,while red shift was found in[Emim]OAc solution.Near-ultraviolet CD analysis showed that[Emim]OAc had caused the change in tertiary structure of cellulase.Then,the conversion of in situ enzymatic hydrolysis of MCC and BC in reaction system containing5%(v/v)[Emim]OAc and 1‰(v/v)Tween 80 were increased by 81.9%and 92%,respectively.SEM analysis revealed that the porosity of both MCC and BC was increased after pretreatment in the above system.