| Epoxides are important organic materials and intermediates for organic synethsis,fine chemical engineering,material science and the other fields.Epoxides can be produced by chemoenzymatic epoxidation reaction using in situ generated peracids.Commonly,hydrogen peroxide is needed in the reaction of in situ production of peracids as substrate and oxidation.Hydrogen peoxide acts as Janus role in biocatalysis.On the one hand,as a ?green? oxgen source hydrogen peoxide was drawn more and more attention to be used in biocatalysis;on the other hand,hydrogen peoxide can lead to decline of activity of biocatalysts partially even all due to their inherent nature.With the demanding of green chemistry,the traditional organic solvents and inoic liquids used in biocatalysis as medium are challenged.Natural deep eutectic solvent(NADESs)has appeared as a new generation of solvent for biocatalysis due to its easily preparation,non-toxic,easily biodegradation,etc.,which could be used as a new type of "green" medium for biocatalysis.Moreover,it is likely to provide a new strategy for overcoming the problem of products safety and environmental damage,thus the researchers gradually pay more attention to NADESs.In addition,studys shown that NADESs are potential stabilizers for enzymes in the process of biotransformation.The other stratgy to improve the resistance of enzymes to hydrogen peroxide could be provided by enzyme engineering technology.Modification of amino acid residues sensitive to hydrogen peroxide in the surface can increase the resistance of enzymes to hydrogen peroxide,however,this strategy needs much works.Operationally,it is hard to improve enzymes resistance to hydrogen perioxide and its catalytic efficiency in the same time.In this study,both solvents engineering and molecular modification methods were employed to increase the stable of enzymes in the reaction and decrease the impact of environment.The behaviors of esterase and lipase in catalyzing of epoxidation in NDESs was studied systemically.The performance of esterase against hydrogen peroxide improved by the rational design mutants was also investigated.This article mainly includes four aspects:(1)Ten kinds of NDESs were prepared by heating method,and their related properties have been characterized by using 1H and 13 C NMR,FT-IF,DSC and TG.Results shown that the liquid solvent still exists in the form of mixture and relatively independent between components.Hydrogen bond interaction was found between the moleculars in NADESs by using FT-IR and it was destroyed when water content exceeds 50%.DSC analysis showed that NDESs belong to Low-Transition-Temperture Mixtures(LTTMs).TG showed that choline chloride/sorbitol solvent was broken down when the temperature was more than 300?.The results showed that NDESs acts as a stabilizer for improvement of enzyme thermal stability.Study of the viscosity of NDESs showed that water has a great influence on the it.The viscosity rapidly reduced with the increasing of water content in solvents.Result showed that NDESs and its aqueous solution viscosity were Newtonian fluids.(2)NDESs can be used as reaction medium for efficient catalysis epoxidation of alkenes.The effects of nine kinds of NDESs on the conversion of epoxidation of alkenes were investigated.The conversion of 72.4% was obtained by Novozym 435 in polyols choline chloride/sorbitol solvent to catalyze the oxidation of octadecene.The optimal conditions were temperature of 40?,octadecene to octanoic acid mole ratio of 1:1,octadecene to hydrogen peroxide mole ratio of 1:2,reaction time of 24 h.The maximum conversion of octadecene oxide was 97% under the optimal conditions.This system can be used in chemoenzymatic epoxidation of different alkenes.And the molecular model simulation was employed in proving lipase catalytic epoxidation mechanism.The thermostability of lipase in choline chloride/sorbitol was found system,which showed choline chloride/sorbitol has the function of the protective enzyme activity.The impact of the choline chloride/sorbitol system was also evaluated in this study.Compared to ionic liquids,E-factor of choline chloride/sorbitol system is only a half time of it.The results showed choline chloride/sorbitol system is moreenvironmental friendly than the ionic liquids.(3)Combination of the mono-and diacylglycerol lipase G with NDESs could produce epoxidized glyceryl trioleate efficiently.This is the first study of carrying out epoxidation of oil in NDESs system.Comparison with the conversion of 8% of epoxidized glyceryl trioleate(EGT)in the control system,the maximum conversion of epoxidized glyceryl trioleate was 86.7% under the optimal conditions in NDESs system.The optimal conditions were temperature of 40?,hydrogen peroxide to C=C bond mole ratio of 3:1,solvent to substrate weight ratio of 1:1.Moreover,there is no hydrolysis products found in the lipase G catalyzed reactions,thus this is a new strategy for preparation of epoxidation oil.Additional,the change secondary structure of lipase G in different media was investigated to explain the high conversion.The results showed that lipase G was more stable in choline chloride/sorbitol system,which could be the reason of higher conversion of epoxidation oil.(4)The resistance performance to the hydrogen peroxide and perhydrolysis activity of essterase can be improved by using molecular modification.Molecular cloning expresses of four thermophilic esterases were carried out.For the stressed,the tolerance ability of hydrogen peroxide of the wild type was PestE>EST1>AFEST>TTEST.The combination of improving the binding ability of substrates with catalytic site and changing the catalytic sites hydrophobic regions could be created the mutant L89R/L40 A.L89R/L40 A showed the better tolerance toward concentrated H2O2 compared with wild-type PestE.By generation of just 9 mutants we have obtained over 80-fold selectivity increase for perhydrolysis: three-fold improvement of absolute perhydrolysis activity,two-fold improvement of thermostability and two-fold improvement of epoxidation oleic acids.Compared to the the traditional replace easily oxidized amino acid method.This strategy has a better guiding significance. |
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