| Glycosides and their aglycones are widely distributed in the plant world,belong to the medicinally and pharmaceutically important compounds,which could play a role in the anti-tumor,antioxidant,fatigue resistance and the prevention and treatment of disease of heart head blood-vessel.However,due to the lack of wild plant resources and the low content,we have to rely on artificial preparation for the demand of these natural functional compounds,such as organic synthesis,tissue culture and enzymatic bioconversion,etc.As is known to all,the preparation methods of chemical synthesis and the cell/tissue suspension culture exist various defects(e.g.,low yield and long cycle time),which could restrict its further development and application in the preparation of the bioactive compounds.It’s no gainsaying that the enzymatic process has a series of significant advantages,such as mild reaction conditions,simple and convenient operation,high efficient and environment friendly.Therefore,the enzymatic bioconversion method is an ideal way,which have great development potential and application prospect for the preparation of glycosides and their aglycones.The enzymatic processes are generally considered to proceed in traditional buffer,but the water is not a suitable solvent medium for some enzymatic reaction,because of the adverse hydrolysis and the poor substrate solubility for some special reaction.For the above consideration,the non-aqueous enzymatic process could effectively promote the reaction conducted which proceed hardly or extremely low efficiency in the traditional water phase,complying with the corresponding requirements in the field of enzyme engineering.Deep Eutectic Solvents(DESs)have gained widespread concern with its unique physical and chemical properties and excellent characteristics since their launch in 2003.DESs have developed into a novel reaction medium for non-aqueous enzymatic reaction,and gradually replaced the use of Ionic liquids(ILs)in recent years.With these considerations in mind,in this thesis,by using the DESs-contained aqueous solutions for enzymatic reaction medium,taking the β-D-glucosidase as the research object for the first time,the effects of different DESs medium on the catalytic properties of the enzyme and the influential mechanism have been discussed preliminarily;then,we systematically analyzed the feasibility and superiority of this DESs reaction system for the enzymatic preparation of bioactive substances through optimizing the process conditions and probing into the large-scale preparation of the model reaction.First,a total of 16 ChCl-based DESs were synthesized,then the enzymatic activity and stability(thermal stability and storage stability)of Almond β-D-glucosidase in above DESs were tested,respectively.As the DESs could play an important role in the enzymological properties,we examed the enzymology characters and dynamic parameters(e.g.,optimum reaction temperature,pH value and Michaelis constant Km)in the four candidate DESs(i.e.,ChCl/G 2:1;ChCl/G 1:2;ChCl/EG 2:1 and ChCl/Glu 2:1).The research suggests that DESs had a significant effect on the catalytic properties of β-D-glucosidase,the enzymatic activity decreased slightly,while the stability was significantly improved,which depended on the choice of the HBDs and the molar ratio of the two.In terms of the HBDs,the polyols-based DESs were superior to amide-based DESs in maintaining the enzymatic activity,and hold the similar result at the molar ratio of 2:1,1:2 and 1:3,but decreased markedly at 1:1.With the presence of DESs,as compared to buffer treatment,there had no obvious alterations for the optimum pH and temperature.This may be attributed to the fact that the structure of enzyme protein has not damaged significantly and still maintain the integrity of the structure in the co-solvent.However,the kinetic parameters of Km and Vmax were decreased to some extent,which indicated that the induced-fit of enzyme to substrate was enhanced,meanwhile,the diffusion of the substrates were inhibited obviously.Second,through integrated screening(e.g.,enzyme activity,stability),ChCl/G(1:2 molar ratio,containing 20 vol% buffer)was employed to the biosynthesis of salidroside.The Chitosan-immobilized β-D-glucosidase was employed for the biosynthesis of salidroside which was simple and convenient,the optimum reaction conditions were determined basing single factor test and response surface methodology(RSM).The optimized conditions are: DESs content of 20 vol%,reaction temperature of 50°C,medium pH of 5.8,reaction time of 100 h,enzyme loading of 45 U/mL and the tyrosol/Dglucose molar ratio of 10,under the above optimal reaction conditions,the maximum productivities of 31.6% was achieved.The purity of salidroside could be reached up more than 75%(m/m)after the column elution with 30 vol% EtOH,moreover,the immobilized β-D-glucosidase could be reused at least five times in ChCl/G by simple filtration without significant decreasing in conversion yield.Overall,the results showed that the procedure could be considered a mild,environmentally friendly,high efficient approach to the economical production of salidroside,along with the simple operation process and no any toxic reagents or hazardous catalysts involved.Third,our present research reported a preparation method of daidzein by enzymatic hydrolysis of daidzin in optimal DESs(ChCl/EG 2:1).Based on the yield of daidzein,the optimum process parameters were determined by single factor test and response surface methodology,the result is as follows: DESs content 30 vol%,reaction time 100 min,reaction temperature 53°C,pH 5.35,enzyme loading 1.0 U/mL,and substrate concentration 300 μg/m L.The maximum yield of 97.53% was achieved under this optimum process conditions,the purity of daidzein crude product reached more than 70%(m/m).What’s more,for the recycling process in large-scale preparation,the DESs medium could be reused 5 times with no significant loss in the yield.The decent biotransformation demonstrated that β-D-glucosidase in DESs medium has very high reactivity towards hydrolysis.The new process is characterized as mild process conditions,high-efficiency,eco-friendly and easy realization of commercial production.Fourth,we investigated the dissolution characteristics of daidzin and daidzein in different DESs medium.The results showed that ChCl/EG(1:2,50 vol%)and ChCl/U(1:2,50 vol%)were the optimum dissolution medium for daidzin and daidzein at room temperature,respectively,and their respective equilibrium solubility were reached 475.4 μg/m L and 993.6 μg/mL.In the process of dissolution,it was found that the concentration of DESs had a significant effect on the solubility of the solute,but there were assumed different dissolution rules for daidzin and daidzein.When discussing the solutes’ mutual influence on solubility,we found that daidzin could promote the solubility of daidzein.Finally,ChCl/U(1:2)/ChCl/EG(1:2)(v/v,80;1:7,100 vol%)was used as the mixed solvent for the dissolution of coexisting solute,and different thermodynamical equilibrium equations were employed for the correlation fitting.The results indicated that the maximum relation degrees was performed by Apelblat equation,the correlation coefficient R2 were all above 0.999.Finally,by using the method of UV-Vis absorption and fluorescence spectra,the effects of different DESs on conformational change of β-D-glucosidase were studied,and the basic mechanism of DESs on the enzymatic properties was analyzed from the angle of enzyme structure.In DESs medium,the UV-Vis absorption and fluorescence spectra of the enzyme were all changed with the maximum wavelength shifted and absorbance varied,the main reason is because of the exposure of the groups of side chains of amino caused by solvent effects,which could lead to the transform of the enzyme proteins.The study found that the alteration of the absorption and emission spectrum in DESs medium could reflect the influence on enzyme activity and stability,it is revealed that the effect of DESs medium on the catalytic properties of the enzyme is mainly due to the destruction of the enzyme protein structure.All in all,we have first studied the function rules and mechanism of DESs medium on β-D-glucosidase,and successfully constructed a new green enzymatic reaction system for the preparation of glycosides and their aglycones compounds,which has the advantages of high efficiency,simple process and environmentally friendly,and displays practical applicable value.Additionally,the study will provide the essential reference and the theoretical basis for the researches of the related academic fields. |
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