Study On Enzymatic Preparation Of Quercetin Using Natural Deep Eutectic Solvents

Quercetin is a class of flavonoids with a variety of physiological functions,mostly distributed in the form of glycosides in the flowers,leaves and fruits of many plants,such as rutin,hypericin,and quercetin.Studies have shown that quercetin has a variety of pharmacological effects,such as anti-oxidant,anti-cancer,anti-inflammatory and anti-viral effects,and has shown great potential for application in the medical field.However,most of the flavonoids are poorly soluble and almost insoluble in water.In modern industry,rutin is usually extracted by alkali-soluble acid precipitation and then it is hydrolyzed to quercetin by inorganic acid.The operation is complicated and the energy consumption is high with low product selectivity and yield,and most of the reagents used are organic solvents and acid-base reagents,which are highly toxic and not environmentally friendly,so it is urgent to develop a green and efficient method for preparing quercetin.Natural eutectic solvents(NADESs)are a new class of green solvents with adjustable physical and chemical properties,which can be obtained by simply mixing their components with heating or lyophilizing.It has a wide range of components,simple preparation,less energy consumption,and is environmentally friendly and biodegradable.Its solvent characteristics can be designed by simply adjusting its components and proportions.Because of its unique physical and chemical properties,NADESs have been widely used in electrochemical,separation and reaction processes.In this paper,NADESs are used to extract rutin from Sophora japonica,and the natural active product quercetin is obtained by rutin degrading enzyme(RDE)prepared from tartary buckwheat.The two processes of extraction and transformation are completed in one system.Then,a strain with RDE activity was obtained through acclimation in moldy buckwheat.It was identified as a Fusarium.spp and applied to whole-cell catalysis to improve the catalytic efficiency.Finally,the RDE obtained from the fungi was subjected to carrier-free immobilization to prepare a rutin-degrading enzyme aggregate(CLEA-RDE),and its thermal stability and mechanical stability were improved through immobilization.The details are as follows:1.Study on the method of preparing quercetin using NADESs.In this study,11 NADESs are selected as extraction media to extract rutin from Sophora japonica,and RDE is extracted from germinated buckwheat to hydrolyze rutin to quercetin.The determination of the solubility of rutin in the NADESs shows that most NADESs have good solubility for rutin with 67～3116 times compared with that in water.The extraction rates of different types of NADESs are significantly different.ChGly has the highest extraction rate,and it can reach 291.57 mg/g after optimized extraction conditions(heating in a 35℃ water bath with a substrate concentration of 1 mg/mL and an enzyme loading of 55 μL).RDE was subsequently added in the extraction system,the rutin conversion rate can reach 8.36 mg·min-1·L-1.The enzymatic properties of RDE have shown that the molecular size of the enzyme is about 60 kD,the optimum pH is 5.0,and the optimum temperature is 50℃;different types and concentrations of metal ions have certain inhibitory effects on the enzyme,but the inhibitory effect are different;the RDE’s Km and Vmax values obtained by double reciprocal mapping are 0.0016 mol/L and 0.015 mol/L,respectively.The experiment proves that the preparation method is simple,feasible,efficient and environmentally friendly,and provides a feasible new method for the preparation of quercetin.2.Study on the preparation of microbial whole-cell catalyst for quercetin.The microorganisms in moldy tartary buckwheat were acclimatized with Chaz’s medium,and the proportion of rutin was gradually increased until it replaced sucrose as the entire carbon source.The purified strain was tested for RDE activity in a liquid Chaz’s medium with rutin as the sole carbon source,and a strain of bacteria and a mold was isolated through domestication.As a result,the conversion rate of the fungus(53.92%)was higher than the bacteria(2.43%).The ecological and biochemical identification of the bacteria was subsequently performed.The colony observation and microscopic observation initially identified it as a Fusarium.After DNA extraction,PCR amplification,the amplified product has a single band and a fragment size of about 600 bp.The sequence was compared with the ITS rDNA sequence recorded in the NCBI,and a strain with a homology of more than 95%was selected to construct a phylogenetic tree.The strain was put into whole-cell catalysis,under the optimal conditions(6 μL whole-cell catalyst were added in 40%NADESs for 36 h),the rutin degradation rate was 369.54μg·min-1·L-1.3.Study on the preparation of CLEA-RDE.In this chapter,RDE is immobilized by the cross-linked enzyme aggregate method.Optimized the immobilization conditions by diluting the purified RDE solution 20-fold,took 4.5 mL and added 0.1 μg BSA,left it at room temperature for 30 minutes,then added 25%glutaraldehyde solution to a final concentration of 0.25%,adjusted the pH to 6,cross-linked slowly under 4℃ for 3 h.CLEA-RDE could be obtained by centrifuging the precipitate,washing and dissolving.The optimized rutin degradation rate was 8.16 mg·min-1·L-1.Subsequently,the structure of CLEA-RDE was characterized,the results from infrared chromatography,scanning electron microscopy,and ultraviolet scanning verified that the formation of the CLEA-RDE was due to the cross-linking effect of glutaraldehyde.Due to the changes in the molecular structure of the enzyme before and after immobilization,the optimal hydrolysis of the CLEA-RDE was re-optimized.Under the optimal conditions(adjust the pH to 5,the reaction was taken under 40℃ for 15 min),the rate of rutin degradation was 10.65 mg·min-1·L-1.Studies on the enzymatic properties of CLEA-RDE showed that the optimum temperature of the enzyme did not change much before and after immobilization,and the optimum pH shifted in the direction of alkali.Both temperature stability and acid-base stability are improved,tolerance tests on organic reagents show that before and after RDE cross-linking,their resistance increases with the increase of the hydrophobicity of organic reagents,and CLEA-RDE’s tolerance under hydrophobic environment is more improved than free-RDE(For example,in ethanol,the residual activity of CLEA-RDE is almost twice that of free-RDE).The kinetic values showed that the affinity between the CLEA-RDE and the substrate decreased,which may be due to the change of the molecular structure of the enzyme and the concealment of the active center of the enzyme.This experiment uses NADESs and RDE to establish a green and efficient preparation method of quercetin,combining whole-cell catalysis and carrier-free immobilization research to expand the source of the catalyst and improve the stability of RDE,providing new ideas for the green and efficient preparation of bioactive molecules.