LU504280B1 - Extraction process for rutin hydrolase from fresh s. japonicum bud - Google Patents

Abstract

The present invention provides an extraction process for rutin hydrolase from fresh S. japonicum bud and relates to the field of rutin hydrolase extraction technology. Based on an extraction process for rutin hydrolase from fresh S. japonicum bud, comprising the following steps: S1: pretreatment of raw materials; S2: extraction of RDE; S3: purification by centrifugal exchange chromatography of RDE; S4: gel filtration chromatography of RDE. The RDE from fresh S. japonicum bud was extracted by a process of ammonium sulphate precipitation, followed by a preliminary elution and purification of the crude enzyme solution by anion-exchange chromatography, and finally a secondary elution and purification by gel filtration chromatography so that the RDE could be extracted.

Description

Extraction process for rutin hydrolase from fresh S. japonicum bud

Technical Field

The present invention relates to the technical field of rutin hydrolase extraction, specifically an extraction process for rutin hydrolase from fresh S. japonicum bud.

Background technology

The flower and flower bud of Styphnolobium japonicum (L.) Schott (S. japonicum) have been used in traditional Chinese medicine (TCM), folk medicine and herbal tea for the treatment of treat hemorrhoids, hematochezia, hematuria, hematemesis, hemorrhinia, uterine or intestinal hemorrhage, arteriosclerosis, headache, hypertension, dysentery, dizziness, and pyodermas illnesses for centuries, especially in China[1,2]. Moreover, S. japonicum have long been used as a traditional medicine and food material in Chinese. Recently, the government of China officially declared that G. elata can be used as a “medicine food homology” plant for food and medicinal applications due to its long edible history in Asian countries and various safety evaluations[3].

The beneficial effects of S. japonicum are due mainly to its flavonoids, such as rutin(RU), iquercetin(QU) and kaempferol(KA), which have been widely identified as the major active constituents and indicators of the quality of S. japonicum[4,5]. Many studies have reported that S. japonicum and/or its main active compounds have anti-tumor, anti-inflammatory, antioxidant, and protective effects on cardiovascular and cerebrovascular diseases[6-10].

For the processing of S. japonicum flower(SJF) and S. japonicum flower bud(SJFB), it is normally collected in the summertime and processed by several steps including drying, removing the branches, root and other contaminations[11]. Processing and drying in the production area are key steps in forming the quality of medicinal materials. Appropriate drying methods are conducive to the storage and transportation of medicinal materials, the retention of effective ingredients, and the reduction of drug toxicity or side effects[12]. Many studies have reported that different processing methods have an impact on the flavonoid components and quality of S. japonicum[13,14], but the comprehensive effects and working mechanisms remain unclear.

Rutin is a glycoside of quercetin that can be hydrolyzed into aglycones and rutose under certain conditions[15]. The importance of endogenous enzymes on the stability of active ingredients has rarely been investigated in the past. Endogenous enzymes may influence the quality and the nutritional value of plants[15]. There may be a rutin hydrolase in the flower of S. japonicum,

which plays an important role in the growth process from flower bud to flower and in the 504280 different processing processes of S. japonicum. Therefore, the purpose of this study was to extract rutin hydrolase from Sophora japonica and studying its properties .

Contents of the invention (I) Technical problem solved

In response to the deficiencies of the prior art, the present invention provides an extraction process for rutin hydrolase from fresh S. japonicum bud, which solves the problem of low extraction activity and purity of rutin hydrolase from fresh S. japonicum bud. (II) Technical solutions

To achieve the above purpose, the present invention is realized by the following technical solution: an extraction process for rutin hydrolase from fresh S. japonicum bud, comprising the following steps:

S1. Pre-treatment of raw materials

Collecting the fresh fresh S. japonicum bud raw material, cooling it with liquid nitrogen and then storing it in a refrigerator at -80°C until use;

S2. Extraction of RDE

A quantitative amount of fresh acacia rice was extracted, added to a quantitative amount of acetate buffer and juiced and mixed, then the rice was filtered through gauze to remove the residue, followed by centrifugation of the filtrate to obtain the supernatant and set aside. = 7.0) until just dissolved, then the dissolved crude enzyme solution was dialyzed in deionized water at 4 °C for 12 h. The dialyzed crude enzyme solution was then concentrated with polyethylene glycol-6000 and the concentrated crude enzyme solution was stored in a refrigerator at 4 °C until use;

S3.RDE ion exchange chromatographic purification

First DEAEcellulose-32 pretreatment and column loading, followed by elution of the crude enzyme solution by anion-exchange chromatography;

S4. RDE gel filtration chromatography

The purified and concentrated enzyme solution obtained in S3 was first pretreated with

SephadexTMG-100 and loaded onto a column, then eluted, collected and concentrated by gel filtration chromatography.

Preferably, in step S2: 0506280 a. 500 mL of 0.02 mol/L acetate buffer (pH=5.0) is added per 100 g of acacia rice; b. removing the raw acacia rice after juicing and mixing it at 4°C for 12h; c. The filtrate after gauze filtration needs to be centrifuged at 12,000g for 30min at 4°C. The insoluble material needs to be removed before taking the supernatant; d. The solid ammonium sulphate powder is added to the supernatant to 80% saturation while stirring, and the process of stirring needs to be carried out continuously at 4°C for 4h; e. The solution precipitated by the ammonium sulphate is centrifuged at 12,000g for 30min at 4°C; f During dialysis, distilled water needs to be changed every 2h, for a total of 5 distilled water changes.

Preferably, in step S3: a. first weigh 50.00 g of DEAEcellulose-32, add 5 times the volume of purified water and soak overnight to remove impurities, soak in 20 times the volume of 0.5 mol/L HCI for 1 h.

Wash DEAEcellulose-32 with purified water to pH=4 or more, then soak in 20 times the volume of 0.5 mol/L NaOH for 1 h. Wash DEAEcellulose-32 with purified water to neutral and set aside; b. Wash the tetrafluorochromatographic column (1.5 x 25 em) with purified water and fix it vertically on an iron stand, leaving a certain height of purified water inside the column, divert the pre-treated DEAEcellulose-32 into the column with a glass rod, open the cork and release the liquid, the fibres slowly settle to the bottom of the column, fill the column to a height of 40 cm, stop loading the column, leave it overnight and wash it with pH =5 in 0.02 mol/L acetate buffer (pH=5) to equilibrate 4 column volumes until the packing height of the chromatographic column is constant; c. 1 mL of the crude enzyme solution obtained by ammonium sulphate precipitation dissolution dialysis concentration was loaded onto a DEAEcellulose-32 anion exchange column and eluted with 0.02 mol/L acetate buffer (pH = 5) containing 1 mol/L. NaCl at 4°C. The elution was carried out until the UV absorption at 280 nm no longer changed, the flow rate was controlled at 9-12 drops/min, and the unbound The unbound and eluted proteins were collected in centrifuge tubes, one tube per 3 mL, and the active RDE was collected by polyamide film assay. The collected RDE was concentrated by dialysis and stored in a refrigerator at 4°C until 204280 use.

Preferably, in step S4: a. 20.00g of SephadexTMG-100 is weighed, 1L of purified water is added, stirred well and then sealed and left to stand at room temperature for 48h to allow the gel to dissolve sufficiently to remove the suspended small particles; b. Wash the tetrafluoro chromatography column (2.5 x 50 cm) clean with purified water and fix it vertically on an iron stand. With a certain height of purified water left in the column, the pretreated SephadexTMG-100 was diverted into the column with a glass rod, the cork was opened, the liquid was released and the gel slowly settled to the bottom of the column, the packing height reached 40 cm, the loading of the column was stopped, left overnight and 4 column volumes were equilibrated with pH=50.02 mol/L acetate buffer until the packing height of the chromatography column was constant; c. 1 mL of enzyme solution purified and concentrated by DEAEcellulose-32 anion exchange column was loaded onto a Sephadex TMG-100 cross-linked dextran gel column at 4°C and eluted with acetate buffer until the UV absorption at 280 nm no longer changed, with the flow rate controlled at 9-12 drops/min. The eluted protein was collected in centrifuge tubes, one tube for every 3 mL collected. The active RDE was detected by polyamide film, and the collected RDE enzyme solution was concentrated by dialysis and stored in a refrigerator at 4°C. (111) Beneficial effects

The present invention provides An extraction process for rutin hydrolase from fresh S. japonicum bud. with the following beneficial effects:

The present invention extracts RDE (rutin hydrolase) from fresh S. japonicum bud by ammonium sulphate precipitation and obtains the crude enzyme solution, and then carries out preliminary elution and purification of the crude enzyme solution by anion exchange chromatography, and finally carries out secondary elution and purification by gel filtration chromatography, so that RDE can be isolated and purified from fresh S. japonicum bud, and the activity of RDE obtained by ammonium sulphate precipitation and column chromatography is

The activity of the RDE obtained by ammonium sulphate precipitation and column chromatography was higher.

Description of the accompanying drawings 0504280

FIG. 1 is an extraction process for rutin hydrolase from fresh S. japonicum bud according to the present invention;

FIG. 2 is chromatography of polyamide films of rutin ( A ) and quercetin ( B ) according to 5 the present invention;

FIG. 3 is a schematic diagram of the standard curve for the determination of RDE enzyme activity;

FIG. 4 is a schematic diagram of the standard curve of the bovine albumin according to the present invention;

FIG. 5 is a schematic diagram of chromatography of polyamide films of ammonium sulfate precipitation protein reaction products (A), inactivated ammonium sulfate precipitation protein reaction products (B), rutin (A) and quercetin (B) according to the present invention;

FIG. 6 is an elution profile of anion exchange chromatography of RDE according to the present invention;

FIG. 7 is a schematic diagram of chromatography of polyamide films of peak inactivated enzyme reaction product (A), peak enzyme solution reaction products (B), rutin (A) and quercetin (B) according to the present invention;

FIG. 8 is an elution profile of gel filtration chromatography of RDE according to the present invention;

FIG. 9 is a schematic diagram of chromatography of polyamide films of peak 1 inactivated enzyme reaction product (A), peak 1 enzyme solution reaction products (B), peak 2 inactivated enzyme reactants (C), peak 2 enzymatic reactants (D), quercetin (E) and rutin (F) according to the present invention;

FIG. 10 is a SDS-PAGE analysis diagram of the purification phase protein sample of ammonium sulfate precipitation protein (A), peranion exchange column protein (B) and supergel filtration column peak 1 protein (C) according to the present invention;

FIG. 11 is a UPLC diagram of rutin (A) according to the present invention;

FIG. 12 is a UPLC map of quercetin (B) according to the present invention;

FIG. 13 is a UPLC map of the purified RDE and FSI reaction of the inactivated RDE reactant (A) according to the present invention; and

FIG. 14 is a UPLC map of the purified RDE and FSI reaction of the RDE reactants 504280 according to the present invention.

Specific embodiments

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention, it being clear that the embodiments described are only a part of the embodiments of the present invention and not all of them. Based on the embodiments in the present invention, all other embodiments obtained without creative labour by a person of ordinary skill in the art fall within the scope of protection of the present invention.

Embodiments:

As shown in Figure 1, embodiments of the present invention provide an extraction process based on An extraction process for rutin hydrolase from fresh S. japonicum bud, comprising the steps of

S1. Raw material pre-treatment

Collecting the fresh fresh S. japonicum bud raw material, cooling it with liquid nitrogen and then storing it in a refrigerator at -80 °C until use;

S2. Extraction of RDE

A quantity of fresh acacia rice was extracted, added to a quantity of acetate buffer and juiced and mixed, then the acacia rice was filtered through gauze and the residue was removed, followed by centrifugation of the filtrate and the supernatant was obtained and set aside, immediately afterwards, solid ammonium sulphate powder was added to the supernatant and stirred, then it was centrifuged and the precipitate was collected, the precipitate was dissolved in 0.02 mol/LTris-HCI buffer solution (pH = 7.0) until just dissolved, then the dissolved crude enzyme solution was dialyzed in deionized water at 4°C for 12h, followed by concentrating the dialyzed crude enzyme solution with polyethylene glycol-6000, after concentration the crude enzyme solution was stored in a refrigerator at 4°C until use, the RDE (rutin hydrolase) in acacia rice was extracted and the crude enzyme solution was obtained by using the process of ammonium sulphate precipitation;

S3.RDE ion exchange chromatographic purification

Firstly, DEAEcellulose-32 pretreatment was performed and the column was loaded, then the crude enzyme solution was eluted by anion-exchange chromatography, the crude enzyme 504280 solution was initially eluted and purified by anion-exchange chromatography, and finally the secondary elution and purification was performed by gel filtration chromatography, so that RDE could be isolated and purified from fresh S. japonicum bud,

S4. RDE gel filtration chromatography

SephadexTMG-100 pretreatment was first performed and the column was loaded. The purified and concentrated enzyme solution obtained in S3 was then eluted, collected and concentrated by gel filtration chromatography, and the RDE obtained by ammonium sulphate precipitation as well as column chromatography was separated with higher activity.

In step S2: a. 500 mL of 0.02 mol/L acetate buffer (pH = 5.0) is added per 100 g of acacia rice; b. Remove the raw acacia rice after juicing and mix it at 4°C for 12h; c. The filtrate after gauze filtration needs to be centrifuged at 12,000g for 30min at 4°C. The insoluble matter needs to be removed before taking the supernatant; d. The solid ammonium sulphate powder is added to the supernatant to 80% saturation while stirring, and the process of stirring needs to be carried out continuously at 4°C for 4h; e. The solution precipitated by the ammonium sulphate is centrifuged at 12,000g for 30min at 4°C; f During dialysis, distilled water needs to be changed every 2h, for a total of 5 distilled water changes.

In step S3: a. First weigh 50.00 g of DEAFcellulose-32, add 5 times the volume of purified water and soak overnight to remove impurities, soak in 20 times the volume of 0.5 mol/L HCI for 1 h.

Wash DEAEcellulose-32 with purified water to pH=4 or more, then soak in 20 times the volume of 0.5 mol/L NaOH for 1 h. Wash DEAEcellulose-32 with purified water to neutral and set aside; b. Wash the tetrafluorochromatographic column (1.5 x 25 em) with purified water and fix it vertically on an iron stand, leaving a certain height of purified water inside the column, divert the pre-treated DEAEcellulose-32 into the column with a glass rod, open the cork and release the liquid, the fibres slowly settle to the bottom of the column, fill the column to a height of 40 cm,

stop loading the column, leave it overnight and wash it with pH =5 in 0.02 mol/L acetate buffer 504280 (pH=5) to equilibrate 4 column volumes until the packing height of the chromatographic column is constant; c. 1 mL of the crude enzyme solution obtained by ammonium sulphate precipitation dissolution dialysis concentration was loaded onto a DEAEcellulose-32 anion exchange column and eluted with 0.02 mol/L acetate buffer (pH = 5) containing 1 mol/L. NaCl at 4°C. The elution was carried out until the UV absorption at 280 nm no longer changed, the flow rate was controlled at 9-12 drops/min and the unbound The unbound and eluted proteins were collected in centrifuge tubes, one tube per 3 mL, and the active RDE was collected by polyamide film assay.

The collected RDE was concentrated by dialysis and stored in a refrigerator at 4°C until use.

In step S4: a. Weigh 20.00g of SephadexTMG-100, add 1L of purified water, stir well and seal, and leave the gel to swell sufficiently at room temperature for 48h to remove the suspended small particles; b. Wash the tetrafluoro chromatography column (2.5 x 50 cm) clean with purified water and fix it vertically on an iron stand. With a certain height of purified water left in the column, the pretreated SephadexTMG-100 was diverted into the column with a glass rod, the cork was opened, the liquid was released, the gel slowly settled to the bottom of the column and the packing height reached 40 cm, the column was stopped, left overnight and 4 column volumes were equilibrated with pH=50.02 mol/L acetate buffer until the packing height of the chromatography column was constant; c. 1 mL of enzyme solution purified and concentrated by DEAEcellulose-32 anion exchange column was loaded onto a Sephadex TMG-100 cross-linked dextran gel column at 4°C and eluted with acetate buffer until the UV absorption at 280 nm no longer changed, the flow rate was controlled at 12 drops/min and the eluted protein was collected in centrifuge tubes, one tube for every 3 mL collected, with The RDE enzyme solution was concentrated by dialysis and stored in a refrigerator at 4°C. The water purifier was a Rephile water purifier and the mixing equipment was a vortex mixer.

Example 2:

As shown in Figure 1 to 14, an extraction process for rutin hydrolase from fresh S.

japonicum bud is provided in the examples of the present invention. The process of TLC 904280 detection of RDE activity is as follows: a capillary tube is used for sampling point in a polyamide film, and the amount of sampling is 10L; at the same time, the standard substances of the rutin and quercetin are set as controls. A developing solvent is ethanol:water = 5:1, and the development distance is 6cm, and a color developing agent is 1% of FeCl3 ethanol solution.

According to the TLC of the standard substances of rutin and quercetin, quercetin formation can be qualitatively detected, as shown in Figure 2, to determine whether the RDE is active.

The steps of determining the RDE activity by UPLC:

A. Precisely weigh 0.5g of dried fresh S. japonicum bud sample powder and prepare five portions in parallel; put them in a conical flask with stopper respectively; add 70% of 8mL ethanol and weigh them; sonicate (power 200W, frequency 40kHz) at 61°C for 30min and weigh them after the temperature returns to room temperature; make up the weight loss with 70% of ethanol and shake well; combine the five prepared extract solutions and use a rotary evaporator to concentrate the extract solution to about 5 mL; then put it into refrigerator with -80°C and freeze it overnight, dry it with a vacuum freeze dryer to obtain FSI; weigh 5 mg of FSI and fix the volume with 20% of ethanol to 10 mL; and configure it into 0.50 mg/mL of FSI solution as a reaction substrate;

B. Precisely weigh 10 mg of rutin standard substance, dissolve it with 20% of 10 mL ethanol and configure it into 1.00 mg/mL of rutin standard substance as the reaction substrate;

C. Chromatographic regulating: chromatographic column:

ThermoFisherScientificAccucore-C18 liquid phase chromatographic column (100x2. 1mm, 2.6um); mobile phase: acetonitrile (A)-1% of acetic acid aqueous solution (B); gradient elution (0-2min, 5% of A; 2-4min, 5-20% of A; 4-8min, 20% of A; 8-12min, 20-32% of A; 12-20min, 32-35% of A; 20-24min, 35-5% of A; 24-28min, 5% of A 5%A; 28-30min, 5-90% of A; 30-42min, 90% of A; 42-45min, 90-5% of A; 45-55min, 5% ofA); flow rate 0.2mL/min; column temperature 30°C; injection volume 1 pL; detection wavelength 260 nm;

D. A standard curve plotting: precisely weigh 10.34 mg of quercetin standard substance and dissolve with 10 mL of methanol and configure into 1.034 mg/mL of quercetin standard solution, and 0.01, 0.02, 0.04, 0.05, 0.1, 0.125, 0.25, 0.5 and 1 mL is fixed to 1 mL, respectively. The determination is performed under the chromatographic conditions of 2.5.2. With the peak area as the vertical coordinate and quercetin concentration as the horizontal coordinate, OriginPro207 17504280 is used for plotting and the linear fitting equation is y=172.6852x-0.79424, R2=0.9997, as shown in Figure 3; and

E. Hydrolysis reaction conditions: take 0.3mL of reaction substrate and add 0.3mL of RDE; react at 40°C in water bath for 30min and quickly add 95% of ethanol for 2mL to terminate the enzyme activity, centrifuge the reaction solution at 12,000g for Smin, remove insoluble matter, take the supernatant and set aside; meanwhile, set inactivating enzyme solution as control, inactivate by water bath at 100°C for Smi; according to 2.2.4 chromatographic conditions to detect and the activity of RDE is calculated according to the standard curve. The enzyme activity of RDE is defined as (U), the amount of enzyme required to generate 1 ug of quercetin per minute under the analytical conditions of rutin as substrate, pH=5.0 and temperature of 40°C.

Determination of protein content:

A. Precisely weigh 10.00 mg of bovine serum protein and fix the volume to 10 mL with 0.15 mol/mL NaCl to obtain 1 mg/mL of protein standard solution. Add 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.007mL of protein standard solution, make up to 0.1mL with saline, add SmL of

Coomassie brilliant blue reagent, shake well, react at room temperature for Smin, measure the absorption value at 595nm, and obtain the standard curve y=0.5147x+0.0876, R?= 0.9920;

C(mg/ 0.1 0.2 0.3 0.4 0.5 0.7 mL)

A595 0.12 0.19 0.24 0.30 0.34 0.40 0.43 96 20 12 70 67 55 25

Figure 1 is a standard curve of the bovine albumin

Steps of dodecyl Sodium Sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE):

Sample solution preparation: radomly take 15 pL of sample solution in a 200 uL PCR tube, add 3 mL of 6x protein reduction sampling buffer, vortex mixing well and then centrifuge.

SDS-PAGE gel electrophoresis:

A. Clean glass plate

B. Preparation of separating gel and spacer gel:

1.5MTris-HCI buffer 2.5 0506280 solution 1.0MTris-HCI buffer 0.63 solution

C. Gel making and sampling: align the glass plates and put them into a glue maker to align and snap them tightly so as not to leak gel, shake and fill the gel immediately after configuring the separating gel, and stop gelling until three-quarters of the glue maker; in order to make the top surface of the separating gel flat, fill the empty space with anhydrous ethanol. After standing for 30min, pour off the upper layer of ethanol on the gel surface and blot the remaining ethanol with filter paper strips, immediately shake the configured spacer gel to fill the gel, and insert the comb into the spacer gel after the spacer gel fills the remaining space; and after the spacer gel solidifies, add enough electrode buffer to perform sample electrophoresis; after adding the sample to the electrophoresis hole, perform electrophoresis; the voltage of the spacer gel is 100V, and the voltage of the separation gel is 120V, and electrophoresis can be stopped when the bottom of the bromophenol blue glass plate is about still 1cm.

RDE extraction and purification:

A. Ammonium sulfate precipitation: the crude enzyme solution precipitated by ammonium sulfate is taken, and 1 mg/mL of rutin standard solution is used as the substrate, and the reaction solution is tested for activity by polyamide film TLC as required in the hydrolysis reaction conditions, and the results are shown in Figure 6. The reaction solution (A) of RDE without high temperature inactivation had quercetin production, indicating that the extracted RDE had obvious activity.

B. Ion exchange chromatography: 1 mL of crude enzyme solution extracted according to the process in step S2 is taken and added to a DEAEcellulose-32 anion exchange column for elution, and the number of tubes (3 mL/tube) is used as the horizontal coordinate, and the absorbance of the collected enzyme solution at 280 nm is used as the vertical coordinate to draw the elution curve, as shown in Figure 7. The enzyme solution at the collected peak TS 504280 concentrated by dialysis, and 1 mg/mL of rutin standard solution as the substrate; and the activity is detected by polyamide film TLC according to the requirements in the hydrolysis reaction conditions. The reaction solution (B) of the RDE at the peak without high temperature inactivation had quercetin production, indicating that the enzyme solution at the outgoing peak is active.

Gel filtration chromatography: 1mL of enzyme concentrate over the anion exchange column is taken and added to the gel column and eluted; with the number of tubes (3mL/tube) as the horizontal coordinate and the absorbance of the collection solution at 280nm as the vertical coordinate, the elution curve is plotted, as shown in Figure 8. The enzyme solution at collection peak 1 and peak 2 is concentrated by dialysis and reacted with mg/mL of rutin standard solution as the substrate at 40°C for 30min, while inactivated enzyme is set as the control; the reaction product is centrifuged and detected by polyamide film TLC for activity. The results are shown in

Figure 9. The reaction solution (B) of peak 1 without hyperthermally inactivated RDE has quercetin production, indicating that the enzyme solution at peak 1 is active, and the reaction solution (D) of peak 2 without hyperthermally inactivated RDE has no quercetin production, indicating that the enzyme solution at peak 2 is not active.

Steps of V Protein Gros Specif Produ Purific

Purification olume | content (mg) s activity | ic activity | ctivity ation fold ( (U) (U/mg (%) (fold) mL) )

Ammoniu 1 14.77 271. 18.36 100.0 1.00 m sulfate 2 16 0 precipitati on

Anion 1 2.72 192. 70.74 70.96 3.85 exchange 6 41 chromatog raphy filtration 106260 chromatograph y

The enzyme solution treated by ammonium sulfate precipitation, anion exchange chromatography and dialysis condensation at peak 1 of gel filtration chromatography is taken and reacted according to the hydrolysis reaction conditions; its activity is measured by UPLC separately, and the concentration of converted quercetin of the sample is known from the peak area and the standard curve of quercetin concentration; and then the enzyme activity is calculated according to the formula of enzyme activity definition. The specific activity of the purified RDE increases from 18.36 U/mg to 103.98 U/mg, a 5.66-fold increase, and the yield is 24.93%.

RDE molecular weight: take the enzyme solution treated by ammonium sulfate precipitation, anion exchange chromatography and gel filtration chromatography peak 1 of dialysis condensation according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) steps for experiments, electrophoresis is used to detect the size of the protein molecular weight, the results are shown in Figure 10, showing the three have a common band, compared with the Maker protein, the size of the band is about 38KDa.

RDE substrate specificity: rutin and quercetin are detected according to the chromatographic conditions, and the results are shown in a figure. The retention time of rutin (A) is 9.180 and that of quercetin (B) is 14.387 under these conditions, indicating that rutin and quercetin are well separated under these chromatographic conditions, and the results are shown in FIGs. 11 and 12.

Rutin is used as a substrate and reacted with the enzyme solution purified by gel filtration chromatography according to the hydrolysis reaction conditions, and the results are shown in

FIGs. 13 and 14. In comparison with the experimental control group, it is found that after 30 min of hydrolysis, the active RDE partially hydrolyzes rutin to quercetin (B), while the rutin content does not change after the reaction with the inactivated treated rutin hydrolase and no quercetin is produced.

Although embodiments of the invention have been shown and described, it will be understood to those of ordinary skill in the art that a variety of variations, modifications, replacements and variants of these embodiments can be made without departing from the

LL . . . 111 . 504280 principles and spirit of the invention, the scope of which is limited by the appended claims an their equivalents.

Claims (4)

CLAIMS LU504280

1. An extraction process for rutin hydrolase from fresh S. japonicum bud, characterized in that it comprises the steps of

S1. Pre-treatment of raw materials Collecting the fresh fresh S. japonicum bud raw material, cooling it with liquid nitrogen and then storing it in a refrigerator at -80°C until use;

S2. Extraction of RDE A quantitative amount of fresh acacia rice was extracted, added to a quantitative amount of acetate buffer and juiced and mixed, then the rice was filtered through gauze to remove the residue, followed by centrifugation of the filtrate to obtain the supernatant and set aside. = 7.0) until just dissolved, then the dissolved crude enzyme solution was dialyzed in deionized water at 4°C for 12 h. The dialysis-treated crude enzyme solution was then concentrated with polyethylene glycol-6000 and the concentrated crude enzyme solution was stored in a refrigerator at 4°C until use; S3 RDE ion exchange chromatographic purification Firstly, DEAEcellulose-32 pretreatment and column loading, followed by elution of the crude enzyme solution by anion-exchange chromatography;

S4. RDE gel filtration chromatography The purified and concentrated enzyme solution obtained in S3 was first pretreated with SephadexTMG-100 and loaded onto a column, then eluted, collected and concentrated by gel filtration chromatography.

2. The extraction process for rutin hydrolase from fresh S. japonicum bud according to claim 1, characterized by: in step S2:

a. 500 mL of 0.02 mol/L acetate buffer (pH=5.0) is added per 100 g of fresh S. japonicum bud;

b. removing the raw acacia rice after juicing and mixing it at 4°C for 12h;

c. The filtrate after gauze filtration needs to be centrifuged at 12,000g for 30min at 4°C. The insoluble matter needs to be removed before taking the supernatant;

d. The solid ammonium sulphate powder is added to the supernatant to 80% saturation while stirring, and the process of stirring needs to be carried out continuously at 4°C for 4h; 0506280 e. The solution precipitated by the ammonium sulphate is centrifuged at 12,000g for 30min at 4°C;

f. During dialysis, distilled water needs to be changed every 2h, for a total of 5 distilled water changes.

3. The extraction process for rutin hydrolase from fresh S. japonicum bud according to claim 1, characterized in step S3 by:

a. first weigh 50.00 g of DEAEcellulose-32, add 5 times the volume of purified water and soak overnight to remove impurities, soak in 20 times the volume of 0.5 mol/L HCI for 1 h. Wash DEAEcellulose-32 with purified water to pH=4 or more, then soak in 20 times the volume of 0.5 mol/L NaOH for 1 h. Wash DEAEcellulose-32 with purified water to neutral and set aside;

b. Wash the tetrafluorochromatographic column (1.5 x 25 em) with purified water and fix it vertically on an iron stand, leaving a certain height of purified water inside the column, divert the pre-treated DEAEcellulose-32 into the column with a glass rod, open the cork and release the liquid, the fibres slowly settle to the bottom of the column, fill the column to a height of 40 cm, stop loading the column, leave it overnight and wash it with pH = 5 in 0.02 mol/L acetate buffer (pH = 5) to equilibrate 4 column volumes until the packing height of the chromatographic column is constant;

c. 1 mL of the crude enzyme solution obtained by ammonium sulphate precipitation dissolution dialysis concentration was loaded onto a DEAEcellulose-32 anion exchange column and eluted with 0.02 mol/L acetate buffer (pH = 5) containing 1 mol/L. NaCl at 4°C. The elution was carried out until the UV absorption at 280 nm no longer changed, the flow rate was controlled at 9-12 drops/min and the unbound The unbound and eluted proteins were collected in centrifuge tubes, one tube per 3 mL, and the active RDE was collected by polyamide film assay. The collected RDE was concentrated by dialysis and stored in a refrigerator at 4°C until use.

4. The extraction process for rutin hydrolase from fresh S. japonicum bud according to claim 1, characterized in that in step S4:

a. 20.00 g of SephadexTMG-100 is weighed, 1 L of purified water is added, stirred well and then sealed and left to stand at room temperature for 48 h to allow the gel to swell sufficiently to remove the small particles in suspension; 0504280 b. Wash the tetrafluoro chromatography column (2.5 x 50 cm) clean with purified water and fix it vertically on an iron stand. With a certain height of purified water left in the column, the pretreated SephadexTMG-100 was diverted into the column with a glass rod, the cork was opened, the liquid was released and the gel slowly settled to the bottom of the column, the packing height reached 40 cm, the loading of the column was stopped, left overnight and 4 column volumes were equilibrated with pH=50.02 mol/L acetate buffer until the packing height of the chromatography column was constant;

c. 1 mL of enzyme solution purified and concentrated by DEAEcellulose-32 anion exchange column was loaded onto a Sephadex TMG-100 cross-linked dextran gel column at 4°C and eluted with acetate buffer until the UV absorption at 280 nm no longer changed, with the flow rate controlled at 9-12 drops/min. The eluted protein was collected in centrifuge tubes, one tube for every 3 mL collected. The active RDE was detected by polyamide film, and the collected RDE enzyme solution was concentrated by dialysis and stored in a refrigerator at 4°C.