Preparation method of 3'-hydroxygenistein

Abstract

The present invention provides a method for preparing 3'-hydroxygenistein, comprising the following steps: constructing a genetically recombinant microorganism that expresses tyrosinase; performing a reaction matrix preparation step, wherein the reaction matrix comprises borate, ascorbic acid and genistein; then inoculating the genetically recombinant microorganism in the reaction matrix to perform a bioconversion reaction step to obtain a bioconversion substance; then adding hydrochloric acid solution to the bioconversion substance to perform a termination reaction step to obtain a reaction solution, wherein the reaction solution comprises 3'-hydroxygenistein. Thus, the method for preparing 3'-hydroxygenistein provided by the present invention can prepare high-purity 3'-hydroxygenistein in large quantities.

Description

Technical Field

[0001] The invention relates to a method for preparing an oxygen-containing organic compound, in particular to a method for preparing 3'-hydroxygenistein. Background Art

[0002] Genistein is a major active factor of soybean isoflavones and has multiple physiological functions. Genistein is similar in structure to the mammalian estrogen-estradiol and has the active group of estrogen-diphenolic hydroxyl group. Therefore, genistein has multiple physiological activities such as estrogen-like activity, such as preventing osteoporosis, improving menopausal syndrome, preventing cancer (such as breast cancer, colon cancer, lung cancer, prostate cancer and skin cancer), preventing Alzheimer's disease, delaying aging, improving menstrual discomfort, lowering cholesterol and regulating blood lipids.

[0003] 3'-Hydroxygenistein (also known as Orobol) is a derivative of genistein. Changes in the isoflavone structure often cause significant changes in the biological activity of the compound. For example, genistein at a concentration of 30μM to 60μM promotes melanocytes to accelerate the production of melanin, but after bioconversion, 3'-hydroxygenistein has a low-dose, high-efficiency skin whitening effect, which has the opposite characteristics of its bioconversion precursor. Other studies have shown that 3'-hydroxygenistein has the activity of inhibiting the growth of T47D tumorigenic breast epithelial cells, as well as inhibiting HIV-1 integrase, anti-inflammatory and liver protection activities.

[0004] However, the low productivity of 3'-hydroxygenistein is still the main factor that hinders its application in health food or pharmaceutical products. Although there are methods to prepare 3'-hydroxygenistein at present, its output is still limited, and the purity of the prepared 3'-hydroxygenistein is not high. Therefore, how to develop a better method to improve the output and purity of 3'-hydroxygenistein has become the development direction of current related industries. Summary of the invention

[0005] The present invention aims to provide a method for preparing 3'-hydroxygenistein, comprising the following steps: constructing a genetically recombinant microorganism, transforming a circular recombinant plasmid into a microbial expression system to obtain a genetically recombinant microorganism, wherein the circular recombinant plasmid comprises a gene that can exhibit tyrosinase activity as shown in sequence identification number 1; performing a reaction matrix preparation step, wherein the reaction matrix comprises borate with a final concentration of 400mM to 600mM, ascorbic acid with a final concentration of 10mM to 40mM, and 500mM of ascorbic acid with a final concentration of 10mM to 40mM. pm to 3000ppm of genistein; performing a bioconversion reaction step: inoculating the genetically recombinant microorganism in the reaction matrix, and culturing at a bioconversion reaction temperature and a bioconversion reaction ventilation volume at a bioconversion reaction stirring speed for a bioconversion reaction time to obtain a bioconversion substance; and then performing a termination reaction step: adding a hydrochloric acid solution to the bioconversion substance, and reacting at a termination reaction temperature and a termination ventilation volume at a termination stirring speed for a termination reaction time to obtain a reaction solution, wherein the reaction solution contains 3'-hydroxygenistein.

[0006] According to the aforementioned method for preparing 3'-hydroxygenistein, the microbial expression system may be yeast; preferably, the yeast is Pichia pastoris.

[0007] According to the aforementioned preparation method of 3'-hydroxygenistein, the reaction matrix preparation step may include the following steps: dissolving genistein in 60 mL of dimethyl sulfoxide (DMSO) to obtain a genistein solution; dissolving ascorbic acid in water to obtain an ascorbic acid solution; dissolving borate in hot water and adjusting the pH value to 8 to 10 to obtain a borate solution; and then adding the genistein solution and ascorbic acid solution to the borate solution to obtain the reaction matrix.

[0008] According to the aforementioned preparation method of 3'-hydroxygenistein, the bioconversion reaction temperature can be 40°C to 60°C; the bioconversion reaction ventilation volume can be 2L / min to 5L / min, and the bioconversion reaction stirring speed can be 200rpm to 400rpm; the bioconversion reaction time can be 250 minutes to 300 minutes.

[0009] According to the aforementioned method for preparing 3'-hydroxygenistein, the final concentration of the hydrochloric acid solution can be 100 mM to 200 mM.

[0010] According to the aforementioned preparation method of 3'-hydroxygenistein, the termination reaction temperature can be 40°C to 60°C; the termination reaction ventilation volume can be 2L / min to 5L / min, and the termination reaction stirring speed can be 200rpm to 400rpm; the termination reaction time can be 5 minutes to 15 minutes.

[0011] According to the aforementioned preparation method of 3'-hydroxygenistein, an extraction step may be further included: adding ethyl acetate to the reaction solution, and repeatedly shaking at a shaking speed for a shaking time 2 to 3 times.

[0012] According to the aforementioned method for preparing 3'-hydroxygenistein, the volume ratio of the reaction solution to ethyl acetate can be 1:1 to 1.5:1.

[0013] According to the aforementioned preparation method of 3'-hydroxygenistein, the shaking speed can be 2000 rpm to 3000 rpm, and each shaking time can be 5 minutes to 20 minutes.

[0014] Therefore, the preparation method of 3'-hydroxygenistein of the present invention uses genetically engineered microorganisms to convert 3'-hydroxygenistein, and its preparation method can be carried out for large-scale industrial production. By controlling the production process such as the starting product and the biological conversion reaction conditions, the produced 3'-hydroxygenistein has stable quality and high purity, thereby solving the problem of low productivity of 3'-hydroxygenistein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a flow chart of the steps of the preparation method of 3'-hydroxygenistein;

[0016] Figure 2 This is a schematic diagram of the results of small-volume fermentation of the strain after selection;

[0017] Figure 3 Schematic diagram of the effect of temperature on yeast culture;

[0018] Figure 4 Schematic diagram of the effect of pH on yeast culture;

[0019] Figure 5 Schematic diagram of the effect of copper ion concentration on yeast culture;

[0020] Figure 6 Schematic diagram of the effect of different carbon sources on yeast culture;

[0021] Figure 7 Schematic diagram of the effect of glucose concentration on yeast culture;

[0022] Figure 8 Schematic diagram of pH stability of tyrosinase in yeast;

[0023] Fig. 9 Schematic diagram of the temperature stability of tyrosinase in yeast;

[0024] Fig.10 Schematic diagram of HPLC chromatography of 3'-hydroxygenistein standard and genistein standard;

[0025] Fig.11 Schematic diagram of HPLC chromatography for yeast conversion of 3'-hydroxygenistein;

[0026] Fig.12 Schematic diagram of the reaction time for yeast to convert 3'-hydroxygenistein;

[0027] Fig.13 Schematic diagram of the conversion rate of yeast to 3'-hydroxygenistein.

[0028] Figure ID

[0029] 100: Preparation method of 3'-hydroxygenistein;

[0030] S1~S4: steps. DETAILED DESCRIPTION

[0031] Preparation method of 3'-hydroxygenistein provided by the invention

[0032] Preparation Example 1

[0033] See also Figure 1 , Figure 1 The present invention is a flow chart of the steps of the preparation method of 3'-hydroxygenistein. Figure 1 In the method 100 for preparing 3'-hydroxygenistein, the method comprises step S1, step S2, step S3 and step S4.

[0034] Step S1 is to construct a genetically recombinant microorganism by transforming a circular recombinant plasmid into a microbial expression system to obtain a genetically recombinant microorganism, wherein the circular recombinant plasmid comprises a gene that can exhibit tyrosinase activity as shown in sequence identification number 1. The microbial expression system can be yeast; preferably, the yeast is Pichia pastoris.

[0035] Step S2 is a step of preparing a reaction matrix, wherein the reaction matrix comprises borate at a final concentration of 400mM to 600mM, ascorbic acid at a final concentration of 10mM to 40mM, and genistein at a final concentration of 500ppm to 3000ppm. Preferably, the reaction matrix preparation step may comprise the following steps: dissolving genistein in 60mL dimethyl sulfoxide (DMSO) to obtain a genistein solution; dissolving ascorbic acid in water to obtain an ascorbic acid solution; dissolving borate in hot water and adjusting the pH value to 8 to 10 to obtain a borate solution; and then adding the genistein solution and the ascorbic acid solution to the borate solution to obtain the reaction matrix.

[0036] Step S3 is a bioconversion reaction step: inoculating the genetically recombinant microorganisms in the reaction matrix, and culturing the bioconversion reaction time at a bioconversion reaction temperature and a bioconversion reaction aeration volume at a bioconversion reaction stirring speed to obtain a bioconversion substance. Preferably, the bioconversion reaction temperature can be 40°C to 60°C; the bioconversion reaction aeration volume can be 2L / min to 5L / min, and the bioconversion reaction stirring speed can be 200rpm to 400rpm; the bioconversion reaction time can be 250 minutes to 300 minutes.

[0037] Step S4 is to perform a termination reaction step: add a hydrochloric acid solution to the bioconversion substance, and react at a termination reaction temperature and a termination ventilation volume with a termination stirring speed for a termination reaction time to obtain a reaction solution, wherein the reaction solution contains 3'-hydroxygenistein. Preferably, the final concentration of the hydrochloric acid solution can be 100mM to 200mM, the termination reaction temperature can be 40°C to 60°C, the termination reaction ventilation can be 2L / min to 5L / min, and the termination reaction stirring speed can be 200rpm to 400rpm, and the termination reaction time can be 5 minutes to 15 minutes.

[0038] In addition, the preparation method 100 of 3'-hydroxygenistein may further include an extraction step: adding ethyl acetate to the reaction solution, and repeatedly shaking at a shaking speed for a shaking time 2 to 3 times. Preferably, the volume ratio of the reaction solution to ethyl acetate may be 1:1 to 1.5:1, the shaking speed may be 2000 rpm to 3000 rpm, and each shaking time may be 5 minutes to 20 minutes.

[0039] The present invention will be further described in detail below in conjunction with specific embodiments, so as to facilitate those skilled in the art to which the present invention belongs, so that they can fully utilize and implement the present invention without excessive interpretation. These embodiments are only used to demonstrate the present invention and indicate how to implement the materials and methods of the present invention, and these embodiments should not be regarded as limiting the scope of the present invention.

[0040] Construction of genetically recombinant microorganisms

[0041] Preparation Example 2

[0042] In this embodiment, a circular recombinant plasmid is first constructed. The expression vector used is pGAPZαA vector (purchased from Thermo Fisher), and the insert is a gene that can show tyrosinase activity as shown in sequence identification number 1, which is derived from the tyrosinase gene body extracted from Bacillus megaterium, and then an artificial gene that can show good tyrosinase activity on yeast is artificially designed. The microbial expression system used is Pichia pastoris X-33 (purchased from Thermo Fisher).

[0043] The insert fragment was cloned into the pGAPZαA vector to obtain the constructed circular recombinant plasmid pGAPZαA-JDTYR. The circular recombinant plasmid pGAPZαA-JDTYR was then transformed into Pichia pastoris X-33 strain (purchased from Thermo Fisher) to form a genetically recombinant microorganism. The transformation method can include but is not limited to chemical methods such as calcium chloride treatment or physical methods such as electroporation.

[0044] After activating the recombinant microorganism constructed above, select a single colony and inoculate it into 5 ml of YPD medium (20 g / L peptone; 10 g / L yeast extract; 20 g / L glucose; 1000 μL / ml zeocin), and place it in a 30°C incubator at 250 rpm for overnight culture. Take 1 mL of the bacterial solution and inoculate it into a 500 mL conical flask containing 50 ml of YPD medium (containing 1000 μL / ml zeocin, 0.1 mM copper sulfate), and culture it at 30°C and 250 rpm for 3 to 4 days. Centrifuge the bacterial solution at 6000 x g and 4°C for 10 minutes to remove the supernatant, and freeze-dry the bacterial cells to obtain freeze-dried bacterial cells.

[0045] Experiment on the Optimal Culture Conditions of Yeast

[0046] Test Example 1

[0047] Expression of recombinant tyrosinase gene in genetically recombinant microorganism (yeast)

[0048] See also Figure 2 , Figure 2 Schematic diagram of the results of small-volume fermentation of the strain after selection. Figure 2In the experiment, the strain was fermented in a small volume in a YPD medium. The contents of the four sample tubes were respectively Pichia X-33 and L-Dopa after genetic recombinant yeast. The amount of Pichia added in the sample tubes was 1%, 0.4%, 0.2%, and 0.1% (weight percentage) from left to right. After the reaction, the darker the color, the higher the amount of L-Dopa converted into melanin. From the preliminary results, it can be seen that the genetically recombinant Pichia X-33 has tyrosinase activity, and the gene that can show tyrosinase activity introduced into Pichia X-33 was successfully introduced and had obvious activation activity.

[0049] Test Example 2

[0050] Effect of temperature on genetically modified microorganisms (yeast)

[0051] Select a single colony and inoculate it into 5mL YPD medium (peptone 20g / L; yeast extract 10g / L; glucose 20g / L; zeocin (bleomycin) 1000μL / ml), and place it in a 30℃ incubator and culture it overnight at 250rpm. Take 1mL of bacterial liquid and inoculate it into a 500mL conical flask with 50ml YPD medium (containing zeocin (bleomycin) 1000μL / ml, copper sulfate 1mM), place it in a 20℃ and 30℃ incubator, and culture it at 250rpm for 4 days.

[0052] See also Figure 3 , Figure 3 Schematic diagram of the effect of temperature on yeast culture. Figure 3 The results show that the enzyme activities of yeast cultured at 20°C and 30°C are 15.8 Unit / mL and 9.8 Unit / mL respectively, and the yeast cultured at 20°C has higher activity.

[0053] Test Example 3

[0054] Effect of pH on genetically recombinant microorganisms (yeast)

[0055] A single colony was selected and inoculated into 5 mL YPD medium (peptone 20 g / L; yeast extract 10 g / L; glucose 20 g / L; zeocin (bleomycin) 1000 μL / ml), and placed in a 30°C incubator and cultured overnight at 250 rpm. 1 mL of bacterial solution was inoculated into a 500 mL conical flask containing 50 ml BMGY medium (50 mM potassium phosphate buffer, peptone 20 g / L; yeast extract 10 g / L; glucose 20 g / L; zeocin (bleomycin) 1000 μL / ml, copper sulfate 1 mM), and placed in a 20°C incubator and cultured at 250 rpm for 4 days.

[0056] The yeast was cultured in pH 5 and pH 6 environments to observe the changes in enzyme activity at different pH values. Figure 4 , Figure 4 Schematic diagram of the effect of pH on yeast culture. Figure 4 The results show that in the environment of pH 5 and pH 6, the enzyme activity of the yeast is 3.2Unit / mL and 3.9Unit / mL respectively, which is not much different.

[0057] Test Example 4

[0058] Effect of copper ion concentration on genetically recombinant microorganisms (yeast)

[0059] A single colony was selected and inoculated into 5 mL YPD medium (peptone 20 g / L; yeast extract 10 g / L; glucose 20 g / L; zeocin (bleomycin) 1000 μL / ml), and placed in a 30°C incubator and cultured overnight at 250 rpm. 1 mL of bacterial solution was inoculated into a 500 mL conical flask containing 50 ml BMGY medium (50 mM potassium phosphate buffer, peptone 20 g / L; yeast extract 10 g / L; glucose 20 g / L; zeocin (bleomycin) 1000 μL / ml, copper sulfate 1 mM), and placed in a 20°C incubator and cultured at 250 rpm for 4 days. 1 mL of bacterial solution was inoculated into 50 mL of YPD medium (containing 1000 μL / ml of zeocin) with different copper ion concentrations (0 mM, 0.1 mM, 1 mM, 2 mM) in a 500 mL conical flask and cultured at 20°C and 250 rpm for 4 days.

[0060] See also Figure 5 , Figure 5 Schematic diagram of the effect of copper ion concentration on yeast culture. Figure 5 The results show that when the yeast is cultured at different copper ion concentrations of 0mM, 0.1mM, 1mM, and 2mM, its activity is 0.43, 1.33, 6.53, and 2.20Unit / mL, respectively. Therefore, the yeast cultured at a copper ion concentration of 1mM has the highest activity.

[0061] Test Example 5

[0062] Effects of different carbon sources on genetically recombinant microorganisms (yeast)

[0063] A single colony was selected and inoculated into 5 mL YPD medium (peptone 20 g / L; yeast extract 10 g / L; glucose 20 g / L; zeocin (bleomycin) 1000 μL / ml), and placed in a 30°C incubator and cultured overnight at 250 rpm. 1 mL of bacterial solution was taken out and inoculated into a 500 mL conical flask containing 50 mL YPD medium (peptone 20 g / L; yeast extract 20 g / L; zeocin (bleomycin) 1000 μL / ml, copper sulfate 1 mM) with 2% (weight percentage) of different carbon sources (glucose, glycerol, sorbitol), and cultured at 20°C and 250 rpm for 4 days.

[0064] See also Figure 6 , Figure 6 Schematic diagram of the effect of different carbon sources on yeast culture. Figure 6 The results show that when the yeast is cultured in 2% glucose, glycerol, and sorbitol, the activities are 5.4, 4.4, and 2.6 Unit / mL, respectively. Therefore, the yeast culture has the highest activity in glucose.

[0065] Test Example 6

[0066] Effects of different concentrations of carbon sources on genetically recombinant microorganisms (yeast)

[0067] A single colony was selected and inoculated into 5 mL YPD medium (peptone 20 g / L; yeast extract 10 g / L; glucose 20 g / L; zeocin (bleomycin) 1000 μL / ml), and placed in a 30°C incubator and cultured overnight at 250 rpm. 1 mL of bacterial solution was taken out and inoculated into 50 mL YPD medium (peptone 20 g / L; yeast extract 10 g / L; zeocin (bleomycin) 1000 μL / ml, copper sulfate 1 mM) in a 500 mL conical flask with different concentrations of glucose carbon source (0 g / L, 20 g / L, 40 g / L, 60 g / L), and cultured at 20°C and 250 rpm for 4 days.

[0068] See also Figure 7 , Figure 7 Schematic diagram of the effect of glucose concentration on yeast culture. Figure 7 From the results shown, it can be seen that when the yeast is cultured at glucose concentrations of 0 g / L, 20 g / L, 40 g / L, and 60 g / L, the activities are 2.9, 5.6, 7.4, and 8.5 Unit / mL, respectively. As the glucose concentration increases, the tyrosinase activity becomes stronger.

[0069] Enzyme activity stability test

[0070] Test Example 7

[0071] Effect of different pH values ​​on the stability of tyrosinase

[0072] Take 0.01g of freeze-dried yeast powder, dilute and dissolve it with different buffer solutions (15mM disodium hydrogen phosphate-sodium dihydrogen phosphate, 50mM sodium chloride, pH 6-8; 15mM glycine-sodium hydroxide, 50mM sodium chloride, pH 9-10) to make the final concentration of reconstitution be 1mg / ml, and take samples at fixed time to detect activity. Experimental method: take 100μL of bacterial solution and add 900μL, 2.5mM L-DOPA, use a spectrophotometer to measure the absorbance at 475nm, and calculate the activity.

[0073] See also Figure 8 , Figure 8 Schematic diagram of pH stability of tyrosinase in yeast. Figure 8 The results show that the yeast tyrosinase can remain stable in the pH 6, 7, 8, 9, and 10 environments for 0 to 6 hours, and has the highest activity at pH 9 to 10. However, after 6 hours, the tyrosinase activity in each pH environment shows a downward trend.

[0074] Test Example 8

[0075] Effect of different temperatures on the stability of tyrosinase

[0076] Take 0.01 g of freeze-dried yeast powder, reconstitute it with buffer solution pH 7.5 to a final concentration of 1 mg / mL, place it at 30℃, 37℃, 40℃, 50℃, and 60℃, and take samples at fixed time points to detect activity. Experimental method: take 100μL of bacterial solution and add 900μL, 2.5mM L-DOPA, use a spectrophotometer to measure the absorbance at 475nm, and calculate the activity.

[0077] See also Fig. 9 , Fig. 9 Schematic diagram of the temperature stability of tyrosinase in yeast. Fig. 9 The results show that the tyrosinase of yeast decreases slowly within 6 hours at 30℃, 37℃, 40℃, 50℃ and 60℃ respectively. It has higher activity at 40℃ and 50℃ and is relatively stable at 50℃. However, after 6 hours, the enzyme activity tends to decrease rapidly at each temperature.

[0078] Based on the above experiments on optimizing the cultivation method of yeast, a single colony was randomly selected from the successfully transformed yeast colonies and inoculated into 5mL YPD medium (20g / L peptone; 10g / L yeast extract; 20g / L glucose; 1000μL / ml zeocin (bleomycin)), and placed in a 30℃ incubator and cultured overnight at 250rpm. Take 1mL of bacterial liquid and inoculate it into a 500mL conical flask with 50mL YPD medium (containing 20g / L peptone; 10g / L yeast extract; 60g / L glucose; 1000μL / ml zeocin (bleomycin), 1mM copper sulfate), place it in a pH 6, 20℃ incubator, and culture it at 250rpm for 4 days to obtain the best tyrosinase activity. The tyrosinase of yeast in this experiment has better tyrosinase activity at pH 9-10 and temperature 50℃.

[0079] Evaluation test of 3'-hydroxygenistein converted by yeast fermentation

[0080] In the method of yeast fermentation conversion of 3'-hydroxygenistein, the conversion reaction is carried out in an environment of pH 9, and the temperature is controlled at 50°C. Under these conditions, the yeast fermentation conversion of 3'-hydroxygenistein is carried out.

[0081] Example 1

[0082] The high performance liquid chromatography (HPLC) analysis method of 3'-hydroxygenistein and genistein was established. The instrument used was a high performance liquid chromatography (LC-2040C 3D, SHIMADZU), and the corresponding column used was a high performance liquid chromatography column (Kinetex 2.6um polar C18, 100x4.6mm). The analysis conditions are shown in Table 1 below.

[0083] Table 1

[0084]

[0085] 0.0100 g of 3'-hydroxygenistein standard and genistein standard were weighed accurately and diluted with DMSO (dimethyl sulfoxide) to 1 mL as the highest stock solution of 100 ppm. Appropriate volumes were then diluted with methanol to prepare 6.25, 12.5, 25, 50, and 100 ppm for analysis by HPLC.

[0086] See also Fig.10 , Fig.10 Schematic diagram of HPLC chromatography of 3'-hydroxygenistein standard and genistein standard. Fig.10 The results show that the retention time (RT) of genistein is 2.8 minutes (min), while the retention time of 3'-hydroxygenistein is 2.6 minutes (min). These two data can be used to determine the efficiency of converting genistein to 3'-hydroxygenistein through yeast fermentation.

[0087] Example 2

[0088] The determination steps of genistein conversion amount are as follows:

[0089] 1. Take 0.1 mL of the bioconversion reaction solution before the reaction (0th minute) and during the reaction, dilute to 1 mL with methanol, and use ultrasonic vibration for 3 minutes. Filter with a 0.22 μm filter membrane until clear, and analyze the sample absorbance (area) by HPLC.

[0090] 2. After the reaction, samples are taken at fixed time (such as 0, 40, 60, 80, 100, 120 min... etc.) to detect the conversion rate.

[0091] 3. Put the obtained absorbance value into the formula and calculate the conversion rate of genistein. The formula is as follows:

[0092] Conversion rate (%) = [(absorbance of sample before reaction - absorbance of sample after reaction) / absorbance of sample before reaction] × 100%

[0093] See also Fig.11 Fig.12 , Fig.11 Schematic diagram of HPLC chromatography for yeast conversion of 3'-hydroxygenistein. Fig.12 Schematic diagram of the reaction time for yeast to convert 3'-hydroxygenistein. Fig.11 and Fig.12 The results shown show that, according to HPLC detection, at the initial reaction time of 0 min, the retention peak of genistein is at 2.8 min, and after another 150 min of reaction, it can be seen that genistein is slowly converted into 3'-hydroxygenistein, and the retention time of 3'-hydroxygenistein is 2.6 min. Finally, after 300 min, the conversion rate can reach 90.91±2.63%. The specific conversion rate is shown in Table 2 below.

[0094] Table 2

[0095]

[0096]

[0097] Example 3

[0098] The determination steps of 3'-hydroxygenistein content in yeast bioconversion are as follows:

[0099] 1. Take 0.01g of sample, dilute to 10mL with methanol, and use ultrasonic vibration for 10 minutes to completely dissolve the sample until it is clear and transparent.

[0100] 2. Take 0.1mL of the sample diluted 10 times and dilute it to 1mL for sample measurement.

[0101] 3. Filter with a 0.22 μm filter membrane until clear and analyze the sample concentration by HPLC.

[0102] 4. Put the obtained concentration into the formula and calculate the 3'-hydroxygenistein content. The formula is as follows:

[0103] Content (%) = [(concentration obtained from calibration curve (μg / mL) × dilution volume (mL) × 10 -6 ) / weighted grams]×100%

[0104] See also Fig.13 , Fig.13 Schematic diagram of the conversion rate of yeast to 3'-hydroxygenistein. Fig.13 The results show that the conversion rate of genistein is 91.18% at 300 min, the purity is more than 25%, and the average yield is about 10 g. The specific purity and yield are shown in Table 3 below.

[0105] Table 3

[0106]

[0107] In summary, the preparation method of 3'-hydroxygenistein of the present invention uses genetically engineered microorganisms to convert 3'-hydroxygenistein, and its preparation method can be produced on a large scale in an industrialized manner. By controlling the production process such as the starting product and the biological conversion reaction conditions, the obtained 3'-hydroxygenistein has stable quality and high purity, thereby solving the problem of low productivity of 3'-hydroxygenistein. In addition, the obtained 3'-hydroxygenistein can be further prepared into a high-yield liquid raw material, and the prepared liquid raw material has high stability, so the 3'-hydroxygenistein prepared by the preparation method of 3'-hydroxygenistein provided by the present invention can be applied to cosmetic compositions, biopharmaceutical compositions, feed compositions, beverage compositions, nutritional supplement compositions, edible compositions, and health edible compositions, and has the potential to be applied to the biomedical health market.

[0108] Although the present invention has been described in detail through the above embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. Any person skilled in the art who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the definition of the claims. Sequence Listing <110> Jordanis International Co., Ltd. <120> Preparation method of 3'-hydroxygenistein <141> 2021-05-10 <150> 110103721 <151> 2021-02-01 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 996 <212> DNA <213> Artificial Sequence <220> <221> gene <223> Gene that expresses tyrosinase activity in yeast (Pichia pastoris) <400> 1 gaattcatgt ctaataaata cagagtccgt aagaatgtcc ttcatttgac cgatactgag 60 aaaagagact ttgtaagaac agtgctgatt ttgaaggaga agggaatcta tgatagatac 120 atcgcttggc atggcgcagc aggtaagttc catactcccc ctggttctga tagaaacgca 180 gctcacatgt cttcagcttt cctgccatgg catagagaat atttactgcg tttcgaaaga 240 gatttgcaat caattaaccc tgaggtcaca ctgccttat gggaatggga aactgatgca 300 caaatgcaag acccaagtca atctcaaatt tggtctgcag actttatggg tggtaatgga 360 aacccaataa aggacttcat agtggatacc ggtccatttg ctgcaggacg ttggacaacc 420 attgatgaac aaggcaatcc ttccggtggt ttgaagagaa actttggtgc aaccaaggaa 480 gcccctactt taccaacaag agatgatgtg ctaaatgcac ttaaaataac ccaatacgac 540 acacctccct gggacatgac cagtcaaaac tctttcagga atcagttgga gggctttatt 600 aatggtccac aactgcacaa tcgtgttcat aggtgggttg gaggccaaat gggagtggtt 660 ccaaccgccc caaatgaccc tgttttcttt cttcaccacg caaacgtgga tagaatttgg 720 gctgtgtggc aaatcgtgca ccgaaaccag aattaccagc ctatgaaaaa cggaccattc 780 ggacaaaatt tcagagaccc aatgtaccca tggaacacta cccctgaaga cgttatgaat 840 catagaaagc ttggctacgt gtatgacatt gaacttcgta aatcaaagag aagttcggta 900 cctcgagccg cggcggccgc cagctttcta gaacaaaaac tcatctcaga agaggatctg 960 aatagcgccg tcgaccatca tcatcatcat cattga 996

Claims

1. A method for preparing 3'-hydroxygenistein, characterized in that: The preparation method comprises: Constructing a genetically recombinant microorganism by transforming a circular recombinant plasmid into a microbial expression system to obtain the genetically recombinant microorganism, wherein the circular recombinant plasmid comprises a gene that can express tyrosinase activity as shown in sequence identification number 1; performing a reaction matrix preparation step, wherein the reaction matrix comprises borate at a final concentration of 400 mM to 600 mM, ascorbic acid at a final concentration of 10 mM to 40 mM, and genistein at a final concentration of 500 ppm to 3000 ppm; The bioconversion reaction step includes inoculating the genetically recombinant microorganism in the reaction matrix, and performing the bioconversion reaction at a temperature of 50° C., a bioconversion reaction aeration of 2 L / min to 5 L / min, a bioconversion reaction stirring speed of 200 rpm to 400 rpm, and a bioconversion reaction time of 300 minutes to obtain a bioconversion substance. as well as The step of performing a termination reaction is as follows: adding a hydrochloric acid solution to the bioconversion substance, and performing a termination reaction at a termination reaction temperature and a termination ventilation volume and a termination stirring speed for a termination reaction time to obtain a reaction solution, wherein the reaction solution contains 3'-hydroxygenistein, wherein The microbial expression system is Pichia pastoris; and The genetically recombinant microorganism has better tyrosinase activity under the condition of pH 9.

2. The method for preparing 3'-hydroxygenistein according to claim 1, characterized in that: The reaction matrix preparation step comprises: dissolving genistein in dimethyl sulfoxide to obtain a genistein solution; dissolving ascorbic acid in water to obtain an ascorbic acid solution; After dissolving the borate in hot water, the pH value is adjusted to 8 to 10 to obtain a borate solution; as well as The genistein solution and the ascorbic acid solution are added to the borate solution to obtain the reaction matrix.

3. The method for preparing 3'-hydroxygenistein according to claim 1, characterized in that: In the termination reaction step: the final concentration of the hydrochloric acid solution is 100mM to 200mM, the termination reaction temperature is 40°C to 60°C, the termination reaction ventilation volume is 2L / min to 5L / min, the termination reaction stirring speed is 200rpm to 400rpm, and the termination reaction time is 5 minutes to 15 minutes.

4. The method for preparing 3'-hydroxygenistein according to claim 1, characterized in that: The method may further include an extraction step: adding ethyl acetate to the reaction solution, and repeatedly shaking the reaction solution at a shaking speed for a shaking time 2 to 3 times.

5. The method for preparing 3'-hydroxygenistein according to claim 4, characterized in that: The volume ratio of the reaction solution to the ethyl acetate is 1:1 to 1.5:

1.

6. The method for preparing 3'-hydroxygenistein according to claim 4, characterized in that: The shaking speed is 2000 rpm to 3000 rpm, and the shaking time is 5 minutes to 20 minutes.

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