Method for synthesizing R-2-(4-hydroxyphenoxy) propionic acid through submerged culture of beauveria bassiana

Abstract

The invention discloses a method for synthesizing R-2-(4-hydroxyphenoxy) propionic acid by submerged culture of beauveria bassiana, and provides a submerged culture method of R-2-(4-hydroxyphenoxy) propionic acid, which comprises the following steps: adding soybean oil and a phase transfer catalyst; the phase transfer catalyst is prepared from one or more of cyclodextrin, tetrabutylammonium bromide, 1-butyl-3-methylimidazolium hexafluorophosphate and polyethylene glycol 1000. By adopting a supplementary material synergistic method, the yield of the R-2-(4-hydroxyphenoxy) propionic acid produced by using the beauveria bassiana ZJB23323 as a production strain through deep culture is remarkably improved.

Description

Technical Field

[0001] The invention relates to a method for synthesizing R-2-(4-hydroxyphenoxy) propionic acid by submerged culture of Beauveria bassiana. Background Art

[0002] In agricultural and forestry weed control, aryloxyphenoxypropionic acid herbicides have been increasing their market share annually due to their high herbicidal activity, broad spectrum, biodegradability, and crop safety. R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA), a key intermediate in the synthesis of phenoxy herbicides, enjoys significant market demand.

[0003] Currently, chemical methods are the primary method for industrial production of R-HPPA. However, these processes involve multiple reaction steps, long cycle times, demanding reaction conditions, numerous byproducts, significant environmental pollution, and low yields. Compared to chemical methods, biocatalytic methods offer advantages such as high stereoselectivity, mild reaction conditions, environmental friendliness, high product purity, and easy product separation and extraction. For example, Beauveria bassiana can catalyze the synthesis of R-2-phenoxypropionic acid (R-PPA) into R-HPPA. This approach meets national energy conservation and emission reduction requirements. Therefore, achieving industrialized biocatalytic production of R-HPPA is of great significance.

[0004] The main production modes in China for synthesizing R-HPPA from R-PPA using the biocatalytic activity of Beauveria bassiana are solid-state culture or liquid static culture, with the maximum yield of liquid static culture reaching 100 g / L. However, in existing fermentation companies, the use of liquid static culture requires specific industrial equipment, and the small height-to-diameter ratio of the equipment will also reduce land utilization, which will greatly increase the company's production costs. Compared with liquid static culture, deep culture, as the main production mode of fermentation companies, does not require the production of specific industrial equipment, which can greatly reduce production costs. However, the existing technology uses deep liquid culture of Beauveria bassiana to produce R-HPPA with low yields. How to provide a method for producing R-HPPA using deep culture of Beauveria bassiana is a technical problem that needs to be solved. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana, characterized in that the method is as follows:

[0006] (1) Beauveria bassiana ZJB23323 was inoculated into a fermentation medium as a production strain to obtain a culture solution containing a high-density bacterial cell of Beauveria bassiana ZJB23323, wherein the fermentation medium was composed of the following components at a final concentration: glucose 20-60 g / L, yeast powder 10-40 g / L, MgSO4 0.5-1.5 g / L, CaCl2

[0007] 0.55-0.95 g / L, K2HPO4 1.5-2.1 g / L, KH2PO4 0.45-1.05 g / L, trace elements 88-250 mg / L, the solvent is water, and the pH is 4.2-9.4; when preparing the fermentation medium, first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes, and then add the trace elements after cooling.

[0008] (2) adding a phase transfer catalyst to the culture solution containing the high-density bacterial cells of Beauveria bassiana ZJB23323 to a final concentration of 5-20 g / L, continuing the fermentation culture at 25-30° C. and 200-300 rpm, and adding vegetable oil and substrate R-PPA once on the 1st to 2nd day, 3rd to 4th day, 5th to 6th day, 7th to 8th day, and 9th to 10th day, respectively, with the amount of vegetable oil added each time being a final concentration of 5-20 g / L, and the amount of R-PPA added each time being a final concentration of 5-20 g / L, and the fermentation culture being carried out for 10-16 days to obtain a fermentation liquid;

[0009] (3) Separating and purifying the fermentation broth in step (2) to obtain R-HPPA.

[0010] Furthermore, the culture method described in step (1) is as follows: a) inoculating Beauveria bassiana ZJB23323 into a plate culture medium, culturing at 28°C for 7 to 13 days to obtain a single colony of Beauveria bassiana ZJB23323, wherein the plate culture medium is composed of the following components at final concentrations: R-PPA 30 to 70 g / L, glucose 10 to 30 g / L, yeast powder 5 to 15 g / L, MgSO4 0.9 to 1.1 g / L, CaCl2 0.65 to 0.85 g / L, K2HPO4 1.7 to 1.9 g / L, KH2PO4 0.65 to 0.85 g / L, agar powder 10 to 40 g / L, trace elements 88 to 250 mg / L, the solvent is water, and the pH is 6.8; when preparing the plate culture medium: first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes n after cooling, adding trace elements; b) inoculating the single colony of Beauveria bassiana ZJB23323 into a seed culture medium at 25-30° C. and 200-300 rpm for 60-96 hours to obtain a seed solution, wherein the final concentration of the seed culture medium is composed of R-PPA 10-30 g / L, glucose 10-30 g / L, yeast powder 5-15 g / L, MgSO4 0.9-1.1 g / L, CaCl2 0.65-0.85 g / L, K2HPO4 1.7-1.9 g / L, KH2PO4 0.65-0.85 g / L, and trace elements 88-250 mg / L, the solvent is water, and the pH is 6.8; when preparing the seed culture medium, first dissolve the components other than the trace elements in water, sterilize at 115° C. for 30 minutes, and then add the trace elements after cooling.

[0011] Furthermore, the seed liquid in step (1) is inoculated into the fermentation medium at a volume inoculation rate of 5-25%, and cultured at 25-30°C and 200-300 rpm for 2-4 days.

[0012] Furthermore, the separation and purification step described in step (3) is as follows: adjusting the pH of the fermentation broth to 5.5, centrifuging, filtering the first supernatant with a 0.22 μm nanofiltration membrane, heating the obtained filtrate to 75° C., adding activated carbon with a final concentration of 3 g / L, stirring for 40 minutes for decolorization, centrifuging, and concentrating the obtained second supernatant by rotary evaporation to 20-30% of the original volume. The obtained concentrate is extracted with ethyl acetate and evaporated to dryness at 120° C. to obtain R-2-(4-hydroxyphenoxy)propionic acid.

[0013] Furthermore, the vegetable oil in step (2) is one or more of soybean oil, peanut oil, rapeseed oil, olive oil, palm oil or sesame oil.

[0014] Furthermore, the phase transfer catalyst in step (2) is one or more of cyclodextrin, tetrabutylammonium bromide, 1-butyl-3-methylimidazolium hexafluorophosphate or polyethylene glycol 1000.

[0015] Furthermore, the trace elements in the fermentation medium, plate medium or seed medium are composed of the following final concentration components: EDTA-2Na·2H2O 50-150 mg / L, FeSO4·7H2O 20-40 mg / L, ZnSO4·7H2O 5-15 mg / L, MnSO4·H2O 5-15 mg / L, NiCl·6H2O 1-5 mg / L, CoCl2 5-15 mg / L, H3BO3 1-5 mg / L and Na2MoO4·H2O 1-5 mg / L.

[0016] Further, the method for synthesizing R-2-(4-hydroxyphenoxy) propionic acid by submerged culture of Beauveria bassiana is as follows:

[0017] (1) Inoculating Beauveria bassiana ZJB23323 into a plate culture medium and culturing at 28° C. for 9 to 11 days to obtain a single colony of Beauveria bassiana ZJB23323; the plate culture medium contains the following components at final concentrations: R-PPA 30 to 70 g / L, glucose 10 to 30 g / L, yeast powder 5 to 15 g / L, MgSO4 0.9 to 1.1 g / L, CaCl2 0.65 to 0.85 g / L, K2HP O41.7~1.9g / L, KH2PO40.65~0.85g / L, agar powder 10~40g / L, trace elements 120~200mg / L, solvent is water, pH 6.8; when preparing the plate culture, first dissolve the components except the trace elements in water, sterilize at 115℃ for 30min, and then add the trace elements; the final concentration of the trace elements in the plate culture medium is: EDTA-2Na·2H2O 75~125mg / L, FeSO4·7H2O 25~35mg / L, ZnSO4·7H2O 5~10mg / L, MnSO4·H2O 5~10mg / L, NiCl·6H2O 2~4mg / L, CoCl25~10mg / L, H3BO31~3mg / L and Na2MoO4·H2O2~3mg / L;

[0018] (2) A single colony of Beauveria bassiana ZJB23323 was inoculated into a seed culture medium and cultured at 26-29°C and 220-280 rpm for 65-90 hours to obtain a seed solution, wherein the seed culture medium is composed of the following components at final concentrations: R-PPA 10-20 g / L, glucose 20-25 g / L, yeast powder 5-15 g / L, MgSO4 0.9-1.1 g / L, CaCl2 0.65-0.85 g / L, K2HPO4 1.7-1.9 g / L, KH2PO4 0.65-0.85 g / L, and trace elements 120-200 mg / L, and the solvent is water with a pH of 6.8. When preparing the seed culture medium, the components except the trace elements were first dissolved in water, sterilized at 115°C for 30 minutes, and then the trace elements were added. The final concentration of the trace elements in the seed culture medium is: EDTA-2Na·2H2O 75~125mg / L, FeSO4·7H2O25~35mg / L, ZnSO4·7H2O 5~10mg / L, MnSO4·H2O 5~10mg / L, NiCl·6H2O2~4mg / L, CoCl25~10mg / L, H3BO31~3mg / L and Na2MoO4·H2O 2~3mg / L;

[0019] (3) The seed liquid is inoculated into the fermentation medium at a volume inoculation rate of 5-20%, and deep culture is carried out at 26-29° C. and 220-280 rpm for 2-3 days to obtain a culture solution containing high-density bacteria, wherein the fermentation medium is composed of the following components at final concentrations: glucose 30-50 g / L, yeast powder 20-30 g / L, MgSO4 0.8-1.2 g / L, CaCl2 0.65-0.8 g / L, K2HPO4 1.6-2.0 g / L, KH2PO4 0.6-0.8 g / L, trace elements 120-200 mg / L, the solvent is water, and the pH is 6.5-7.5; when preparing the fermentation medium, first dissolve the components except the trace elements in water, sterilize at 115° C. for 30 minutes, and then add the trace elements after cooling; the final concentration of the trace elements in the fermentation medium is: EDTA-2Na·2H2O 75~125mg / L, FeSO4·7H2O25~35mg / L, ZnSO4·7H2O 5~10mg / L, MnSO4·H2O 5~10mg / L, NiCl·6H2O2~4mg / L, CoCl25~10mg / L, H3BO31~3mg / L and Na2MoO4·H2O 2~3mg / L;

[0020] (4) adding a phase transfer catalyst to the culture medium of the high-density bacteria to a final concentration of 5-15 g / L, and adding vegetable oil and substrate R-PPA once on days 1-2, 3-4, 5-6, 7-8, and 9-10, respectively, with the amount of vegetable oil added each time being a final concentration of 5-20 g / L, and the amount of R-PPA added each time being a final concentration of 5-15 g / L, culturing and fermenting at 26-29° C. and 220-280 rpm for 11-14 days to obtain a fermentation broth, wherein the phase transfer catalyst is cyclodextrin or tetrabutylammonium bromide, and the vegetable oil is soybean oil or peanut oil;

[0021] (5) The fermentation broth was adjusted to pH 5.5 and centrifuged. The first supernatant obtained was filtered with a 0.22 μm nanofiltration membrane. The filtrate was heated to 75°C, activated carbon with a final concentration of 3 g / L was added, and the mixture was stirred for 40 min for decolorization. The second supernatant obtained was concentrated by rotary evaporation to 20-30% of the original volume. The concentrated solution was extracted with ethyl acetate and evaporated to dryness at 120°C to obtain R-HPPA.

[0022] The present invention provides a submerged culture method for R-HPPA. By adding soybean oil and a phase transfer catalyst and adopting a fed-batch synergistic method during the fermentation process, the yield of R-HPPA produced by submerged culture using Beauveria bassiana ZJB23323 as a production strain is significantly increased. The method is also suitable for industrial production scale expansion and has the advantages of simple and stable production process. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effect of single addition of different vegetable oils on R-HPPA yield when R-PPA was added once without adding phase transfer catalyst

[0024] Figure 2 Effect of adding different amounts of soybean oil on the yield of R-HPPA when no phase transfer catalyst was added and R-PPA was added once

[0025] Figure 3 Effects of single addition of different phase transfer catalysts on R-HP PA yield when no vegetable oil is added and R-PPA is added once

[0026] Figure 4 Effects of adding different phase transfer catalysts on R-HPPA yield under single addition of soybean oil and R-PPA Figure 5 Effect of adding soybean oil in batches on the yield of R-HPPA when R-PPA is added once without adding phase transfer catalyst

[0027] Figure 6 Effect of adding R-PPA in batches on the yield of R-HPPA when soybean oil is added once without adding phase transfer catalyst

[0028] Figure 7 The effect of adding soybean oil and phase transfer catalyst on the yield of R-HPPA was investigated by using a fed-batch synergistic method. DETAILED DESCRIPTION

[0029] To make the above-mentioned objectives, features, and advantages of the present invention more readily apparent, the following detailed description of the specific embodiments of the present invention is provided in conjunction with specific examples. However, it should be noted that the numerous specific details set forth in the following description are provided solely for a thorough understanding of the present invention and do not represent the complete technical approach underlying the present invention. Therefore, they should not be construed as limiting the overall technical solution of the present invention.

[0030] Comparative Example 1: Effect of single addition of different vegetable oils on R-HPPA yield when no phase transfer catalyst is added and R-PPA is added once

[0031] Experimental Materials:

[0032] Plate culture medium: R-PPA 50 g / L, glucose 20 g / L, yeast powder 10 g / L, MgSO4 0.977 g / L, CaCl2 0.755 g / L, K2HPO4 1.8 g / L, KH2PO4 0.75 g / L, agar powder 20 g / L, trace elements 165 mg / L, solvent is water, pH 6.8. When preparing the plate culture medium, first dissolve the ingredients except the trace elements in water, sterilize at 115 ° C for 30 minutes, cool, and then add the trace elements. The final concentrations of trace elements in the plate culture medium are: EDTA-2Na·2H2O 100 mg / L, FeSO4·7H2O 30 mg / L, ZnSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, NiCl·6H2O 3 mg / L, CoCl2 8 mg / L, H3BO3 2 mg / L, Na2MoO4·H2O 2 mg / L;

[0033] Seed culture medium: 20 g / L R-PPA, 20 g / L glucose, 10 g / L yeast extract, 0.977 g / L MgSO₄, 0.755 g / L CaCl₂, 1.8 g / L K₂HPO₄, 0.75 g / L KH₂PO₄, 165 mg / L trace elements, water, pH 6.8. To prepare the seed culture medium, first dissolve all ingredients except the trace elements in water, sterilize at 115°C for 30 min, and then cool before adding the trace elements. The final concentrations of trace elements in the seed culture medium are: EDTA-2Na·2H2O 100 mg / L, FeSO4·7H2O 30 mg / L, ZnSO4·7H2O 10 mg / L, MnSO4·H2O 10 mg / L, NiCl·6H2O 3 mg / L, CoCl2 8 mg / L, H3BO3 2 mg / L, Na2MoO4·H2O 2 mg / L;

[0034] Fermentation medium: glucose 40g / L, yeast powder 10g / L, MgSO4 0.977g / L, CaCl2 0.755g / L, K2HPO4 1.8g / L, KH2PO4 0.75g / L, trace elements 165mg / L, solvent is water, pH 6.8. When preparing the fermentation medium, first dissolve the ingredients except the trace elements in water, sterilize at 115°C for 30 minutes, and then add the trace elements after cooling. The final concentration of trace elements in the fermentation medium is: EDTA-2Na·2H2O 100mg / L, FeSO4·7H2O 30mg / L, ZnSO4·7H2O 10mg / L, MnSO4·H2O 10mg / L, NiCl·6H2O 3mg / L, CoCl2 8mg / L, H3BO 32mg / L, Na2MoO4·H2O 2mg / L;

[0035] The above experimental materials are all commercially available products.

[0036] Beauveria bassiana ZJB23323 used in the embodiments of the present invention is deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 2023584, a deposit date of April 21, 2023, and an address of Wuhan University, Wuhan, China, 430072.

[0037] The production of R-HPPA by Beauveria bassiana ZJB23323 is achieved through submerged liquid culture. The specific operation is as follows:

[0038] Step 1: inoculate Beauveria bassiana ZJB23323 onto a plate culture medium and culture at 28°C for 10 days to obtain a single colony;

[0039] Step 2: Inoculate the single colony in step 1 into the seed culture medium, and culture at 250 rpm at 28°C for 72 hours to obtain the seed solution.

[0040] Step 3: 5 ml of the seed solution from step 2 was added to 45 ml of fermentation medium, and submerged culture was performed at 28° C. and 250 rpm for 3 days to obtain a culture solution of high-density bacteria (high-density bacteria of Beauveria bassiana ZJB23323).

[0041] Step 4: Add 50 g / L R-PPA and 40 g / L vegetable oil to the culture medium obtained in step 3. Set up six parallel control experiments, and the added vegetable oils are soybean oil, peanut oil, rapeseed oil, olive oil, palm oil, and sesame oil. The blank control is to add 40 g / L glucose instead of vegetable oil. Fermentation is carried out at 28°C and 250 rpm for 12 days. After the fermentation is completed, the R-HPPA yield in the fermentation broth is measured.

[0042] The fermentation broth was adjusted to pH 5.5 and centrifuged. The obtained supernatant was filtered through a 0.22 μm nanofiltration membrane. The obtained filtrate was heated to 75° C., activated carbon with a final concentration of 3 g / L was added, and the mixture was stirred for 40 minutes for decolorization. The obtained supernatant was concentrated by rotary evaporation to 25% of the original volume. The obtained concentrate was extracted with ethyl acetate and evaporated to dryness at 120° C. to obtain R-2-(4-hydroxyphenoxy)propionic acid.

[0043] The R-HPPA content in the fermentation broth was determined using HPLC. Sample pretreatment: 1 ml of fermentation broth was placed in a 1.5 ml centrifuge tube and centrifuged at 14,000 rpm for 2 minutes. The precipitate was discarded and the supernatant was collected. The supernatant was filtered through a 0.22 μm microfiltration membrane and analyzed using a Thermo Fisher HPLC. HPLC mobile phase preparation: Aqueous phosphoric acid was prepared to pH 2, then mixed with acetonitrile in a 3:2 ratio, shaken, filtered through the membrane, and ultrasonically degassed for 15 minutes. HPLC analysis conditions: Column: C18, column temperature: 30°C, flow rate: 1 ml / min, detection wavelength: 220 nm, injection volume: 5 μL.

[0044] The final corresponding R-HPPA yields were 8.23 g / L, 17.17 g / L, 15.01 g / L, 6.32 g / L, 13.15 g / L, 11.66 g / L, and 5.47 g / L, respectively. Figure 1 As shown. Figure 1 It can be seen that the addition of soybean oil and peanut oil has the greatest effect on the yield of R-HPPA, among which the addition of soybean oil has the highest R-HPPA yield.

[0045] Comparative Example 2: Effect of adding different amounts of soybean oil on the yield of R-HPPA when no phase transfer catalyst is added and R-PPA is added once

[0046] 50 g / L R-PPA and soybean oil were added to the culture solution obtained in step 3 of Comparative Example 1. Six parallel tests were conducted, with the amounts of soybean oil being 0, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g, and 60 g / L, respectively. The culture and fermentation were carried out at 28°C and 250 rpm for 12 days. After the fermentation, the R-HPPA yield in the fermentation solution was determined according to the method in Comparative Example 1. The final corresponding R-HPPA yields were 8.12 g / L, 10.62 g / L, 12.94 g / L, 14.62 g / L, 16.68 g / L, 18.67 g / L, and 16.62 g / L, respectively. The results are shown in FIG. Figure 2 As shown, from Figure 2 It can be seen that when no phase transfer catalyst was added and 50 g / L soybean oil was added once, the yield of R-HPPA was the highest.

[0047] Comparative Example 3: Effect of adding different phase transfer catalysts on R-HPPA yield when no vegetable oil is added and R-PPA is added once:

[0048] To the culture solution obtained in step 3 of Comparative Example 1, 50 g / L R-PPA and 5 g / L of phase transfer catalyst were added, which were cyclodextrin, tetrabutylammonium bromide, 1-butyl-3-methylimidazolium hexafluorophosphate, and polyethylene glycol 1000, respectively. The culture was cultured and fermented at 28 ° C and 250 rpm for 12 days. After the fermentation, the R-HPPA yield in the fermentation broth was measured. The final corresponding R-HPPA yields were 13.06 g / L, 10.95 g / L, 3.44 g / L, and 8.06 g / L, respectively. The results are as follows Figure 3 As shown. Figure 3 It can be seen that the addition of cyclodextrin and tetrabutylammonium bromide promotes the increase in R-HPPA production, among which the addition of cyclodextrin has the highest R-HPPA production.

[0049] Comparative Example 4: Effect of adding different phase transfer catalysts on R-HPP A yield under the condition of single addition of soybean oil and R-PPA:

[0050] To the culture broth obtained in step 3 of Comparative Example 1, 50 g / L R-PPA and 40 g / L soybean oil were added, along with various phase transfer catalysts, with final concentrations of 0, 5 g / L, 10 g / L, 15 g / L, and 20 g / L, respectively. A blank control group consisted of only 45 ml of fermentation medium. After fermentation with 50 g / L R-PPA, the R-HPPA yield in the fermentation broth was measured. Fermentation was carried out at 28°C and 250 rpm for 12 days, and the R-HPPA yield in the fermentation broth was measured after fermentation.

[0051] See Table 1.

[0052] Experimental results:

[0053] Table 1: Effect of adding different phase transfer catalysts simultaneously with soybean oil on R-HPPA yield (g / L)

[0054]

[0055]

[0056] The results are shown in Table 1 and Figure 4 , from Table 1 and Figure 4 The results show that the addition of a phase transfer catalyst simultaneously with soybean oil affects R-HPPA yield. The addition of cyclodextrin and tetrabutylammonium bromide significantly increases R-HPPA yield. The highest R-HPPA yield was achieved when 10 g / L tetrabutylammonium bromide was added.

[0057] Comparative Example 5: Effect of adding soybean oil in batches on R-HPPA yield when no phase transfer catalyst is added and R-PPA is added once

[0058] R-PPA 50 g / L was added to the culture solution obtained in step 3 of Comparative Example 1, and soybean oil was added in batches, 10 g / L was added each time on the 1st, 3rd, 5th, 7th, and 9th days, 12.5 g / L was added each time on the 1st, 3rd, 5th, and 7th days, 16.7 g / L was added each time on the 1st, 3rd, and 5th days, and 25 g / L was added each time on the 1st and 3rd days. The blank control group was added with 50 g / L of glucose on the 1st day. ， The fermentation was carried out at 28°C and 250 rpm for 12 days, and the R-HPPA yield in the fermentation broth was measured. The final corresponding R-HPPA yields were 31.34 g / L, 25.33 g / L, 18.32 g / L, 12.45 g / L, and 11.52 g / L, respectively. Figure 5 As shown. Figure 5 It can be seen that the yield of R-HPPA is the highest when soybean oil is added in batches at an addition rate of 10 g / L every 2 days.

[0059] Comparative Example 6: Effect of adding R-PPA in batches on R-HPPA yield when soybean oil is added once without adding a phase transfer catalyst

[0060] R-PPA 50 g / L was added to the culture solution obtained in step 3 of Comparative Example 1 in batches, and 10 g / L was added each time on the 1st, 3rd, 5th, 7th, and 9th days, 12.5 g / L was added each time on the 1st, 3rd, 5th, and 7th days, 16.7 g / L was added each time on the 1st, 3rd, and 5th days, and 25 g / L was added each time on the 1st and 3rd days. The blank control group was R-PPA 50 g / L added on the 1st day. ， The fermentation was carried out at 28°C and 250 rpm for 12 days, and the R-HPPA yield in the fermentation broth was measured. The final corresponding R-HPPA yields were 21.86 g / L, 18.53 g / L, 11.29 g / L, 7.60 g / L, and 8.23 g / L, respectively. Figure 6 As shown. Figure 6 It can be seen that the yield of R-HPPA is the highest when the substrate is added in batches at an addition rate of 10 g / L every 2 days.

[0061] Example 1: Effect of adding soybean oil and phase transfer catalyst on R-HPPA yield using a fed-batch synergistic approach

[0062] Take the culture solution obtained in step 3 of Comparative Example 1, add 20g / L R-PPA to the culture solution on days 1, 3, 5, 7, and 9, add 10g / L tetrabutylammonium bromide on day 1, and add 10g / L soybean oil to the high-density cells on days 1, 3, 5, 7, and 9. After 14 days of culture and fermentation, the R-HP PA production in the fermentation solution was measured. The results are as follows: Figure 7 As shown. Figure 7It can be seen that the final yield of R-HPPA reached 100 g / L.

[0063] In summary, the present invention can significantly increase the yield of R-HPPA synthesized by submerged culture of Beauveria bassiana by adding a phase transfer catalyst, adding soybean oil in batches, and adding the substrate R-PPA in batches during the fermentation conversion process.

Claims

1. A method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana, characterized in that: The method is as follows: (1) Beauveria bassiana ZJB23323 was inoculated into a fermentation medium as a production strain to obtain a culture solution containing a high-density bacterial cell of Beauveria bassiana ZJB23323, wherein the fermentation medium was composed of the following components at final concentrations: glucose 20-60 g / L, yeast powder 10-40 g / L, MgSO4 0.5-1.5 g / L, CaCl2 0.55-0.95 g / L, K2HPO4 1.5-2.1 g / L, KH2PO4 0.45-1.05 g / L, trace elements 88-250 mg / L, the solvent is water, pH 4.2-9.4; when preparing the fermentation medium, first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes, and then add the trace elements after cooling. (2) adding a phase transfer catalyst to the culture solution containing the high-density bacterial cells of Beauveria bassiana ZJB23323 to a final concentration of 5-20 g / L, continuing the fermentation culture at 25-30° C. and 200-300 rpm, and adding vegetable oil and substrate R-2-phenoxypropionic acid once on the 1st to 2nd day, 3rd to 4th day, 5th to 6th day, 7th to 8th day, and 9th to 10th day, respectively, with the amount of vegetable oil added each time being a final concentration of 5-20 g / L, and the amount of R-2-phenoxypropionic acid added each time being a final concentration of 5-20 g / L, and the fermentation culture being carried out for 10-16 days to obtain a fermentation liquid; (3) separating and purifying the fermentation broth in step (2) to obtain R-2-(4-hydroxyphenoxy)propionic acid.

2. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana according to claim 1, wherein: The culture method of step (1) comprises the following steps: a) inoculating Beauveria bassiana ZJB23323 onto a plate culture medium, culturing at 28° C. for 7 to 13 days, and obtaining a single colony of Beauveria bassiana ZJB23323, wherein the plate culture medium comprises the following components at final concentrations: 30 to 70 g / L of R-2-phenoxypropionic acid, 10 to 30 g / L of glucose, 5 to 15 g / L of yeast powder, 0.9 to 1.1 g / L of MgSO4, 0.65 to 0.85 g / L of CaCl2, 1.7 to 1.9 g / L of K2HPO4, 0.65 to 0.85 g / L of KH2PO4, 10 to 40 g / L of agar powder, 88 to 250 mg / L of trace elements, and solvent. The plate culture medium is prepared by first dissolving the components except the trace elements in water, sterilizing at 115° C. for 30 minutes, cooling, and then adding the trace elements; b) inoculating a single colony of Beauveria bassiana ZJB23323 into a seed culture medium, and culturing at 25-30° C. and 200-300 rpm for 60-96 hours to obtain a seed solution, wherein the final concentration of the seed culture medium is 10-30 g / L of R-2-phenoxypropionic acid, 10-30 g / L of glucose, 5-15 g / L of yeast powder, 0.9-1.1 g / L of MgSO4, 0.65-0.85 g / L of CaCl2, 1.7-1.9 g / L of K2HPO4, and 1.7-2.9 g / L of KH2PO4. 0.65-0.85 g / L, trace elements 88-250 mg / L, solvent is water, pH 6.8; when preparing the seed culture medium, first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes, cool, and then add the trace elements.

3. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana according to claim 1, wherein: The seed liquid in step (1) is inoculated into the fermentation medium at a volume inoculation rate of 5-25%, and cultured at 25-30°C and 200-300 rpm for 2-4 days.

4. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana according to claim 1, wherein: The separation and purification step described in step (3) is as follows: adjusting the pH of the fermentation broth to 5.5, centrifuging, filtering the obtained first supernatant with a 0.22 μm nanofiltration membrane, heating the obtained filtrate to 75° C., adding activated carbon with a final concentration of 3 g / L, stirring for 40 minutes for decolorization, centrifuging, and concentrating the obtained second supernatant by rotary evaporation to 20-30% of the original volume. The obtained concentrate is extracted with ethyl acetate and evaporated to dryness at 120° C. to obtain R-2-(4-hydroxyphenoxy)propionic acid.

5. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana as claimed in claim 1, characterized in that: The vegetable oil in step (2) is one or more of soybean oil, peanut oil, rapeseed oil, olive oil, palm oil or sesame oil.

6. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana as claimed in claim 1, characterized in that The phase transfer catalyst in step (2) is one or more of cyclodextrin, tetrabutylammonium bromide, 1-butyl-3-methylimidazolium hexafluorophosphate or polyethylene glycol 1000.

7. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana according to claim 1 or 2, characterized in that The trace elements in the fermentation medium, plate medium or seed medium are composed of the following final concentration components: EDTA-2Na·2H2O 50-150 mg / L, FeSO4·7H2O 20-40 mg / L, ZnSO4·7H2O 5-15 mg / L, MnSO4·H2O 5-15 mg / L, NiCl·6H2O 1-5 mg / L, CoCl2 5-15 mg / L, H3BO3 1-5 mg / L and Na2MoO4·H2O 1-5 mg / L.

8. The method for synthesizing R-2-(4-hydroxyphenoxy)propionic acid by submerged culture of Beauveria bassiana as claimed in claim 1, characterized in that: The method is as follows: (1) Inoculating Beauveria bassiana ZJB23323 into a plate culture medium and culturing at 28°C for 9 to 11 days to obtain a single colony of Beauveria bassiana ZJB23323; the plate culture medium contains the following components at final concentrations: R-2-phenoxypropionic acid 30 to 70 g / L, glucose 10 to 30 g / L, yeast powder 5 to 15 g / L, MgSO4 0.9 to 1.1 g / L, CaCl2 0.65 to 0.85 g / L, K2 HPO4 1.7-1.9 g / L, KH2PO4 0.65-0.85 g / L, agar powder 10-40 g / L, trace elements 120-200 mg / L, solvent is water, pH 6.8; when preparing the plate culture, first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes, and then add the trace elements after cooling; the final concentration of the trace elements in the plate culture medium is: EDTA-2Na·2H2O 75~125mg / L, FeSO4·7H2O 25~35mg / L, ZnSO4·7H2O 5~10mg / L, MnSO4·H2O 5~10mg / L, NiCl·6H2O 2~4mg / L, CoCl25~10mg / L, H3BO31~3mg / L and Na2MoO4·H2O 2~3mg / L; (2) A single colony of Beauveria bassiana ZJB23323 was inoculated into a seed culture medium and cultured at 26-29°C and 220-280 rpm for 65-90 hours to obtain a seed solution, wherein the seed culture medium comprises the following components at final concentrations: R-2-phenoxypropionic acid 10-20 g / L, glucose 20-25 g / L, yeast powder 5-15 g / L, MgSO4 0.9-1.1 g / L, and CaCl2 0.65-0. 85g / L, K2HPO41.7-1.9g / L, KH2PO40.65-0.85g / L, trace elements 120-200mg / L, the solvent is water, pH 6.8; when preparing the seed culture medium, first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes, and then add the trace elements after cooling; the final concentration of the trace elements in the seed culture medium is: E; EDTA-2Na·2H2O 75-125mg / L, FeSO4·7H2O 25-35mg / L, ZnSO4·7H2O 5-10mg / L, MnSO4·H2O 5~10mg / L, NiCl·6H2O 2~4mg / L, CoCl25~10mg / L, H3BO31~3mg / L and Na2MoO4·H2O 2~3mg / L; (3) The seed liquid is inoculated into a fermentation medium at a volume inoculation rate of 5-20%, and deep culture is carried out at 26-29°C and 220-280 rpm for 2-3 days to obtain a culture medium containing high-density bacteria, wherein the fermentation medium is composed of the following components at final concentrations: glucose 30-50 g / L, yeast powder 20-30 g / L, MgSO4 0.8-1.2 g / L, CaCl2 0.65-0.8 g / L, K2HPO4 1.6-2.0 g / L, KH2PO4 0.6-0.8 g / L, trace elements 120-200 mg / L, the solvent is water, pH 6.5-7.5; when preparing the fermentation medium, first dissolve the components except the trace elements in water, sterilize at 115°C for 30 minutes, and then add the trace elements after cooling; the final concentration of the trace elements in the fermentation medium is: EDTA-2Na·2H2O 75-125 mg / L, FeSO4·7H2O 25~35mg / L, ZnSO4·7H2O 5~10mg / L, MnSO4·H2O 5~10mg / L, NiCl·6H2O 2~4mg / L, CoCl25~10mg / L, H3BO31~3mg / L and Na2MoO4·H2O2~3mg / L; (4) adding a phase transfer catalyst to the culture medium of the high-density bacteria to a final concentration of 5-15 g / L, and adding vegetable oil and substrate R-2-phenoxypropionic acid once on the 1st to 2nd day, 3rd to 4th day, 5th to 6th day, 7th to 8th day, and 9th to 10th day, respectively, the amount of the vegetable oil added each time is a final concentration of 5-20 g / L, the amount of the R-2-phenoxypropionic acid added each time is a final concentration of 5-15 g / L, and culturing and fermenting at 26-29° C. and 220-280 rpm for 11-14 days to obtain a fermentation broth, the phase transfer catalyst is cyclodextrin or tetrabutylammonium bromide, and the vegetable oil is soybean oil or peanut oil; (5) The fermentation broth was adjusted to pH 5.5 and centrifuged. The obtained first supernatant was filtered through a 0.22 μm nanofiltration membrane. The obtained filtrate was heated to 75° C., activated carbon with a final concentration of 3 g / L was added, and the mixture was stirred for 40 min for decolorization. The mixture was centrifuged and the obtained second supernatant was concentrated by rotary evaporation to 20-30% of the original volume. The obtained concentrate was extracted with ethyl acetate and evaporated to dryness at 120° C. to obtain R-2-(4-hydroxyphenoxy)propionic acid.

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