CN113881715A - Biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid - Google Patents
Biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid Download PDFInfo
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Abstract
The invention provides a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid (DHPPA), which comprises the following steps: inoculating a spore suspension of beauveria bassiana to a seed culture medium for culture to obtain a fermented seed solution; inoculating the strain into a solid fermentation culture medium for solid culture, monitoring the content of a precursor R- (+) -2-phenoxypropionic acid (PPA) in the solid fermentation culture medium, and controlling the concentration of the PPA in the solid fermentation culture medium to be within the range of 10-40 g/kg. According to the invention, the precursor is supplemented for many times, and the supplement is carried out according to the result feedback of PPA content detection by high performance liquid chromatography, so that the toxicity of PPA to beauveria bassiana is reduced, based on the solid fermentation of the PPA content feedback supplement, the yield of DHPPA can reach 70.53g/kg solid fermentation culture medium, and the conversion rate of the precursor PPA reaches 99.84%.
Description
Technical Field
The invention relates to the technical field of bioengineering, and particularly relates to a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid.
Background
R- (+) -2- (4-hydroxyphenoxy) propionic acid (DHPPA) is an important chiral intermediate for synthesizing 4- (aryloxyphenoxy) propionic acid herbicides, and is used for synthesizing herbicides such as fenoxaprop-p-ethyl, clodinafop-propargyl (for wheat fields), quizalofop-p-ethyl, cyhalofop-butyl (for paddy fields), metamifop-ethyl, fluazifop-p-butyl and the like, and the herbicides have a symmetrical structure and have a plurality of excellent characteristics such as low toxicity, high selectivity, safety to crops, easiness in degradation and the like, so that the research on the high-efficiency synthesis method of the DHPPA has important significance.
The chemical synthesis method for synthesizing DHPPA has the defects of difficult control of reaction process, high energy consumption, more byproducts, large environmental pollution and the like. The biological synthesis preparation of DHPPA has the advantages of mild reaction conditions, few reaction steps, convenient separation and extraction and the like, and is widely concerned in recent years. The biological synthesis preparation of DHPPA is to specifically convert R- (+) -2-phenoxypropionic acid into DHPPA by utilizing the microbial production of hydroxylating enzyme. The microorganisms which can hydroxylate R- (+) -2-phenoxypropionic acid at present are: beauveria bassiana, Aspergillus niger, Aspergillus flavus, streptomyces, paecilomyces farinosus and the like, wherein the Beauveria bassiana shows better catalytic performance in the process of biologically synthesizing DHPPA. Compared with liquid fermentation or biotransformation after liquid fermentation, the solid fermentation mode has the characteristics of low fermentation raw material cost, less wastewater discharge and capability of preventing filamentous bacteria from forming clusters to influence oxygen transfer in the fermentation process by using the solid fermentation raw material as a carrier of the filamentous bacteria. Although the solid fermentation DHPPA has a plurality of advantages, the key problem that high concentration of substrates or precursors commonly existing in the biocatalytic reaction has toxic effect on cells is still not solved, and the biosynthesis of DHPPA is influenced by high concentration of R- (+) -2-phenoxypropionic acid.
Therefore, it is urgently needed to develop a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid to overcome the toxicity problem of the precursor at high concentration in solid fermentation.
Disclosure of Invention
The invention aims to provide a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, which adopts precursor feedback supplement solid state fermentation to catalyze precursor R- (+) -2-phenoxypropionic acid (PPA) to synthesize R- (+) -2- (4-hydroxyphenoxy) propionic acid, abandons the continuous fed-batch mode according to the characteristics of solid fermentation, adopts the mode of supplementing the precursor for many times, and effectively controls the PPA concentration within the range of 10-40g/kg solid state fermentation culture medium according to the result feedback of PPA content detection by high performance liquid chromatography, thereby reducing the toxicity of PPA to cells.
The invention provides a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, which comprises the following steps:
inoculating Beauveria bassiana to a PDA slant surface for slant surface culture to obtain PDA slant surface spores;
washing off the PDA slant spores by using sterile water to prepare a spore suspension, and then inoculating the spore suspension into a seed culture medium for seed culture to obtain a fermented seed solution;
inoculating the fermentation seed liquid into a solid state fermentation culture medium for solid state culture, monitoring the content of the precursor PPA in the solid state fermentation culture medium, and controlling the concentration of the precursor PPA in the solid state fermentation culture medium to be in the range of 10-40g/kg by adding the precursor PPA into the solid state fermentation culture medium.
Further, when the precursor PPA is added, the precursor PPA is added in the form of a PPA solution having a pH of 7 to 12 and a concentration of 45 to 55% (m/V).
Further, the adding times of the precursor PPA are 1-6 times.
Furthermore, the addition amount of the precursor PPA is 10-20g/kg of solid fermentation medium each time.
Further, the monitoring of the content of the precursor PPA in the solid-state fermentation medium includes the following steps of adding the precursor PPA into the solid-state fermentation medium to control the concentration of the precursor PPA in the solid-state fermentation medium to be in the range of 10-40 g/kg:
sampling the solid fermentation medium every day, and detecting the concentration of the precursor PPA by high performance liquid chromatography;
when the concentration of the PPA precursor is lower than 10-40g/kg of solid fermentation medium, beginning to supplement PPA so as to control the concentration of the PPA precursor to be 10-40g/kg of solid fermentation medium;
and finally stopping feeding when the total PPA addition amount is 50-80g/kg of solid state fermentation medium.
Further, the slant culture conditions are as follows: the temperature is 27-29 deg.C, and the time is 3-7 days.
Further, the steps of washing the PDA slant spores with sterile water to obtain a spore suspension, inoculating the spore suspension into a seed culture medium for seed culture to obtain a fermented seed solution specifically include:
washing PDA slant spore with sterile water to obtain spore with amount of 107-109Inoculating 1-3% spore suspension into a seed culture medium by mass percent for seed culture to obtain a fermented seed liquid.
Further, the seed culture conditions are as follows: the temperature is 25-30 ℃, the rotating speed is 200-.
Further, the fermentation seed liquid is inoculated into a solid state fermentation culture medium according to the mass fraction of 5-10%, and the solid state culture conditions are as follows: the temperature is 25-30 deg.C, and the time is 5-21 d.
Further, the formula of the seed culture medium is as follows: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50ml/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L, and sterilizing at 121 ℃ for 20min after preparation.
Further, the formula of the solid state fermentation medium is as follows: 200-400g/kg of dry materials, wherein the dry materials comprise at least one of bran, rice hull, rice bran, corn grits and rice; 25-50ml/kg of trace element solution, and adding water to ensure that the ratio of material to water is 1: (1.5-2.5), adding 6-32ml of PPA solution, wherein the concentration of the PPA solution is 500g/L, and adjusting the pH of the solution to 7 by using NaOH; mixing, and sterilizing at 121 deg.C for 20 min.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the invention provides a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, which adopts precursor feedback supplement solid state fermentation to catalyze precursor R- (+) -2-phenoxypropionic acid (PPA) to synthesize R- (+) -2- (4-hydroxyphenoxy) propionic acid, abandons the continuous fed-batch mode according to the characteristics of solid fermentation, adopts the mode of supplementing the precursor for many times, and effectively controls the PPA concentration within the range of 10-40g/kg solid state fermentation culture medium, thereby reducing the toxicity of PPA to cells.
Meanwhile, the conversion efficiency of synthesizing R- (+) -2- (4-hydroxyphenoxy) propionic acid by catalyzing precursor R- (+) -2-phenoxypropionic acid (PPA) is high: compared with the control DHPPA yield of only 1.45 g/kg solid state fermentation medium by adding the precursor PPA at one time without feedback, the PPA conversion rate is 2.84%; according to the invention, based on solid fermentation of PPA content feedback feed, the yield of DHPPA can reach 70.53g/kg solid fermentation medium, and the conversion rate of precursor PPA reaches 99.84%.
The invention adopts a precursor solid fermentation method, and has the advantages of mild reaction conditions, few reaction steps, convenient product separation and extraction and the like. Compared with liquid fermentation or biotransformation after liquid fermentation, the method has the characteristics of low fermentation raw material cost, less wastewater discharge and capability of preventing filamentous bacteria from agglomerating to influence oxygen transfer in the fermentation process by using the solid fermentation raw material as a carrier of the filamentous bacteria.
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In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a liquid chromatography plot of a PPA and DHPPA standard;
FIG. 2 is the result of feed-back in example 1; and (delta): a PPA concentration; a tangle-solidup: DHPPA concentration;
FIG. 3 is the result of feed-back feeding in example 2; and (delta): a PPA concentration; a tangle-solidup: DHPPA concentration;
FIG. 4 is a chromatogram for detection of samples taken during solid fermentation of precursor feed in example 2; FIG. 4A is a liquid chromatography measurement of a sample taken after 5d fermentation in example 4, which was fed immediately after detection; FIG. 4B is a liquid chromatography detection chart of samples taken after 6d of fermentation in example 4; FIG. 4C is a liquid chromatography detection chart of samples taken after 7d of fermentation in example 4;
FIG. 5 is a comparison of PPA conversion for the feedback feed and the non-feed groups of example 3; □: PPA concentration in non-feedback fed-batch group; and (delta): feed-back fed-batch PPA concentration; ■: non-feedback fed-batch DHPPA concentration; a tangle-solidup: feedback fed-batch DHPPA concentration;
FIG. 6 is the result of feed-back in example 4; and (delta): a PPA concentration; a tangle-solidup: DHPPA concentration;
FIG. 7 shows the results of the feed-back in example 5. And (delta): a PPA concentration; a tangle-solidup: DHPPA concentration;
FIG. 8 is a flow chart of a process for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to an embodiment of the present invention;
FIG. 9 is a schematic diagram of a method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to an embodiment of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be obtained by an existing method.
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
the problems existing in the prior art are as follows: the solid fermentation DHPPA has a plurality of advantages, but the key problem that a high-concentration substrate or precursor usually existing in a biocatalytic reaction has a toxic effect on cells is still not solved, and the biosynthesis DHPPA can be influenced by the high-concentration R- (+) -2-phenoxypropionic acid;
through analysis, the problem can be effectively solved by adopting a fed-batch mode during liquid fermentation culture, such as the fed-batch of hypoxanthine during adenosine fermentation and the fed-batch of anthranilic acid during L-tryptophan fermentation. However, in practical operation, it is difficult to adopt fed-batch method for precursor solid-state fermentation or solid-state fermentation, on one hand, because the solid fermentation medium has low water content and the fed-batch amount needs to be strictly controlled, the solid fermentation medium is easy to be fed-batch excess and becomes semi-solid state fermentation or liquid fermentation, and the significance of solid fermentation is lost. On the other hand, it is difficult to accurately measure fermentation parameters such as pH, dissolved oxygen and carbon dioxide content, etc. to perform pH-stat feedback fedbatch, DO-stat feedback fedbatch or CER (CO) because the substrate of the solid fermentation medium is not uniform2Emission rate) method to accurately control the flow rate.
The inventor of the application finds that the method for synthesizing the R- (+) -2- (4-hydroxyphenoxy) propionic acid by catalyzing the precursor R- (+) -2-phenoxypropionic acid (PPA) through precursor feedback feeding solid-state fermentation, abandons the continuous feeding and feeding mode according to the characteristics of solid fermentation, adopts the mode of feeding the precursor for multiple times, and feeds the time for adding the PPA according to the result of PPA content detection by high performance liquid chromatography, so that the concentration of the PPA can be effectively controlled within the range of 10-40g/kg of solid-state fermentation culture medium, and the toxicity of the PPA to cells is reduced while the conversion rate of the PPA is kept high.
According to an exemplary embodiment of the present invention, there is provided a method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid, as shown in fig. 8, the method including:
step S1, inoculating Beauveria bassiana to a PDA slant surface for slant surface culture to obtain PDA slant surface spores; as an alternative embodiment, in step S1, the condition of slant culture is: the temperature is 27-29 deg.C, and the time is 3-7 days.
Step S2, washing the PDA slant spores with sterile water to prepare a spore suspension, and then inoculating the spore suspension into a seed culture medium for seed culture to obtain a fermented seed solution; the step S2 specifically includes:
washing PDA slant spore with sterile water to obtain spore with amount of 107-109Inoculating 1-3% spore suspension into a seed culture medium by mass percent for seed culture to obtain a fermented seed liquid. The seed culture conditions are as follows: the temperature is 25-30 ℃, the rotating speed is 200-. The culture condition is favorable for obtaining more fermentation seed liquid more quickly;
the formula of the seed culture medium is as follows: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50ml/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L, and sterilizing at 121 ℃ for 20min after preparation.
And step S3, inoculating the fermentation seed liquid into a solid state fermentation culture medium for solid state culture, wherein in the solid state culture, the content of the precursor PPA in the solid state fermentation culture medium is monitored, and the concentration of the precursor PPA in the solid state fermentation culture medium is controlled within the range of 10-40g/kg by adding the precursor PPA into the solid state fermentation culture medium.
In the embodiment of the invention, the time for adding the PPA precursor is fed back according to the result of PPA content detection by high performance liquid chromatography, the concentration of PPA is effectively controlled within the range of 10-40g/kg of solid fermentation medium, and the toxicity of PPA to cells can be reduced. If the PPA concentration is lower than 10g/kg of solid fermentation medium, the yield is not high; if the PPA concentration is higher than 40g/kg of the solid fermentation culture medium, the PPA is easy to poison cells;
in the step S3, the fermentation seed solution is inoculated to a solid fermentation medium at a mass fraction of 5-10%, and the solid culture conditions are as follows: the temperature is 25-30 deg.C, and the time is 5-21 d.
Wherein the weight of the PPA initially added into the solid state fermentation culture medium is 7.5-80g per kg of the solid state fermentation culture medium; specifically, the formula of the solid fermentation medium is as follows: 200-400g/kg of dry materials, wherein the dry materials comprise at least one of bran, rice hull, rice bran, corn grits and rice; 25-50ml/kg of trace element solution, and adding water to ensure that the ratio of material to water is 1: (1.5-2.5); then adding 6-32ml of PPA solution, wherein the concentration of the PPA solution is 500g/L, and adjusting the pH of the solution to 7 by using NaOH; mixing, and sterilizing at 121 deg.C for 20 min.
As a preferred embodiment, the dry material comprises 100% of bran on a mass fraction basis; under the solid-state culture medium, the PPA conversion rate is 99.84%;
as a preferred embodiment, the dry material comprises 70% of bran and 30% of rice hull by mass fraction; under this solid state medium, the conversion of PPA was 85.66%;
as a preferred embodiment, the dry materials comprise 40% of bran, 40% of corn flour and 30% of rice hull by mass fraction; under the solid-state culture medium, the PPA conversion rate is 98.17%;
as a preferred embodiment, the dry matter comprises, in mass fraction, 70% of bran and 30% of corn flour; under the solid-state culture medium, the PPA conversion rate is 98.76%;
as a preferred embodiment, the dry materials comprise 17.5 to 37.5 percent of bran, 17.5 to 52.5 percent of rice bran, 7.5 to 22.5 percent of corn flour and 15 to 22.5 percent of rice hull; under the solid culture medium, the PPA conversion rate is 83.13% -93.70%;
as an alternative embodiment, the time for adding the PPA precursor is fed back by the result of detecting the content of the PPA by high performance liquid chromatography; sampling from the solid fermentation medium every day, feeding when the concentration of the precursor PPA is lower than 10-40g/kg of the solid fermentation medium, and supplementing PPA to control the concentration of the precursor PPA to be in the range of 10-40g/kg of the solid fermentation medium.
In the technical proposal, the device comprises a base,
as an alternative embodiment, PPA consumed to a concentration of ≤ 10g/kg can be fed;
as an alternative embodiment, PPA is consumed to a concentration of ≤ 20g/kg and fed;
as an alternative embodiment, PPA is consumed to a concentration of 30g/kg or less to start feeding;
as an alternative embodiment, PPA is consumed to a concentration of 40g/kg or less to start feeding;
preferably, feeding is started when the sample detecting PPA is consumed to a concentration of 20g/kg or less;
more preferably, when the sample detection PPA concentration is less than or equal to 10g/kg, the PPA solution is added; thus the conversion of PPA is higher;
monitoring the content of the precursor PPA in the solid-state fermentation medium during the solid-state culture, adding the precursor PPA into the solid-state fermentation medium, and controlling the concentration of the precursor PPA in the solid-state fermentation medium to be in the range of 10-20g/kg, so that the conversion rate of PPA is higher;
more preferably, the precursor feed-back feed controls the solid fermentation at a PPA concentration which is lower than 20g/kg throughout; thus the conversion of PPA is higher;
as an alternative embodiment, the number of times of adding the precursor PPA is 1-6 times. The PPA amount of the precursor is 10-20g/kg of solid fermentation medium each time, if the adding times is more than 6, the concentration of the substrate is too high, the strain is difficult to convert 80 g/kgPPA, and the waste of the substrate is caused; the adding times are determined according to the detected feedback condition; for example, FIG. 7 of example 5 shows that the precursor remains after 2 additions, and thus only 2 additions are used.
As an alternative embodiment, the feeding is stopped when the total amount of the PPA added finally is 50-80g/kg of solid fermentation medium; the PPA addition amount is 50-80g/kg of solid fermentation medium: the PPA is completely converted within 21 days and the conversion rate is high; if the total amount of the PPA is less than 50g/kg, the substrate conversion concentration is not high, and if the total amount of the PPA is more than 80g/kg, the PPA is difficult to completely convert within 21 days; the PPA is added in a total amount of 50-80g/kg for solid fermentation, and the concentration of the PPA precursor is controlled in a range of 10-40g/kg of solid fermentation medium by feedback feeding, so that the conversion rate under the condition of high-concentration substrate precursor can be kept high; the final total amount of PPA added, i.e., the initial amount of PPA added in the solid medium, was also calculated;
as an alternative embodiment, when the precursor PPA is added, the precursor PPA is prepared as a PPA solution with pH of 7-12 and concentration of 45-55% (m/V) and added because: if the pH is less than 7, the conversion rate is low, and PPA can be crystallized; if the pH is more than 12, it is difficult to adjust the pH of the solid medium to 6 to 8, and microorganisms are difficult to grow; if the concentration is less than 45 percent, the PPA concentration is too low, more liquid needs to be added when the PPA is added, so that the water control of the solid culture medium becomes difficult, and if the concentration is more than 55 percent, the PPA solution is added too little, so that the PPA and the solid culture medium are difficult to be uniformly mixed, and simultaneously, less alkaline components are brought in;
the inoculation mass fraction in the inoculation amount in the example of the present invention refers to the ratio of the mass of the inoculated bacterial liquid to the volume of the culture medium. In the invention, PPA refers to R- (+) -2-phenoxypropionic acid, and DHPPA refers to R- (+) -2- (4-hydroxyphenoxy) propionic acid;
the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to the present application will be described in detail with reference to examples and experimental data. In the following examples, Beauveria bassiana B2660, which is deposited in China center for type culture Collection at 6.4.2021 by the applicant and has a preservation number of CCTCC NO: m2021337. The strain is named Beauveria bassiana B2660(Beauveria bassiana B2660).
Example 1 solid fermentation with precursor feed-back controlled PPA concentration below 30g/kg
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 7 days at the temperature of 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108Inoculating the spore suspension with the inoculation amount of 2% into 50mL of seed culture medium, and culturing for 48h at 28 ℃ under the condition of shaking table 200r/min to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50ml/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. The seed culture medium is prepared and sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 5%. The solid state fermentation culture medium comprises: 100g of dry materials, wherein the dry materials are bran; 15mL of trace element solution, 18mL of PPA solution (the concentration of the mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by NaOH), water is added, the final solid fermentation material is 300g, and the mixture is sterilized at 121 ℃ for 20min after being mixed.
Solid fermentation 14d at 28 ℃. Sampling and detecting are carried out at regular time every day, when the PPA concentration of the sample detection is lower than 20g/kg of solid culture medium, 6mL of PPA solution is supplemented (the mother liquor concentration is 500g/L, the pH is adjusted to be 7.5), the total volume of the final fed PPA is controlled to be 30mL, and the fermentation is finished after 14 days. During sampling, 1g of fermentation substrate is added into 9ml of ultrapure water for extraction, then the mixture is centrifuged at 4000r/min for 15min, the supernatant is filtered by a 0.22 mu m filter membrane, and the content of PPA and DHPPA in the filtrate is detected by liquid chromatography, and the result is shown in figure 2.
In FIG. 2 it can be seen that in fermentation 3d, the PPA concentration had fallen below 20g/kg solid medium, the medium was fed with an amount of PPA feed of about 10g/kg solid medium relative to the medium, and the PPA concentration in the medium returned to the initial precursor concentration after feeding. After 3 times of feeding within 14 days of fermentation, the content of DHPPA gradually increases within 14 days, and finally 39.88g/kg of solid medium is achieved, and the PPA conversion rate is 65.45%.
Example 2 solid fermentation with precursor feed-back controlled PPA concentration below 20g/kg
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 7 days at the temperature of 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108Inoculating spore suspension of each mL into 50mL of seed culture medium according to the inoculation amount of 2%, and culturing for 48h at 30 ℃ under the condition of a shaking table of 200r/min to serve as fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50ml/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. After the seed culture medium is prepared, the seed culture medium is sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 5%. The solid fermentation medium is: 100g of dry materials, wherein the dry materials are bran; 15mL of trace element solution, 18mL of PPA solution (the concentration of the mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by KOH), water is added, the final solid fermentation material is 300g, and the mixture is sterilized at 121 ℃ for 20min after being mixed.
Solid fermentation 14d at 28 ℃. Sampling and detecting every other day, when the PPA concentration of the sample detection is lower than 10g/kg, adding 6mL of PPA solution (the mother solution concentration is 500g/L, the pH is adjusted to 7.5), controlling the total volume of the final PPA fed to be 30mL, and finishing the fermentation after 14 days. During sampling, 1g of fermentation substrate is added with 9ml of ultrapure water for extraction, then centrifugation is carried out for 15min at 4000r/min, after a supernatant is filtered by a 0.22-micron filter membrane, the content of PPA and DHPPA in a filtrate is detected by liquid chromatography, the solid fermentation result is shown in figure 3, and the liquid chromatography detection graph of a part of samples in the fermentation process is shown in figure 4.
In FIG. 3, it is seen that the PPA content was already lower than 10g/kg solid medium at fermentation time 5d, and PPA feeding was performed at a concentration of 10g/kg solid medium. 3 times of feeding are carried out within 14 days of fermentation, the DHPPA content of the product reaches 51.7g/kg solid medium at 14 days, and the PPA conversion rate is 85.55%. A comparison of example 1 and example 2 shows that maintaining the substrate PPA content at a lower concentration of 0-20g/kg is more favorable for solid fermentation of DHPPA.
Example 3 solid fermentation with precursor feed-back controlled PPA concentration Whole course below 20g/kg
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 6 days at 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108Inoculating spore suspension of each ml into 50ml of seed culture medium according to the inoculation amount of 2%, and culturing for 40h at 28 ℃ under the condition of a shaking table at 200r/min to serve as fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50mL/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. After the seed culture medium is prepared, the seed culture medium is sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 5%. The solid fermentation medium is: 100g of dry materials, wherein the dry materials are bran; 15ml of trace element solution, 6ml of PPA solution (the concentration of the mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by NaOH), water is supplemented, the final weight of the solid fermentation material is 300g, and the solid fermentation material is sterilized for 20min at 121 ℃ after being mixed.
Solid fermentation 14d at 28 ℃. Sampling and detecting every other day, when the PPA concentration of the sample detection is lower than 10g/kg, adding 6mL of PPA solution (the mother solution concentration is 500g/L, the pH is adjusted to 7.5), controlling the total volume of the final PPA fed to be 30mL, and finishing the fermentation after 14 days. During sampling, 1g of fermentation substrate is added into 9ml of ultrapure water for extraction, then the mixture is centrifuged at 4000r/min for 15min, the supernatant is filtered by a 0.22 mu m filter membrane, and the content of PPA and DHPPA in the filtrate is detected by liquid chromatography, and the result is shown in figure 5.
As can be seen from FIG. 5, the accumulation of DHPPA was rapid in the initial stage of fermentation, and the final DHPPA concentration reached 55.26g/kg solid medium with a PPA conversion of 78.00% after 6 feedings within 14 days.
Example 4 solid fermentation with PPA concentration controlled by precursor feed feedback and total PPA addition of 70g/kg
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 7 days at the temperature of 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108Inoculating spore suspension of each ml into 50ml of seed culture medium according to the inoculation amount of 2%, and culturing for 43h at 28 ℃ under the condition of shaking table 200r/min to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50mL/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. After the seed culture medium is prepared, the seed culture medium is sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 10%. The solid fermentation medium is: 133g of dry materials, wherein the dry materials are bran and corn flour according to a mass ratio of 7: 3, preparing the mixture; adding 18ml of trace element solution, adding 32ml of PPA solution (the concentration of the mother solution is 500g/L, the pH is adjusted to 7.5), supplementing water, and sterilizing at 121 ℃ for 20min after mixing, wherein the final weight of the solid fermentation material is 400 g.
Solid fermentation at 28 ℃ for 21 d. Sampling and detecting every other day, when the PPA concentration detected by the sample is lower than 10g/kg, adding 8mL of PPA solution (the concentration of mother liquor is 500g/L, the pH of the solution is adjusted to 12 by KOH), controlling the total volume of the final PPA feed to be 24mL, and ending the fermentation after 21 days. During sampling, 1g of fermentation substrate is added into 9ml of ultrapure water for extraction, then the mixture is centrifuged at 4000r/min for 15min, the supernatant is filtered by a 0.22 mu m filter membrane, and the content of PPA and DHPPA in the filtrate is detected by liquid chromatography, and the result is shown in figure 6.
As can be seen from FIG. 6, the accumulation of DHPPA was slightly slow during the initial fermentation period, and 3 feeding times were carried out within 21 days, so that the final DHPPA concentration reached 70.53g/kg solid medium, and the PPA conversion rate was 99.84%.
Example 5 solid fermentation with PPA concentration controlled by precursor feed feedback and total PPA addition of 80g/kg
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 6 days at 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108Inoculating spore suspension of each ml into 50ml of seed culture medium according to the inoculation amount of 2%, and culturing for 44h at 28 ℃ under the condition of shaking table 200r/min to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50mL/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. After the seed culture medium is prepared, the seed culture medium is sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 5%. The solid fermentation medium is: 133g of dry materials, wherein the dry materials are a mixture of bran, corn flour and rice hulls according to a mass ratio of 4:3: 3; adding 20ml of trace element solution, adding 32ml of PPA solution (the concentration of mother liquor is 500g/L, the pH value of the solution is adjusted to 7 by NaOH), supplementing water, and sterilizing at 121 ℃ for 20min after mixing.
Solid fermentation at 28 ℃ for 21 d. Sampling every other day for detection, when the PPA concentration of the sample detection is lower than 10g/kg, adding 16mL of PPA solution (the mother solution concentration is 500g/L, the pH is adjusted to 9), controlling the total volume of the final PPA fed to be 32mL, and ending the fermentation after 21 days. During sampling, 1g of fermentation substrate is added into 9ml of ultrapure water for extraction, then the mixture is centrifuged at 4000r/min for 15min, the supernatant is filtered by a 0.22 mu m filter membrane, and the content of PPA and DHPPA in the filtrate is detected by liquid chromatography, and the result is shown in figure 7.
As can be seen from FIG. 7, only 2 feedings were performed within 21 days, and PPA was difficult to be reconverted to DHPPA in the late fermentation stage, so that some precursor remained. The final DHPPA concentration reached 62.97g/kg solid medium, with a PPA conversion of 77.54%.
Comparative example 1 Primary non-feedback feed supplement solid fermentation conversion PPA of precursor
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 7 days at the temperature of 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108 Inoculating 2% of spore suspension into 50mL of seed culture medium according to the inoculation amount, and culturing for 48h at 28 ℃ under the condition of a shaking table at 200r/min to serve as fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50mg/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. The seed culture medium is prepared and then sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 10%. The solid fermentation medium is: 100g of dry materials, wherein the dry materials are bran; 15mL of trace element solution, 18mL of PPA solution (the concentration of the mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by KOH), water is added, the final solid fermentation material is 300g, and the mixture is sterilized at 121 ℃ for 20min after being mixed.
Solid fermentation 14d at 28 ℃. The PPA adding mode is as follows: 30g/kg of PPA was added, and then 20g/kg of PPA was added once on the 5 th day of fermentation, and 12mL of PPA solution was added (mother liquor concentration 500g/L, pH adjusted to 7.5). Sampling for solid fermentation at 7 th and 14 th days, extracting 1g fermentation substrate with 9mL of ultrapure water, centrifuging at 4000r/min for 15min, filtering supernatant with 0.22 μm filter membrane, and detecting PPA content of filtrate by liquid chromatography.
Comparative example 2 Primary non-feedback feed supplement solid fermentation conversion PPA of precursor
In this comparative example, the PPA addition was: the solid medium was initially charged with 30g/kg PPA and then supplemented once with 20g/kg PPA on day 6 of fermentation, all other steps being as in comparative example 1.
Comparative example 3 Primary non-feedback feed supplement solid fermentation conversion PPA of precursor
In this comparative example, the PPA addition was: the solid medium was initially charged with 30g/kg of PPA and then supplemented once with 20g/kg of PPA on day 7 of fermentation, all other steps being as in comparative example 1.
Comparative example 4 Primary non-feedback feed-batch solid fermentation conversion of precursor PPA
In this comparative example, 50g/kg of PPA was initially added to the solid medium in one portion, and the other steps were the same as in comparative example 1.
Experimental example 1 search for solid fermentation Material
(1) Slant culture: beauveria bassiana 2660 is taken as a strain, inoculated on a PDA slant and cultured for 7 days at the temperature of 28 ℃;
(2) seed culture: washing PDA slant spore with sterile water to obtain spore amount of 108Inoculating the spore suspension with the inoculation amount of 2% into 50mL of seed culture medium, and culturing for 48h at 28 ℃ under the condition of shaking table 200r/min to obtain fermentation seed liquid;the seed culture medium comprises the following components in percentage by weight: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50ml/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, and sodium molybdate dihydrate 5 mg/L. Sterilizing the prepared seed culture medium for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation culture medium according to the mass fraction of 10%. The solid state fermentation culture medium comprises: 100g of dry materials, wherein the dry materials are mixture of bran, rice bran and wheat bran, corn flour and wheat bran (the specific formula of different solid fermentation raw materials is shown in table 1); 15mL of trace element solution, 18mL of PPA solution (the concentration of the mother solution is 500g/L, the pH of the solution is adjusted to 8 by NaOH), water is supplemented, the final weight of the solid fermentation material is 300g, and the solid fermentation material is sterilized at 121 ℃ for 20min after being mixed.
Solid fermentation at 28 deg.C for 7 d. Samples are taken from the solid fermentation culture medium at the fermentation stages 0, 3, 5 and 7d respectively, 9ml of ultrapure water is added into 1g of the taken samples for leaching, and then the samples are centrifuged at 4000r/min for 15 min. The pH of the supernatant was measured by a pH meter, followed by filtration through a 0.22 μm filter, and the content of PPA and the content of DHPPA as a product were measured from the filtrate by liquid chromatography to calculate the conversion of PPA as a precursor, which was 1-PPA day on which the content was measured/total amount of PPA added × 100%. The liquid chromatography conditions were: the chromatographic column is Amethyl C18-H (4.6mm multiplied by 250mm, 5 μm) and adopts the Seiki science and technology; the mobile phase is acetonitrile-phosphoric acid water solution (pH value is 2) ═ 4: 6; the flow rate is 1.0 ml/min; the detector is a PDA detector; the detection wavelength is 220 nm; the column temperature is 30 ℃; the amount of sample was 5. mu.l. The results of liquid chromatography detection of the precursor PPA and the product DHPPA standard are shown in FIG. 1.
The results of the solid fermentation conversion of PPA are shown in Table 1.
TABLE 1D PPA conversion at different solid fermentation feedstock compositions
As can be seen from Table 1, many formulations of solid fermentation raw materials, such as formulations numbered 1, 5, 7, 13 and 14, can achieve a PPA conversion rate of more than 90% at 3% (i.e. 30g/kg solid medium), and particularly, the PPA conversion rate is as high as 99.84% when pure bran is used as a dry material for solid fermentation. Therefore, the solid fermentation raw materials of the above examples and comparative examples adopt experimental group 1, 5 or 7, and for comparison, the solid fermentation raw materials of the above examples and comparative examples adopt the formulation of experimental group 1.
Experimental example 2 comparison of different fermentation methods
The PPA conversion rates of examples 1-5 and comparative examples 1-4 were summarized as shown in table 2.
TABLE 2
From the data in table 2, it can be seen that:
in the non-feedback feeding of comparative examples 1 to 4, the PPA conversion rate is lower than 75%; in comparative examples 1 to 4, the conversion rate of PPA as a precursor was higher than that of Control (CK) to which 5% PPA was added at a time in each of the non-feedback feeding on day 5, day 6, and day 7, and the conversion rate was higher in the non-feedback feeding for 5 to 7 days as the feeding time was later in a certain time range.
As can be seen from examples 1 to 5,
when the PPA concentration of the sample detection is lower than 10g/kg, the PPA solution is supplemented to control the PPA concentration of the precursor in the range of 10-40g/kg of solid fermentation medium, and the conversion rate of PPA is higher;
the precursor feedback material feeding controls the PPA concentration to be lower than 20g/kg in the whole process of solid fermentation, and the conversion rate of PPA is higher;
the PPA adding times are 1-6 times, and the adding times are determined according to feedback; wherein, the number of times of PPA addition is 3-6, so that the conversion rate of PPA under high-concentration substrate precursors can be kept higher;
the PPA is added in a total amount of 50-80g/kg for solid fermentation, so that the conversion rate under the condition of high-concentration substrate precursors can be kept high;
in conclusion, the method for synthesizing DHPPA by solid fermentation and catalysis of the feedback feed precursor provided by the invention can avoid the poisoning or inhibition of high-concentration precursor on cells, and provides a new idea for improving the yield of DHPPA synthesized by a biological method.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (10)
1. A method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid, comprising:
inoculating Beauveria bassiana to a PDA slant surface for slant surface culture to obtain PDA slant surface spores;
washing off the PDA slant spores by using sterile water to prepare a spore suspension, and then inoculating the spore suspension into a seed culture medium for seed culture to obtain a fermented seed solution;
inoculating the fermentation seed liquid into a solid state fermentation culture medium for solid state culture, monitoring the content of the precursor PPA in the solid state fermentation culture medium, and controlling the concentration of the precursor PPA in the solid state fermentation culture medium to be in the range of 10-40g/kg by adding the precursor PPA into the solid state fermentation culture medium.
2. The method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the precursor PPA is added after being prepared as a PPA solution having a pH of 7 to 12 and a concentration of 45 to 55% (m/V).
3. The method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the number of times of addition of PPA, the precursor, is 1 to 6.
4. The method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 3, wherein the amount of PPA precursor added is 10-20g/kg of solid fermentation medium at a time.
5. The method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the content of PPA precursor in the solid-state fermentation medium is monitored, and the PPA precursor is added to the solid-state fermentation medium to control the concentration of PPA precursor in the solid-state fermentation medium to be in the range of 10-40g/kg, and the method specifically comprises the following steps:
sampling the solid fermentation medium every day, and detecting the concentration of the precursor PPA by high performance liquid chromatography;
when the concentration of the PPA precursor is lower than 10-40g/kg of solid fermentation medium, beginning to supplement PPA so as to control the concentration of the PPA precursor to be 10-40g/kg of solid fermentation medium;
and finally stopping feeding when the total PPA addition amount is 50-80g/kg of solid state fermentation medium.
6. The biosynthetic method of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the slant culture conditions are: the temperature is 27-29 deg.C, and the time is 3-7 days.
7. The method for the biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the PDA slant spores are washed with sterile water to obtain a spore suspension, and the spore suspension is inoculated into a seed culture medium for seed culture to obtain a fermented seed solution, which comprises:
washing PDA slant spore with sterile water to obtain spore with amount of 107-1091-3% of spore suspension by mass fraction, inoculating the spore suspension into a seed culture medium for seed culture, wherein the conditions of the seed culture are as follows: the temperature is 25-30 ℃, the rotating speed is 200-.
8. The biosynthetic method agent of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the fermentation seed liquid is inoculated to a solid fermentation culture medium according to the mass fraction of 5-10%, and the solid culture conditions are as follows: the temperature is 25-30 deg.C, and the time is 5-21 d.
9. The biosynthetic method of R- (+) -2- (4-hydroxyphenoxy) propionic acid of claim 1, wherein the seed culture medium is prepared from the following components: 20g/L of glucose, 10g/L of yeast powder, 2g/L of magnesium sulfate heptahydrate, 1g/L of calcium chloride dihydrate, 1.8g/L of dipotassium phosphate, 0.75g/L of potassium dihydrogen phosphate and 50ml/L of trace element solution. The composition and content of the trace element solution are as follows: EDTA-2Na 2000mg/L, ferrous sulfate heptahydrate 600mg/L, zinc sulfate heptahydrate 200mg/L, manganese sulfate monohydrate 150mg/L, boric acid 30mg/L, cobalt chloride hexahydrate 20mg/L, copper chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L, and sterilizing at 121 ℃ for 20min after preparation.
10. The biosynthetic method agent of R- (+) -2- (4-hydroxyphenoxy) propionic acid of claim 1, wherein the solid state fermentation medium has a formula of: 200-400g/kg of dry materials, wherein the dry materials comprise at least one of bran, rice hull, rice bran, corn grits and rice; 25-50ml/kg of trace element solution, and adding water to ensure that the ratio of material to water is 1: (1.5-2.5), adding 6-32ml of PPA solution, wherein the concentration of the PPA solution is 500g/L, and adjusting the pH of the solution to 7 by using NaOH; mixing, and sterilizing at 121 deg.C for 20 min.
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