CN113881715B - 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 spore suspension of beauveria bassiana into a seed culture medium for culture to obtain fermentation seed liquid; and inoculating the mixture into a solid state fermentation culture medium for solid state culture, monitoring the content of a precursor R- (+) -2-phenoxypropionic acid (PPA) in the solid state fermentation culture medium, and controlling the concentration of the PPA in the solid state fermentation culture medium to be in the range of 10-40 g/kg. The method adopts a mode of supplementing the precursor for multiple times, feeds back and supplements the feed according to the result of detecting the PPA content by high performance liquid chromatography, reduces the poison of PPA to beauveria bassiana, feeds back the solid fermentation of the feed back based on the PPA content, ensures that the DHPPA yield can reach 70.53g/kg of solid fermentation medium, and ensures that the PPA conversion rate of the precursor reaches 99.84 percent.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a biosynthesis method of R- (+) -2- (4-hydroxy phenoxy) propionic acid.
Background
R- (+) -2- (4-hydroxy phenoxy) propionic acid (DHPPA) is an important chiral intermediate for synthesizing 4- (aryloxy phenoxy) 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), oxazoxamide, haloxyfop-butyl and the like, and the herbicides have symmetrical structures, have low toxicity, high selectivity, are safe to crops, are easy to degrade and the like, so that the research on the efficient synthesis method of the DHPPA has important significance.
The reaction process for synthesizing DHPPA by a chemical synthesis method is difficult to control, and has the defects of high energy consumption, more byproducts, large environmental pollution and the like. The biosynthesis of DHPPA has the advantages of mild reaction conditions, few reaction steps, convenient separation and extraction and the like, and has been paid attention to in recent years. Biosynthesis to produce DHPPA is the specific conversion of R- (+) -2-phenoxypropionic acid to DHPPA using the production of hydroxylase-producing microorganisms. Microorganisms which are currently capable of hydroxylating R- (+) -2-phenoxypropionic acid are: beauveria bassiana, aspergillus niger, aspergillus flavus, streptomyces, paecilomyces farinosus and the like, wherein beauveria bassiana shows better catalytic performance when the DHPPA is biosynthesized. Compared with liquid fermentation or biological conversion after liquid fermentation, the solid fermentation mode has the characteristics of low cost of fermentation raw materials, less wastewater discharge, and capability of being used as a carrier of the filamentous bacteria to avoid the influence of the agglomeration of the filamentous bacteria on oxygen transmission in the fermentation process. Although there are many advantages of solid state fermentation DHPPA, the key problem that high concentration of substrate or precursor often present in biocatalytic reactions has toxic effect on cells is still not solved, and high concentration of R- (+) -2-phenoxypropionic acid also affects biosynthesis of DHPPA.
Therefore, there is an urgent need to develop a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid to overcome the toxicity problem of high concentration precursors in solid fermentation.
Disclosure of Invention
The invention aims to provide a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, which adopts a method for synthesizing R- (+) -2- (4-hydroxyphenoxy) propionic acid by solid state fermentation of precursor feedback feed-back catalytic precursor R- (+) -2-phenoxypropionic acid (PPA), and adopts a mode of continuously feeding feed-back for multiple times according to the characteristics of solid state fermentation, wherein the addition time of the precursor PPA is fed back according to the result of detecting PPA content by high performance liquid chromatography, the concentration of PPA is effectively controlled within the range of 10-40g/kg solid state fermentation medium, and the toxicity of PPA to cells is reduced.
The invention provides a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, which comprises the following steps:
inoculating beauveria bassiana Beauveria bassiana to a PDA inclined plane for inclined plane culture to obtain PDA inclined plane spores;
washing the PDA inclined spores with sterile water to obtain spore suspension, and inoculating the spore suspension into a seed culture medium for seed culture to obtain fermentation seed liquid;
inoculating the fermentation seed liquid into a solid state fermentation medium for solid state culture, wherein the solid state culture is carried out, the content of the precursor PPA in the solid state fermentation medium is monitored, and the concentration of the precursor PPA in the solid state fermentation medium is controlled to be in the range of 10-40g/kg by adding the precursor PPA into the solid state fermentation medium.
Further, when the precursor PPA is added, the precursor PPA is prepared into a PPA solution with pH of 7-12 and concentration of 45-55% (m/V) for addition.
Further, the number of times of adding the precursor PPA is 1-6.
Further, the amount of PPA precursor added is 10-20g/kg solid state fermentation medium per time.
Further, the monitoring of the content of the precursor PPA in the solid state fermentation medium is performed by adding the precursor PPA to 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 10-40g/kg, and specifically comprises:
sampling from the solid fermentation medium every day, and detecting the concentration of the precursor PPA by high performance liquid chromatography;
starting to replenish PPA when the concentration of the precursor PPA is lower than 10-40g/kg of solid state fermentation medium so as to control the concentration of the precursor PPA to be 10-40g/kg of solid state fermentation medium;
and stopping feeding when the PPA is added to the solid state fermentation medium with the total amount of 50-80 g/kg.
Further, the conditions of the slant culture are as follows: the temperature is 27-29 ℃ and the time is 3-7d.
Further, the PDA inclined spores are washed by sterile water to prepare spore suspension, and then the spore suspension is inoculated into a seed culture medium for seed culture, so as to obtain fermentation seed liquid, which specifically comprises the following steps:
washing PDA inclined plane spores with sterile water to obtain spores with spore amount of 10 7 -10 9 And inoculating the spore suspension with the mass fraction of 1-3% into a seed culture medium for seed culture to obtain fermentation seed liquid.
Further, the seed culture conditions are as follows: the temperature is 25-30deg.C, the rotation speed is 200-300r/min, and the time is 36-48h.
Further, the fermentation seed liquid is inoculated into a solid state fermentation culture medium according to the mass fraction of 5-10%, and the conditions of the solid state culture are as follows: the temperature is 25-30deg.C, and the time is 5-21d.
Further, the formula of the seed culture medium is as follows: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50ml/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L, and sterilized at 121℃for 20min after being prepared.
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 husk, rice bran, corn grits and rice; 25-50ml/kg of microelement solution, and adding water to make the feed water ratio 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 NaOH; sterilizing at 121deg.C for 20min.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
the invention provides a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, which adopts a method for synthesizing R- (+) -2- (4-hydroxyphenoxy) propionic acid by solid state fermentation and catalysis of a precursor, namely R- (+) -2-phenoxypropionic acid (PPA), by adopting a precursor feedback feeding method, according to the characteristics of solid state fermentation, a continuous flow feeding mode is abandoned, a mode of repeatedly feeding precursor is adopted, the time of adding the precursor PPA is fed back according to the result of detecting the PPA content by high performance liquid chromatography, the PPA concentration is effectively controlled within the range of 10-40g/kg solid state fermentation medium, and the poison of PPA to cells is reduced.
Meanwhile, the conversion efficiency of synthesizing the R- (+) -2- (4-hydroxy phenoxy) propionic acid from the catalytic precursor R- (+) -2-Phenoxy) Propionic Acid (PPA) is high: the PPA conversion was 2.84% compared to a control DHPPA yield of only 1.45 g/kg solid state fermentation medium with non-fed-back one-time addition of precursor PPA; the invention is based on the solid fermentation of PPA content feedback feed, the DHPPA yield can reach 70.53g/kg solid fermentation medium, and the precursor PPA conversion rate reaches 99.84%.
The invention adopts the solid fermentation method of the precursor, and has the advantages of mild reaction conditions, fewer reaction steps, convenient separation and extraction of the product, and the like. Compared with liquid fermentation or biological conversion after liquid fermentation, the method has the characteristics of low cost of fermentation raw materials, less wastewater discharge, and capability of preventing the aggregation of the filamentous bacteria in the fermentation process from affecting the transmission of oxygen by taking the solid fermentation raw materials as carriers of the filamentous bacteria.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a liquid chromatographic analysis of PPA and DHPPA standards;
FIG. 2 is the feedback feed of example 1; delta: PPA concentration; and (2) the following steps: DHPPA concentration;
FIG. 3 is the result of feedback feeding in example 2; delta: PPA concentration; and (2) the following steps: DHPPA concentration;
FIG. 4 is a detection chromatogram of a sample taken during a precursor fed-back solid fermentation in example 2; FIG. 4A is a liquid chromatography test chart of the sample obtained after 5d fermentation in example 4, and feeding immediately after sample detection; FIG. 4B is a liquid chromatography test chart of the sample obtained after fermentation for 6d in example 4; FIG. 4C is a liquid chromatography detection chart of the sample obtained after 7d fermentation in example 4;
FIG. 5 is a comparison of PPA conversion for the feedback fed and non-fed batch of example 3; and ∈: non-feedback feed supplement group PPA concentration; delta: feeding back the PPA concentration of the feeding group; ■ : non-feedback feed supplement group DHPPA concentration; and (2) the following steps: feeding back the DHPPA concentration of the feeding group;
FIG. 6 is the result of feedback feeding in example 4; delta: PPA concentration; and (2) the following steps: DHPPA concentration;
FIG. 7 is the result of feedback feeding in example 5. Delta: PPA concentration; and (2) the following steps: DHPPA concentration;
FIG. 8 is a flow chart of a method for 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 synthesizing R- (+) -2- (4-hydroxyphenoxy) propionic acid according to an embodiment of the present invention.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning 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. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
problems of the prior art: the solid fermentation of DHPPA has a plurality of advantages, but the key problem that high concentration of substrate or precursor commonly existing in biocatalysis reaction has toxic effect on cells is still not solved, and high concentration of R- (+) -2-phenoxypropionic acid also affects biosynthesis of DHPPA;
according to analysis, the problem can be effectively solved by adopting a fed-batch mode in the liquid fermentation culture, such as fed-batch of hypoxanthine in the adenosine fermentation and fed-batch of anthranilic acid in the L-tryptophan fermentation. However, in the actual operation, the solid state fermentation of the precursor or the solid state fermentation adopts the mode of feeding the precursor, on the one hand, because the water content of the solid state fermentation medium is low, the feeding amount needs to be strictly controlled, and the solid state fermentation or even the liquid fermentation is easily changed from the feeding amount, thereby losing the meaning of the solid state fermentation. On the other hand, due to the non-uniformity of the substrate of the solid state fermentation medium, it is difficult to accurately measure fermentation parameters such as pH, dissolved oxygen, carbon dioxide content, etc., to perform pH-stat feedback feeding, DO-stat feedback feeding, or CER (CO) 2 Discharge rate) to precisely control the fed-batch amount.
Experiments of the inventor of the application find that the method for synthesizing the R- (+) -2- (4-hydroxyphenoxy) propionic acid by adopting the precursor feedback feed-back solid-state fermentation catalytic precursor R- (+) -2-phenoxypropionic acid (PPA) is carried out by adopting a continuous flow feed-back feeding mode and adopting a repeated precursor feeding mode according to the solid fermentation characteristics, the adding time of the precursor PPA is carried out according to the result feedback of the PPA content detected by the high performance liquid chromatography, the PPA concentration can be effectively controlled within the range of 10-40g/kg solid-state fermentation medium, and the poison of PPA to cells is reduced while the conversion rate of PPA is kept high.
According to an exemplary embodiment of the present invention, there is provided a biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid, as shown in fig. 8, comprising:
step S1, inoculating beauveria bassiana Beauveria bassiana to a PDA inclined plane for inclined plane culture to obtain PDA inclined plane spores; as an alternative embodiment, in the step S1, the conditions of the slant culture are as follows: the temperature is 27-29 ℃ and the time is 3-7d.
S2, washing the PDA inclined spores with sterile water to obtain spore suspension, and inoculating the spore suspension into a seed culture medium for seed culture to obtain fermentation seed liquid; the step S2 specifically includes:
washing PDA inclined plane spores with sterile water to obtain spores with spore amount of 10 7 -10 9 And inoculating the spore suspension with the mass fraction of 1-3% into a seed culture medium for seed culture to obtain fermentation seed liquid. The seed culture conditions are as follows: the temperature is 25-30 ℃, the rotating speed is 200-300r/min, and the time is 36-48h. Under the culture condition, more fermentation seed liquid can be obtained more quickly;
the formula of the seed culture medium is as follows: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50ml/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L, and sterilized at 121℃for 20min after being prepared.
And S3, inoculating the fermentation seed liquid into a solid state fermentation medium for solid state culture, wherein in the solid state culture, the content of the precursor PPA in the solid state fermentation medium is monitored, and the concentration of the precursor PPA in the solid state fermentation medium is controlled to be in the range of 10-40g/kg by adding the precursor PPA into the solid state fermentation medium.
In the embodiment of the invention, the adding time of the precursor PPA is fed back according to the result of detecting the PPA content by high performance liquid chromatography, the concentration of PPA is effectively controlled within the range of 10-40g/kg solid state fermentation medium, and the poison of PPA to cells can be reduced. If the PPA concentration is lower than 10g/kg of solid state fermentation medium, the yield is not high; if the PPA concentration is higher than 40g/kg of solid state fermentation medium, toxicity to cells is easy to occur;
in the step S3, the fermentation seed liquid is inoculated into a solid state fermentation culture medium according to the mass fraction of 5-10%, and the conditions of the solid state culture are as follows: the temperature is 25-30deg.C, and the time is 5-21d.
Wherein PPA is initially added into the solid state fermentation culture medium, and the mass of PPA is 7.5-80 g/kg/solid state fermentation culture medium; specifically, the formula of the solid state fermentation base is as follows: 200-400g/kg of dry materials, wherein the dry materials comprise at least one of bran, rice husk, rice bran, corn grits and rice; 25-50ml/kg of microelement solution, and adding water to make the feed water ratio 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 NaOH; sterilizing at 121deg.C for 20min.
As a preferred embodiment, the dry ingredients comprise 100% bran by mass fraction; under the solid state culture medium, PPA conversion is 99.84%;
as a preferred embodiment, the dry material comprises 70% bran and 30% rice hull in mass fraction; under the solid medium, PPA conversion was 85.66%;
as a preferred embodiment, the dry material comprises 40% bran, 40% corn flour and 30% rice hull by mass fraction; under the solid medium, PPA conversion was 98.17%;
as a preferred embodiment, the dry ingredients comprise 70% bran and 30% corn flour in mass fraction; under the solid state culture medium, PPA conversion rate is 98.76%;
as a preferred embodiment, the dry material comprises 17.5-37.5% bran, 17.5-52.5% rice bran, 7.5% -22.5 corn flour and 15-22.5% rice husk by mass fraction; under the solid culture medium, the PPA conversion rate is 83.13% -93.70%;
as an alternative embodiment, the time of adding the precursor PPA is fed back by the result of detecting the PPA content by high performance liquid chromatography; sampling from the solid fermentation medium every day, and starting feeding when the concentration of the precursor PPA is lower than 10-40g/kg of the solid fermentation medium, wherein the concentration of the precursor PPA is controlled within the range of 10-40g/kg of the solid fermentation medium by feeding PPA.
In the technical proposal, the utility model has the advantages that,
as an alternative embodiment, PPA consumption to a concentration of 10g/kg or less may begin feeding;
as an alternative embodiment, PPA is consumed to a concentration of less than or equal to 20g/kg to begin feeding;
as an alternative embodiment, PPA consumption is reduced to a concentration of 30g/kg or less and feeding is started;
as an alternative embodiment, PPA is consumed to a concentration of less than or equal to 40g/kg to begin feeding;
preferably, feeding is started when the sample detects PPA consumption to a concentration of less than or equal to 20 g/kg;
more preferably, when the concentration of PPA detected by the sample is less than or equal to 10g/kg, the PPA solution is supplemented; thus, the conversion rate of PPA is higher;
in the solid state culture, monitoring the content of the precursor PPA in the solid state fermentation medium, 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 feedback feed controls the solid fermentation at PPA concentrations of less than 20g/kg throughout; thus, the conversion rate of PPA is higher;
as an alternative embodiment, the precursor PPA is added 1 to 6 times. If the quantity of the precursor PPA added each time is 10-20g/kg of solid state fermentation medium and the addition times are more than 6, the concentration of the substrate is too high, and the strain is difficult to convert 80 g/kgPPA, so that the substrate is wasted; the adding times are determined according to the detected feedback condition; for example, in FIG. 7 of example 5, 2 additions of precursor have a residue and therefore only 2 additions are used.
As an alternative embodiment, the feeding is stopped when the final PPA addition amount is 50-80g/kg total solid state fermentation medium; the PPA is added in a total amount of 50-80g/kg of solid state fermentation medium: so that 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 PPA is more than 80g/kg, PPA is difficult to be completely converted within 21 days; the PPA is added to the total amount of 50-80g/kg for solid fermentation, and the concentration of the precursor PPA is controlled within the range of 10-40g/kg of solid fermentation medium through feedback feeding, so that the conversion rate of the precursor with high concentration substrate can be kept higher; the total amount of the final PPA addition, namely the total PPA addition, namely the initial PPA addition in the solid medium is also calculated;
as an alternative embodiment, the precursor PPA is added as prepared as a PPA solution having a pH of 7-12 and a concentration of 45-55% (m/V) because: if the pH is less than 7, the conversion rate is low, and PPA can crystallize out; if the pH is more than 12, it is difficult to adjust the pH of the solid medium to 6-8, and microorganisms are difficult to grow; if the concentration is less than 45%, the concentration of PPA is too low, more liquid is needed to be added when PPA is added, so that the water content of the solid culture medium becomes difficult to control, and if the concentration is more than 55%, the concentration is too high, the concentration of PPA solution is too little, so that PPA and the solid culture medium are difficult to mix uniformly, and meanwhile, the brought alkaline components are less;
the inoculation mass fraction in the inoculum size of the example of the invention refers to the ratio of the mass of the inoculated inoculum solution to the volume of the medium. PPA in the invention refers to R- (+) -2-phenoxypropionic acid, and DHPPA refers to R- (+) -2- (4-hydroxyphenoxy) propionic acid;
a method for 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 Beauveria bassiana B2660, and the applicant was preserved in China Center for Type Culture Collection (CCTCC) at 2021, with the preservation number of CCTCC NO: m2021337. Its classification is named beauveria bassiana B2660 (Beauveria bassiana B2660).
Example 1 solid fermentation with precursor feedback feed control PPA concentration below 30g/kg
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 7 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension with the inoculum size of 2% into 50mL seed culture medium, and culturing at 28deg.C under shaking table 200r/min for 48 hr to obtain fermentation seed liquid;the seed culture medium comprises the following components in percentage by weight: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50ml/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L. The seed culture medium is prepared and sterilized at 121 ℃ for 20min.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation medium according to the mass fraction of 5%. The solid state 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 mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by NaOH), 300g of the final solid fermentation material is supplemented with water, and the mixture is sterilized at 121 ℃ for 20min after mixing.
Solid fermentation was carried out at 28℃for 14d. Sampling and detecting every day at regular time, when the PPA concentration detected by the sample is lower than 20g/kg of solid medium, supplementing 6mL of PPA solution (the mother liquor concentration is 500g/L, the pH is adjusted to 7.5), controlling the total volume of PPA of the final feed to be 30mL, and ending the fermentation after 14 days. 1g of fermentation substrate is taken during sampling, 9ml of ultrapure water is added for extraction, then 4000r/min is used for centrifugation for 15min, 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.
As can be seen in FIG. 2, the PPA concentration in the 3d fermentation, which had been below 20g/kg solid state medium, was fed to the medium in an amount of about 10g/kg relative to the medium and after feeding the PPA concentration in the medium was returned to the initial precursor concentration. 3 times of feeding are carried out within 14 days of fermentation, the content of DHPPA gradually rises within 14 days, and finally 39.88g/kg of solid medium is reached, and PPA conversion rate is 65.45%.
EXAMPLE 2 precursor feedback feed control of solid fermentation with PPA concentration below 20g/kg
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 7 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension with the inoculum size of 2% into 50mL seed culture medium, culturing at 30deg.C under shaking table 200r/min for 48 hr to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50ml/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/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 medium according to the mass fraction of 5%. The solid fermentation base is as follows: 100g of dry materials, wherein the dry materials are bran; 15mL of trace element solution, 18mL of PPA solution (the concentration of mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by KOH), 300g of the final solid fermentation material is supplemented with water, and the mixture is sterilized at 121 ℃ for 20min.
Solid fermentation was carried out at 28℃for 14d. Sampling and detecting every other day, when the PPA concentration detected by the sample is lower than 10g/kg, supplementing 6mL of PPA solution (the mother liquor concentration is 500g/L, the pH is adjusted to 7.5), controlling the total volume of PPA of the final feed to be 30mL, and ending the fermentation after 14 days. Taking 1g of fermentation substrate, adding 9ml of ultrapure water for extraction, centrifuging at 4000r/min for 15min, filtering supernatant with 0.22 μm filter membrane, detecting PPA and DHPPA content of filtrate by liquid chromatography, and solid fermentation results are shown in figure 3, and liquid chromatography detection diagram of part of the sample in the fermentation process is shown in figure 4.
In FIG. 3, it is seen that the PPA content was already below 10g/kg of solid medium at 5d of fermentation, at which point PPA feed was available at a concentration of 10g/kg of solid medium. 3 times of feeding are carried out within 14 days of fermentation, the DHPPA content of the product reaches 51.7g/kg of solid medium at 14d, and the PPA conversion rate is 85.55%. Comparison of example 1 with example 2 shows that maintaining the substrate PPA content at a lower concentration of 0-20g/kg is more advantageous for the solid state fermentation of DHPPA.
Example 3 solid fermentation with precursor feedback feed control of PPA concentration to a full Process of less than 20g/kg
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 6 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension of each ml into 50ml seed culture medium according to the inoculum size of 2%, and culturing at 28deg.C under shaking table 200r/min for 40 hr to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50mL/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/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 medium according to the mass fraction of 5%. The solid fermentation base is as follows: 100g of dry materials, wherein the dry materials are bran; 15ml of trace element solution and 6ml of PPA solution (the concentration of mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by NaOH), 300g of the final solid fermentation material is supplemented with water, and the mixture is sterilized at 121 ℃ for 20min.
Solid fermentation was carried out at 28℃for 14d. Sampling and detecting every other day, when the PPA concentration detected by the sample is lower than 10g/kg, supplementing 6mL of PPA solution (the mother liquor concentration is 500g/L, the pH is adjusted to 7.5), controlling the total volume of PPA of the final feed to be 30mL, and ending the fermentation after 14 days. 1g of fermentation substrate is taken during sampling, 9ml of ultrapure water is added for extraction, then 4000r/min is used for centrifugation for 15min, 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 is faster at the initial stage of fermentation, 6 times of feeding are carried out within 14 days, and finally the concentration of DHPPA reaches 55.26g/kg of solid medium, and the PPA conversion rate is 78.00%.
EXAMPLE 4 precursor feedback feed control of PPA concentration and PPA Total addition 70g/kg solid fermentation
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 7 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension of each ml into 50ml seed culture medium according to the inoculum size of 2%, and culturing at 28deg.C under shaking table 200r/min for 43 hr to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50mL/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/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 medium according to the mass fraction of 10%. The solid fermentation base is as follows: 133g of dry materials, wherein the dry materials are bran and corn flour according to the mass ratio of 7:3 a configured mixture; 18ml of trace element solution and 32ml of PPA solution (the concentration of mother solution is 500g/L, the pH is adjusted to 7.5), 400g of the final solid fermentation material is added with water, and the mixture is sterilized for 20min at 121 ℃.
Solid fermentation was carried out at 28℃for 21d. 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 mother solution concentration is 500g/L, the pH of the solution is adjusted to 12 by KOH), controlling the total volume of PPA of the final feed to be 24mL, and ending the fermentation after 21 days. 1g of fermentation substrate is taken during sampling, 9ml of ultrapure water is added for extraction, then 4000r/min is used for centrifugation for 15min, 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 at the initial stage of fermentation, and 3 feeds were performed within 21 days, so that the final DHPPA concentration reached 70.53g/kg of solid medium, and the PPA conversion was 99.84%.
Example 5 precursor feedback feed control of PPA concentration and PPA Total addition 80g/kg solid fermentation
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 6 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension of each ml into 50ml seed culture medium according to the inoculum size of 2%, and culturing at 28deg.C under shaking table 200r/min for 44 hr to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50mL/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/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 medium according to the mass fraction of 5%. The solid fermentation base is as follows: 133g of dry materials, wherein the dry materials are a mixture of bran, corn flour and rice husk according to a mass ratio of 4:3:3; 20ml of trace element solution and 32ml of PPA solution (the concentration of mother solution is 500g/L, the pH of the solution is adjusted to 7 by NaOH), 400g of the final solid fermentation material is supplemented with water, and the mixture is sterilized at 121 ℃ for 20min.
Solid fermentation was carried out at 28℃for 21d. Sampling and detecting every other day, when the PPA concentration detected by the sample is lower than 10g/kg, supplementing 16mL of PPA solution (the mother liquor concentration is 500g/L, the pH is adjusted to 9), controlling the total volume of final feed PPA to be 32mL, and ending the fermentation after 21 days. 1g of fermentation substrate is taken during sampling, 9ml of ultrapure water is added for extraction, then 4000r/min is used for centrifugation for 15min, 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 feeds were performed within 21 days, and PPA was difficult to be reconverted to DHPPA in the late fermentation period, so that there was some precursor residue. The final DHPPA concentration reached 62.97g/kg solid medium and PPA conversion was 77.54%.
Comparative example 1 conversion of the precursor to PPA by one-time non-feedback fed-batch solid fermentation
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 7 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension with the inoculum size of 2% into 50mL seed culture medium, and culturing at 28deg.C under shaking table 200r/min for 48 hr to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: 20g/L glucose, 10g/L yeast powder, 2g/L magnesium sulfate heptahydrate, 1g/L calcium chloride dihydrate, 1.8g/L dipotassium hydrogen phosphate, 0.75g/L potassium dihydrogen phosphate and 50mg/L trace element solution. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L. The seed culture medium is sterilized at 121 ℃ for 20min after preparation.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation medium according to the mass fraction of 10%. The solid fermentation base is as follows: 100g of dry materials, wherein the dry materials are bran; 15mL of trace element solution, 18mL of PPA solution (the concentration of mother solution is 500g/L, the pH of the solution is adjusted to 7.5 by KOH), 300g of the final solid fermentation material is supplemented with water, and the mixture is sterilized at 121 ℃ for 20min.
Solid fermentation was carried out at 28℃for 14d. The PPA adding mode is as follows: PPA 30g/kg was added, and then PPA 20g/kg was supplemented once on day 5 of fermentation, 12mL of PPA solution (mother liquor concentration 500g/L, pH 7.5) was supplemented. Sampling solid fermentation at 7 and 14d, adding 9mL of ultrapure water into 1g of fermentation substrate, extracting, centrifuging at 4000r/min for 15min, filtering the supernatant with a 0.22 μm filter membrane, and detecting PPA content of the filtrate by liquid chromatography.
Comparative example 2 conversion of the precursor to PPA by one-time non-feedback fed-batch solid fermentation
In this comparative example, PPA was added as follows: the solid medium was initially supplemented with 30g/kg PPA and then 20g/kg PPA was supplemented once on day 6 of fermentation, the other steps being the same as in comparative example 1.
Comparative example 3 conversion of PPA by one-time non-feedback fed-batch solid fermentation of precursor
In this comparative example, PPA was added as follows: the solid medium was initially supplemented with 30g/kg PPA and then 20g/kg PPA was supplemented once on day 7 of fermentation, the other steps being the same as in comparative example 1.
Comparative example 4 conversion of the precursor to PPA by one-time non-feedback fed-batch solid fermentation
In this comparative example, 50g/kg 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 of solid fermentation Material
(1) Slant culture: taking beauveria bassiana Beauveria bassiana 2660 as a strain, inoculating the strain to a PDA inclined plane, and culturing for 7 days at 28 ℃;
(2) Seed culture: washing PDA inclined plane spores with sterile water to obtain spore amount 10 8 Inoculating spore suspension with the inoculum size of 2% into 50mL seed culture medium, and culturing at 28deg.C under shaking table 200r/min for 48 hr to obtain fermentation seed liquid; the seed culture medium comprises the following components in percentage by weight: glucose 20g/L, yeast powder 10g/L, magnesium sulfate heptahydrate 2g/L, calcium chloride dihydrate 1g/L, dipotassium hydrogen phosphate 1.8g/L, potassium dihydrogen phosphate 0.75g/L and trace element solution 50ml/L. The trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L. The seed culture medium is sterilized for 20min at 121 ℃.
(3) Solid state fermentation: inoculating the cultured fermentation seed liquid into a solid fermentation medium according to the mass fraction of 10%. The solid state fermentation medium is: 100g of dry materials, wherein the dry materials are bran, rice bran, corn bran and rice hull (the specific formula of the composition of different solid fermentation raw materials is shown in table 1); 15mL of trace element solution, 18mL of PPA solution (the concentration of mother solution is 500g/L, the pH of the solution is adjusted to 8 by NaOH), 300g of the final solid fermentation material is supplemented with water, and the mixture is sterilized at 121 ℃ for 20min.
Solid fermentation was carried out at 28℃for 7d. Samples were taken from the solid fermentation medium at fermentation times 0, 3, 5, and 7d, respectively, and 1g of the sample was extracted by adding 9ml of ultrapure water, followed by centrifugation at 4000r/min for 15min. The pH value of the supernatant was measured with a pH meter, followed by filtration with a 0.22 μm filter, and the PPA content and the product DHPPA content of the filtrate were measured by liquid chromatography to calculate the conversion of the precursor PPA, PPA conversion=1-PPA content detected on the day/PPA total addition amount X100%. The liquid chromatography conditions were: the chromatographic column is Amethylst C18-H (4.6mm.times.250mm, 5 μm), and is of Seisakusho technology; the mobile phase is acetonitrile-phosphoric acid aqueous solution (pH value is 2) =4:6; the flow rate is 1.0ml/min; the detector is a PDA detector; the detection wavelength is 220nm; column temperature is 30 ℃; the sample was introduced in an amount of 5. Mu.l. The liquid chromatography detection results of the precursor PPA and the product DHPPA standard are shown in figure 1.
The results of solid state fermentation to convert PPA are shown in Table 1.
TABLE 1D PPA conversion at different compositions of solid fermentation feedstock
As can be seen from Table 1, many solid fermentation raw material formulations, such as those of experiment numbers 1,5,7, 13 and 14, can lead to PPA conversion of 3% (i.e. 30g/kg solid medium) higher than 90%, especially pure bran as dry material of solid fermentation, and PPA conversion as high as 99.84%. Thus, the solid fermentation raw materials of the above examples and comparative examples were used in experimental groups 1,5 or 7, and for convenience of comparison, the solid fermentation raw materials of the above examples and comparative examples were each formulated in experimental group 1.
Experimental example 2 comparison of different fermentation methods
PPA conversion rates for examples 1-5 and comparative examples 1-4 are summarized as shown in table 2.
TABLE 2
As can be seen from the data in table 2:
the PPA conversion was less than 75% for the primary non-feedback feeds of comparative examples 1-4; in comparative examples 1-4, the conversion rate of the precursor PPA was higher than that of the Control (CK) to which 5% PPA was added at one time on either the 5 th, 6 th or 7 th day of non-feedback feeding, and the conversion rate was higher for 5-7 days of feeding as the feeding time was later in a certain time range for the non-feedback feeding.
As can be seen from examples 1-5,
when the concentration of PPA detected by a sample is lower than 10g/kg, supplementing PPA solution to control the concentration of PPA of the precursor to be in the range of 10-40g/kg solid state fermentation medium, so that the conversion rate of PPA is higher;
the precursor feedback feed controls the PPA concentration to be lower than 20g/kg in the whole process for solid fermentation, and the PPA conversion rate is higher;
the PPA adding times are 1-6 times, and the adding times are determined specifically according to feedback; wherein, the PPA adding times are 3-6 times, which can keep the conversion rate of PPA higher under the high concentration substrate precursor;
the PPA added total amount is 50-80g/kg for solid fermentation, so that the conversion rate of the substrate precursor with high concentration can be kept high;
in summary, the method for synthesizing the DHPPA by the feedback feed supplement precursor solid fermentation catalysis can avoid poisoning or inhibiting the high-concentration precursor on cells, and provides a new thought for improving the yield of synthesizing the DHPPA by a biological method.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Finally, it is also 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. It is therefore intended that the following claims be interpreted as including the 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 modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
1. A method of biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid, the method comprising:
inoculating beauveria bassiana Beauveria bassiana to a PDA inclined plane for inclined plane culture to obtain PDA inclined plane spores;
washing the PDA inclined spores with sterile water to obtain spore suspension, and inoculating the spore suspension into a seed culture medium for seed culture to obtain fermentation seed liquid;
inoculating the fermentation seed liquid into a solid fermentation medium for solid state culture, sampling from the solid fermentation medium every day, and detecting the concentration of the precursor PPA by high performance liquid chromatography;
starting to replenish PPA when the concentration of the precursor PPA is lower than 10-40g/kg of solid state fermentation medium so as to control the concentration of the precursor PPA to be 10-40g/kg of solid state fermentation medium; when the precursor PPA is added, preparing the precursor PPA into PPA solution with pH of 7-12 and concentration of 45-55% m/V for adding; the addition amount of the precursor PPA is 10-20g/kg of solid state fermentation medium each time;
stopping feeding when the PPA is added to the solid state fermentation culture medium with the total amount of 50-80 g/kg;
wherein, the formula of the solid state fermentation culture medium is as follows: 200-400g/kg of dry materials, wherein the dry materials comprise at least one of bran, rice husk, rice bran, corn grits and rice; 25-50 g ml/kg of microelement solution, and adding water to make the ratio of feed water to water be 1: (1.5-2.5), adding PPA solution 6-32ml, wherein the concentration of the PPA solution is 500g/L, and adjusting the pH of the solution to 7 by NaOH; sterilizing at 121deg.C for 20min.
2. The method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the number of PPA additions of the precursor is 1-6.
3. The method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the conditions of the slant culture are: the temperature is 27-29 ℃ and the time is 3-7d.
4. The method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein said washing of PDA oblique spores with sterile water to obtain spore suspension, and then inoculating said spore suspension into seed culture medium for seed culture to obtain fermented seed liquid, specifically comprising:
washing PDA inclined plane spores with sterile water to obtain spores with spore amount of 10 7 -10 9 Inoculating spore suspension with mass fraction of 1-3% into seed culture medium for seed culture, wherein the seed culture conditions are as follows: the temperature is 25-30 ℃, the rotating speed is 200-300r/min, and the time is 36-48h, so as to obtain the fermentation seed liquid.
5. The method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid according to claim 1, wherein the fermentation seed liquid is inoculated into a solid state fermentation medium at a mass fraction of 5-10%, and the conditions of the solid state culture are as follows: the temperature is 25-30deg.C, and the time is 5-21d.
6. The method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid of claim 1, wherein the seed medium is formulated as follows: glucose 20. 20g/L, yeast powder 10. 10g/L, magnesium sulfate heptahydrate 2. 2g/L, calcium chloride dihydrate 1. 1g/L, dipotassium hydrogen phosphate 1.8. 1.8g/L, potassium dihydrogen phosphate 0.75. 0.75g/L and trace element solution 50. 50 ml/L; the trace element solution comprises the following components in percentage by weight: 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, cupric chloride dihydrate 40mg/L, nickel chloride dihydrate 40mg/L, sodium molybdate dihydrate 5mg/L, and sterilized at 121deg.C 20 after being prepared
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3910024A1 (en) * | 1989-03-28 | 1990-10-11 | Basf Ag | METHOD FOR THE FERMENTATIVE PRODUCTION OF 2- (4-HYDROXIPENOXI-) PROPIONIC ACID |
| DE19814528A1 (en) * | 1998-04-01 | 1999-10-07 | Basf Ag | Method for increasing the POPS hydroxylation rate |
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108823098A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain |
| CN109536392A (en) * | 2018-12-29 | 2019-03-29 | 浙江工业大学 | Aspergillus versicolor ZJB16085 and the application for synthesizing R-2- (4- hydroxyphenoxy) propionic acid |
| CN109609389A (en) * | 2018-12-29 | 2019-04-12 | 浙江工业大学 | Penicillium oxalicum ZJB16086 and its application for synthesizing R-2- (4- hydroxyphenoxy) propionic acid |
| CN113151015A (en) * | 2021-05-13 | 2021-07-23 | 湖北工业大学 | Beauveria bassiana B2660 and application thereof in synthesizing p-hydroxybenzoic acid by using benzoic acid under full-cell biological catalysis |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITRM20030556A1 (en) * | 2003-11-28 | 2005-05-29 | Uni Degli Studi Di Roma To R Vergata | METHOD FOR THE QUANTITATIVE AND SEMIQUANTITATIVE DETERMINATION OF L-PHENYLALANINE, L-THYROSINE, L-3,4-DIHYDROXYPENYLALANINE AND THEIR CORRESPONDING KETOIDS WITH BIOLOGICAL FLUIDS AND ITS DIAGNOSTIC KIT. |
| CN107118973B (en) * | 2017-01-20 | 2019-10-22 | 厦门欧米克生物科技有限公司 | A method of utilizing micro-organisms parahydroxyben-zaldehyde |
-
2021
- 2021-09-15 CN CN202111080415.0A patent/CN113881715B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3910024A1 (en) * | 1989-03-28 | 1990-10-11 | Basf Ag | METHOD FOR THE FERMENTATIVE PRODUCTION OF 2- (4-HYDROXIPENOXI-) PROPIONIC ACID |
| DE19814528A1 (en) * | 1998-04-01 | 1999-10-07 | Basf Ag | Method for increasing the POPS hydroxylation rate |
| CN108486209A (en) * | 2018-03-22 | 2018-09-04 | 浙江工业大学 | A kind of method of high-flux fast screening high yield R-2- (4- hydroxyphenoxies) propionic acid bacterial strain |
| CN108823098A (en) * | 2018-06-30 | 2018-11-16 | 浙江工业大学 | A kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain |
| CN109536392A (en) * | 2018-12-29 | 2019-03-29 | 浙江工业大学 | Aspergillus versicolor ZJB16085 and the application for synthesizing R-2- (4- hydroxyphenoxy) propionic acid |
| CN109609389A (en) * | 2018-12-29 | 2019-04-12 | 浙江工业大学 | Penicillium oxalicum ZJB16086 and its application for synthesizing R-2- (4- hydroxyphenoxy) propionic acid |
| CN113151015A (en) * | 2021-05-13 | 2021-07-23 | 湖北工业大学 | Beauveria bassiana B2660 and application thereof in synthesizing p-hydroxybenzoic acid by using benzoic acid under full-cell biological catalysis |
Non-Patent Citations (2)
| Title |
|---|
| Yuan-Shan Wang et al..Production of (R)-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana ZJB16007 in solid state fermentation using rice bran.《Preparative Biochemistry & Biotechnology》.2020,第50卷(第8期),第781-787页. * |
| 郭西鹏等.球孢白僵菌基于前体物固态发酵合成D-HPPA.《农药》.2022,第61卷(第12期),第868-872页. * |
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