CN114540440A - Method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis - Google Patents
Method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis Download PDFInfo
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- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 28
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- 102000004674 D-amino-acid oxidase Human genes 0.000 claims abstract description 27
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- 239000000758 substrate Substances 0.000 claims abstract description 25
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 claims abstract description 24
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- 239000002518 antifoaming agent Substances 0.000 claims description 13
- 238000003756 stirring Methods 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 6
- 230000004186 co-expression Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical group N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 abstract description 28
- 108090000790 Enzymes Proteins 0.000 abstract description 28
- 230000000694 effects Effects 0.000 abstract description 18
- 230000035484 reaction time Effects 0.000 abstract description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 15
- 239000001301 oxygen Substances 0.000 description 15
- 229910052760 oxygen Inorganic materials 0.000 description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
- 230000003197 catalytic effect Effects 0.000 description 8
- 239000012467 final product Substances 0.000 description 6
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- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- JRBJSXQPQWSCCF-UHFFFAOYSA-N 3,3'-Dimethoxybenzidine Chemical compound C1=C(N)C(OC)=CC(C=2C=C(OC)C(N)=CC=2)=C1 JRBJSXQPQWSCCF-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical group [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
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- 229930182818 D-methionine Natural products 0.000 description 2
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- SXFSQZDSUWACKX-UHFFFAOYSA-N 4-methylthio-2-oxobutanoic acid Chemical compound CSCCC(=O)C(O)=O SXFSQZDSUWACKX-UHFFFAOYSA-N 0.000 description 1
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 1
- 102000016901 Glutamate dehydrogenase Human genes 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
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- 239000004009 herbicide Substances 0.000 description 1
- 150000004715 keto acids Chemical class 0.000 description 1
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- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- ASHGTUMKRVIOLH-UHFFFAOYSA-L potassium;sodium;hydrogen phosphate Chemical compound [Na+].[K+].OP([O-])([O-])=O ASHGTUMKRVIOLH-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention discloses a method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis, which comprises the following steps: adding a coexpression recombinant escherichia coli wet thallus and a pH regulator into racemic glufosinate-ammonium serving as a substrate to regulate the pH of a reaction system to be 6.5-9.0, and carrying out the reaction process under the conditions of sealing, ventilation and pressurization; the recombinant Escherichia coli can co-express D-amino acid oxidase and catalase; the reaction process at least comprises the following two stages: a first reaction stage, wherein the reaction system is controlled to be in a first ventilation quantity and a first pressure state; in the second reaction stage, the reaction system is controlled to be in a second ventilation rate and a second pressure state; the first ventilation volume is greater than the second ventilation volume and the first pressure is greater than the second pressure. The invention keeps higher enzyme catalysis activity of the D-amino acid oxidase in the reaction system by controlling the ventilation volume and pressure of different reaction stages in the reaction process, shortens the catalytic reaction time and improves the enzyme catalysis conversion efficiency.
Description
Technical Field
The invention relates to the technical field of biocatalysis, in particular to a method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis.
Background
2-carbonyl-4- (hydroxymethylphosphono) butanoic acid (PPO) is an important keto acid intermediate for the asymmetric synthesis of optically pure L-glufosinate-an organophosphorus herbicide with low toxicity, broad spectrum and safety, and has twice the herbicidal activity of racemic glufosinate-ammonium. The asymmetric amination of PPO takes transaminase or glutamate dehydrogenase as a biocatalyst to generate L-PPT, has the advantage of maximum theoretical yield of 100 percent, and becomes a promising strategy for industrial production of L-PPT. In view of the strict stereoselectivity, mild reaction conditions, high recovery and simple separation and purification process, DAAO deamination of racemic glufosinate, which retains the herbicidally active L-isomer due to its strict D-isomer enantioselectivity, would be an ideal choice.
DAAO deamination reaction of racemic glufosinate-ammonium is an oxygen consumption reaction, whether enough dissolved oxygen can be provided in the catalytic process or not has a decisive influence on the reaction process, and the dissolved oxygen in liquid is mainly related to the driving force of oxygen transfer (C-C) besides the property of the liquidL) And volumetric oxygen mass transfer coefficient (K)La) In connection with the actual conditions, high-power aeration and agitation acceleration are generally employed to improve the oxygen supply efficiency. However, large scale reactions in industrial production do not provide high power aeration and high speed agitation, which limits the large scale industrial application of the DAAO deamination reaction of racemic glufosinate.
Disclosure of Invention
In order to overcome the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a process for the pressure-catalyzed preparation of 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid.
Considering that a large-scale industrial reactor can conveniently provide certain tightness and high pressure resistance, the invention provides a method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid (PPO) by a D amino acid oxidase catalyzed method under a pressurized condition from the perspective of reaction kinetics.
In order to solve the problems, the invention adopts the following technical scheme:
a process for the pressure catalytic preparation of 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid comprising the steps of:
adding a co-expression recombinant escherichia coli wet thallus and a pH regulator into racemic glufosinate-ammonium serving as a substrate to regulate the pH of a reaction system to be 6.5-9.0, and carrying out the reaction process under the conditions of sealing, ventilation and pressurization;
the recombinant escherichia coli can co-express D-amino acid oxidase and catalase;
the reaction process at least comprises the following two stages:
a first reaction stage, wherein the reaction system is controlled to be in a first ventilation quantity and a first pressure state;
in the second reaction stage, the reaction system is controlled to be in a second ventilation quantity and a second pressure state;
the first ventilation is greater than the second ventilation, and the first pressure is greater than the second pressure.
Preferably, the reaction temperature in the reaction process is 25-45 ℃.
Preferably, a defoaming agent is also added into the reaction system.
More preferably, the addition amount of the defoaming agent accounts for 0.05-5% of the total volume of the reaction system.
Preferably, the concentration of the racemic glufosinate-ammonium in the reaction system is 50-500 mmol/L, and the addition amount of the recombinant escherichia coli is 5-20 g/L.
Preferably, the pH adjuster is ammonia water.
Preferably, the first ventilation volume and the second ventilation volume are both controlled to be 0.1-5 vvm.
Preferably, the first pressure and the second pressure are both controlled to be 0.02-1.5 MPa.
More preferably, the first ventilation amount and the second ventilation amount are controlled to be 0.1-2 vvm, and the first pressure and the second pressure are controlled to be 0.02-0.1 MPa.
Preferably, the stirring speed in the reaction process is controlled to be 50-500 rpm.
Compared with the prior art, the invention has the technical effects that:
the method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis improves the driving force of oxygen transfer and the volumetric oxygen mass transfer coefficient in the reaction process in a pressure mode, and avoids the defects of high-power ventilation and strong stirring; in the reaction process, the D-amino acid oxidase in the reaction system keeps higher enzyme catalytic activity by controlling the ventilation volume and pressure of different reaction stages in the reaction process, thereby shortening the catalytic reaction time and improving the enzyme catalytic conversion efficiency. The method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis has the prospect of industrial large-scale production and application.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
It will be appreciated by those skilled in the art that the objects and advantages that can be achieved with the present invention are not limited to the specific details set forth above, and that these and other objects that can be achieved with the present invention will be more clearly understood from the detailed description that follows.
Drawings
FIG. 1 is a schematic diagram of an enzyme-catalyzed reaction provided by an embodiment of the present invention;
FIG. 2 is a graph showing a comparison of the enzymatic activities of D-amino acid oxidases under different reaction conditions, which are provided in examples of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art.
Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
The enzyme activity of D amino acid oxidase is easily generated by byproduct hydrogen peroxide H in the reaction process2O2The activity is lost by oxidation and the hydrogen peroxide is decomposed into water and oxygen by catalase expressed by the co-expression strain in the course of the reaction. Meanwhile, on a gas-liquid interface, certain activity is lost when the enzyme is contacted with oxygen for a long time, namely, the continuous aeration can generate negative influence on a protein structure (namely, the activity of the enzyme). In a specific enzymatic reaction experiment, the enzyme activity of the D-amino acid oxidase is lost later in the actual reaction process, resulting in a prolonged period of time for complete conversion of the substrate, so the embodiment of the present invention provides a pressure-catalyzed process for producing 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid, comprising the steps of:
adding a coexpression recombinant escherichia coli wet thallus and a pH regulator to regulate the pH of a reaction system to 6.5-9.0 by using racemic glufosinate-ammonium (D, L-glufosinate-ammonium, D, L-PPT) as a substrate, and carrying out the reaction process under the conditions of sealing, ventilation and pressurization;
wherein, the recombinant Escherichia coli can co-express D-amino acid oxidase (DAAO) and catalase;
and the reaction process at least comprises the following two stages:
a first reaction stage, wherein the reaction system is controlled to be in a first ventilation quantity and a first pressure state;
in the second reaction stage, the reaction system is controlled to be in a second ventilation rate and a second pressure state;
the first ventilation volume is larger than the second ventilation volume, and the first pressure is larger than the second pressure.
The first reaction stage refers to the early stage of the reaction, and the second reaction stage refers to the late stage of the reaction, i.e. the ventilation and pressure in the early stage of the reaction are controlled to be higher than those in the late stage of the reaction in the reaction process. In the reaction process, the method combines the reaction kinetics, and adopts the means of gradually reducing the ventilation volume and reducing the pressure of a reaction system to ensure that the D-amino acid oxidase is always kept at a higher enzyme activity level in the reaction process so as to eliminate the negative influence of a high-pressure environment and a high dissolved oxygen environment on the activity of the D-amino acid oxidase.
The principle of the enzyme-catalyzed reaction of the present invention is shown in FIG. 1. The method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis improves the driving force of oxygen transfer and the volumetric oxygen mass transfer coefficient in the reaction process in a pressure mode, and avoids the defects of high-power ventilation and strong stirring; in the reaction process, the D-amino acid oxidase in the reaction system keeps higher enzyme catalytic activity by controlling the ventilation volume and pressure of different reaction stages in the reaction process, thereby shortening the catalytic reaction time and improving the enzyme catalytic conversion efficiency. The method for preparing 2-carbonyl-4- (hydroxymethyl phosphonyl) butyric acid by pressure catalysis has the prospect of industrial large-scale production and application.
Preferably, the first ventilation amount and the second ventilation amount are controlled to be 0.1-5 vvm. Preferably, the first pressure and the second pressure are both controlled to be 0.02-1.5 MPa. More preferably, the first ventilation amount and the second ventilation amount are both controlled to be 0.1-2 vvm, namely 3-60L/min of ventilation is carried out in a 30L reaction system. The first pressure and the second pressure are both controlled to be 0.02-0.1 MPa.
In the specific embodiment of the present invention, the reaction process is divided into three stages according to the development of the reaction process:
in the first reaction stage, namely the early stage of the reaction, the reaction system is controlled to be in a first ventilation quantity and a first pressure state;
in the second reaction stage, namely the middle reaction stage, controlling the reaction system to be in a second gas ventilation amount and a second pressure state;
and in the third reaction stage, namely the later reaction stage, controlling the reaction system to be in a third gas passing amount and a third pressure state.
Wherein, the ventilation volume and the pressure in the early reaction stage, the middle reaction stage and the later reaction stage are arranged according to the descending order. Before the reaction, the dissolved oxygen level of the reaction system is maintained at 7.4-8.0 mg/L. After the reaction starts, the initial reaction rate is high, and the dissolved oxygen value in the reaction system is maintained at a low level of 0-1 mg/L; when the dissolved oxygen value of the reaction system is increased back to about 3.0-9.0 mg/L, corresponding means of reducing the ventilation and reducing the pressure can be carried out to reduce the ventilation to 1/2-3/4 and the pressure to 1/3-2/3, so that on one hand, the energy consumption is reduced, and on the other hand, the enzyme activity can be maintained.
The aeration in the embodiment of the invention refers to introducing air, and the recombinant escherichia coli adopted in the embodiment is obtained by the preliminary research and construction of a subject group and is the recombinant escherichia coli co-expressing the D-amino acid oxidase and catalase mutant. (the strain preparation process is disclosed in Journal of Biotechnology 325(2021) 372-379.)
In order to enable the catalytic reaction enzyme to be in a higher enzyme catalytic activity level, the reaction temperature in the reaction process is preferably 25-45 ℃.
The shearing agitation during the reaction causes bubbles to be generated in the reaction system, and in order to eliminate the influence of the bubbles, it is preferable that a defoaming agent is further added to the reaction system. More preferably, the addition amount of the defoaming agent accounts for 0.05-5% of the total volume of the reaction system. Further, the addition amount of the defoaming agent accounts for 0.1-2.5% of the total volume of the reaction system. Preferably, the stirring speed in the reaction process is controlled to be 50-500 rpm. Further, the stirring speed in the reaction process is controlled to be 200-400 rpm.
Preferably, the concentration of the racemic glufosinate-ammonium in the reaction system is 50-500 mmol/L, and the addition amount of the co-expression recombinant escherichia coli is 5-20 g/L. More preferably, the concentration of the racemic glufosinate-ammonium in the reaction system is 300-500 mmol/L, and the addition amount of the co-expression recombinant escherichia coli is 10-15 g/L.
Preferably, the embodiment of the present invention uses ammonia water as a pH adjuster.
The following is a further description with reference to specific examples.
Example 1: constant ventilation and pressure
Carrying out enzyme catalysis reaction in a 30L reaction kettle, wherein the concentration of the substrate D and the L-type glufosinate-ammonium is 400mmol/L, and the adding amount of the coexpression recombinant escherichia coli is 15 g/L. The pH value is regulated and controlled to be 8.0, the reaction temperature is 30 ℃, and the dosage of the defoaming agent is 0.1 percent. The aeration rate was 0.5 vvm, the pressure was 0.05MPa, and the stirring speed was 250 rpm.
When the reaction time is 12 hours, the conversion rate of the substrate D-type glufosinate-ammonium is 94.9%. After 24 hours of reaction, the substrate conversion rate is 98.7%, and the final product yield can reach 45.8%.
Example 2: low aeration constant pressure conditions
Carrying out enzyme catalysis reaction in a 30L reaction kettle, wherein the concentration of the substrate D and the L-type glufosinate-ammonium is 400mmol/L, and the adding amount of the coexpression recombinant escherichia coli is 15 g/L. The pH value is regulated and controlled to be 8.0, the reaction temperature is 30 ℃, and the dosage of the defoaming agent is 0.1 percent. The aeration rate was 0.25 vvm, the pressure was 0.075MPa, and the stirring speed was 250 rpm.
When the reaction time is 12 hours, the conversion rate of the substrate D-type glufosinate-ammonium is 96.8%. After 24 hours of reaction, the conversion rate of the substrate is 99.3 percent, and the yield of the final product can reach 46.5 percent.
Example 3: high aeration constant pressure conditions
Carrying out enzyme catalysis reaction in a 30L reaction kettle, wherein the concentration of the substrate D and the L-type glufosinate-ammonium is 400mmol/L, and the adding amount of the coexpression recombinant escherichia coli is 15 g/L. The pH value is regulated and controlled to be 8.0, the reaction temperature is 30 ℃, and the dosage of the defoaming agent is 0.1 percent. The aeration rate was 1.0 vvm, the pressure was 0.075MPa, and the stirring speed was 250 rpm.
When the reaction time is 12 hours, the conversion rate of the substrate D-type glufosinate-ammonium is 96.1%. After 24 hours of reaction, the substrate conversion rate is 99.1%, and the final product yield can reach 46.4%.
Example 4: variable ventilation constant pressure condition
Carrying out enzyme catalysis reaction in a 30L reaction kettle, wherein the concentration of the substrate D and the L-type glufosinate-ammonium is 400mmol/L, and the adding amount of the coexpression recombinant escherichia coli is 15 g/L. The pH value is regulated and controlled to be 8.0, the reaction temperature is 30 ℃, and the dosage of the defoaming agent is 0.1 percent. Setting the ventilation quantity to be 1.0 vvm when the reaction is carried out for 0-4 h; the ventilation volume is reduced to 0.5 vvm within 4-8 h; the ventilation volume is reduced to 0.25 vvm within 8-12 h, the pressure is always kept at 0.075MPa, and the stirring speed is 250 rpm.
After the reaction is carried out for 12 hours, the substrate D-type glufosinate-ammonium is completely converted, and the final product yield can reach 46.9 percent
Example 5: variable gas and pressure conditions
Carrying out enzyme catalysis reaction in a 30L reaction kettle, wherein the concentration of the substrate D and L-type glufosinate-ammonium is 400mmol/L, and the adding amount of the coexpression recombinant escherichia coli is 15 g/L. The pH value is regulated and controlled to be 8.0, the reaction temperature is 30 ℃, and the dosage of the defoaming agent is 0.1 percent. Setting the ventilation quantity at 1.0 vvm and the pressure at 0.1MPa when the reaction is carried out for 0-4 h; the ventilation volume is reduced to 0.5 vvm within 4-8 h, and the pressure is 0.075 MPa; the ventilation volume is reduced to 0.25 vvm within 8-12 h, the pressure is 0.05MPa, and the stirring speed is 250 rpm.
After 10 hours of reaction, the substrate D-type glufosinate-ammonium is completely converted, and the yield of the final product reaches 47.2%.
Comparative example 1: constant aeration normal pressure condition
Carrying out enzyme catalysis reaction in a 30L reaction kettle, wherein the concentration of the substrate D and the L-type glufosinate-ammonium is 400mmol/L, and the adding amount of the coexpression recombinant escherichia coli is 15 g/L. The pH value is regulated and controlled to be 8.0, the reaction temperature is 30 ℃, and the dosage of the defoaming agent is 0.1 percent. The aeration rate was 0.5 vvm, the pressure was normal pressure, and the stirring speed was 250 rpm.
When the reaction time is 12 hours, the conversion rate of the substrate D-type glufosinate-ammonium is 90.1%. After 24 hours of reaction, the substrate conversion rate is 92.4%, and the final product yield can reach 41.5%.
Example 1 and comparative example 1 show that the reaction conversion rate is faster under pressurized conditions than in conventional reactions, i.e., the reaction is conducted under atmospheric pressure conditions.
Compared with examples 1 to 5, in the reaction process, the reaction can realize complete conversion of the substrate in a shorter time by adopting the variable-ventilation and variable-pressure condition compared with the constant-ventilation and constant-pressure condition.
The enzyme activities of the D-amino acid oxidases of the examples of the present invention under different reaction conditions were tested, and the D-amino acid oxidase under a constant aeration pressure condition (comparative example 1), the D-amino acid oxidase under a constant aeration pressure condition (example 1), and the D-amino acid oxidase under a variable aeration pressure condition (example 5) were selected as test subjects.
D-amino acid oxidase activity was determined by the peroxidase-o-dianisidine method (cited in the literature Biochemical information I, Boehringer Mannheim GmbH, Biochemical, Germany, 1973, pp. 41). The reaction mixture included buffer-substrate (D-methionine, 10M) solution, sodium potassium phosphate buffer (pH 8.0, 0.2M), o-dianisidine, catalase (300U/cm) suspended in ammonium sulfate solution (3.2M)3) And a sampled reaction solution containing D-amino acid oxidase to a final volume of 1 cm3. The enzyme-free mixture was pre-incubated at 30 ℃ and the reaction was started for 10 min by adding a reaction solution containing D-amino acid oxidase (100. mu.L) which oxidized the substrate D-methionine to produce hydrogen peroxide as a by-product and 2-oxo-4-methylthiobutanoic acid as a product. The brown color formed by the reaction of hydrogen peroxide and o-dianisidine (10M) was determined by spectrophotometer at 436 nm. The test results are shown in fig. 2.
Under the same system, the initial value of the specific enzyme activity of the recombinant escherichia coli wet cells is about 120U/g DCW. As seen from FIG. 2, the loss of enzyme activity was minimal at constant aeration pressure, and 81.0U/g DCW remained after 12 hours; under a constant-ventilation constant-pressure mode (0.5 vvm, 0.05 Mpa), the enzyme activity loss is large, and only 37.9U/g DCW is obtained at 12 hours; under the mode of changing air and pressure, the enzyme activity is 59.6U/g DCW within 12 h. The invention shows that the enzyme activity loss of the D-amino acid oxidase can be reduced by adopting a variable-aeration variable-pressure mode (compared with constant aeration constant pressure) in the reaction process, so that the D-amino acid oxidase always maintains higher enzyme catalytic activity in the whole reaction process, and a reaction substrate can be completely catalytically converted in shorter reaction time.
The present invention is not limited to the above-described specific embodiments, and various modifications and variations are possible. Any modifications, equivalents, improvements and the like made to the above embodiments in accordance with the technical spirit of the present invention should be included in the scope of the present invention.
Claims (10)
1. A process for the pressure-catalyzed preparation of 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid comprising the steps of:
adding a coexpression recombinant escherichia coli wet thallus and a pH regulator into racemic glufosinate-ammonium serving as a substrate to regulate the pH of a reaction system to be 6.5-9.0, and carrying out the reaction process under the conditions of sealing, ventilation and pressurization;
the recombinant escherichia coli can co-express D-amino acid oxidase and catalase;
the reaction process at least comprises the following two stages:
a first reaction stage, wherein the reaction system is controlled to be in a first ventilation quantity and a first pressure state;
in the second reaction stage, the reaction system is controlled to be in a second ventilation rate and a second pressure state;
the first ventilation volume is greater than the second ventilation volume and the first pressure is greater than the second pressure.
2. The method for preparing 2-carbonyl-4- (hydroxymethylphosphono) butyric acid by pressure catalysis according to claim 1, wherein the reaction temperature of the reaction process is 25-45 ℃.
3. The process for pressure-catalyzed preparation of 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid as claimed in claim 1, wherein an antifoaming agent is further added to the reaction system.
4. The method for preparing 2-carbonyl-4- (hydroxymethylphosphono) butyric acid by pressure catalysis as claimed in claim 3, wherein the amount of the antifoaming agent added is 0.05-5% of the total volume of the reaction system.
5. The method for preparing 2-carbonyl-4- (hydroxymethylphosphono) butyric acid by pressure catalysis according to claim 1, wherein the concentration of the racemic glufosinate-ammonium in the reaction system is 50-500 mmol/L, and the addition amount of the co-expression recombinant Escherichia coli is 5-20 g/L.
6. The method for preparing 2-carbonyl-4- (hydroxymethylphosphono) butyric acid by pressure catalysis according to claim 1, wherein the pH regulator is ammonia.
7. The method for preparing 2-carbonyl-4- (hydroxymethylphosphono) butyric acid by pressure catalysis according to claim 1, wherein the first aeration amount and the second aeration amount are controlled to be 0.1 to 5 vvm.
8. The pressure-catalyzed process for preparing 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid as claimed in claim 7, wherein the first pressure and the second pressure are controlled to be 0.02 to 1.5 MPa.
9. The pressure-catalyzed process for preparing 2-carbonyl-4- (hydroxymethylphosphono) butanoic acid according to claim 8, wherein the first aeration amount and the second aeration amount are controlled to be 0.1 to 2vvm, and the first pressure and the second pressure are controlled to be 0.02 to 0.1 MPa.
10. The method for preparing 2-carbonyl-4- (hydroxymethylphosphono) butyric acid by pressure catalysis as claimed in claim 1, wherein the stirring speed during the reaction process is controlled to be 50-500 rpm.
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