CN115786290A - Method for converting S adenosylmethionine into ACC (ACC) by using magnetic nano immobilized enzyme - Google Patents
Method for converting S adenosylmethionine into ACC (ACC) by using magnetic nano immobilized enzyme Download PDFInfo
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Abstract
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for converting S adenosylmethionine into ACC (ACC) by using magnetic nano immobilized enzyme, which comprises the following steps: cultivating the seeds under certain conditions to obtain etiolated seedlings; extracting 1-aminocyclopropanecarboxylate synthetase (ACS) solution from etiolated seedlings; the extract is treated by ammonium persulfate salting-out method and ultrafiltration method to obtain high-purity 1-aminocyclopropanecarboxylate synthetase (ACS); obtaining magnetic nano immobilized 1-aminocyclopropanecarboxylic acid synthetase by a glutaraldehyde crosslinking method from high-purity 1-aminocyclopropanecarboxylic acid synthetase (ACS); the magnetic nano immobilized 1-aminocyclopropanecarboxylate synthetase is utilized to carry out continuous cyclic biotransformation on S-adenosylmethionine in a coenzyme solution system to synthesize the 1-aminocyclopropanecarboxylic acid. The 1-aminocyclopropane-1-carboxylic acid synthetase is extracted and prepared from plant etiolated seedlings, and the magnetic nano immobilized enzyme prepared by the cross-linking method can be recycled, so that the cost is saved.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for converting S adenosylmethionine into ACC (ACC) by using magnetic nano immobilized enzyme.
Background
Adams and Yang, in 1979, suggested that 1-aminocyclopropane-1-carboxylic Acid (ACC) was the direct precursor for ethylene biosynthesis. And preliminarily clarified by a large number of experiments, the biosynthesis of ethylene starts from methionine (Met), 1-aminocyclopropane-1-carboxylic Acid (ACC) is taken as a key intermediate, and Ethylene (ET) is synthesized under the catalysis of another rate-limiting enzyme ACC oxidase (ACO). Since then, a number of studies have shown that ACC has a significant growth regulating effect on most plants.
Research shows that ACC has excellent physiological regulation effect on plant, animal, such as silkworm, mouse, etc. and is one new kind of animal and plant double growth regulator. In the medical field, ACC also has a great potential for medical use, for example, conners and Washburn et al have found that ACC has a tumor cell inhibiting effect. In addition, some natural and synthetic ACC derivatives are useful as bactericides, fungicides, gametocides, enzyme inhibitors, nerve stimulants, and the like. Due to the great application value and prospect of ACC and derivatives thereof in biology, medicine, agricultural science and the like, many chemists are actively engaged in the research of synthetic methods of the compounds.
Currently, ACC is mainly obtained by chemical synthesis. The synthesis routes of ACC have tens of routes, but they are summarized in the following three categories: glycine derivative-1,2-dibromoethane cyclization method, alanine derivative-diazomethane method or special precursor intramolecular cyclization synthesis method. However, the methods have the disadvantages of complicated steps, low yield and high production cost, and the heavy metal participates in the reaction process, thereby causing huge damage to the environment; therefore, the biological synthesis method for preparing ACC becomes the development direction in the future. At present, there is no report on the production of ACC by biosynthetic methods, which is still blank in the art.
Disclosure of Invention
In view of the above, the invention provides a method for synthesizing ACC by converting S-adenosylmethionine with a magnetic nano immobilized enzyme, so as to solve the problems of complicated steps, low yield, high production cost, participation of heavy metals in the reaction process and great damage to the environment in the method for synthesizing ACC in the prior art.
In order to achieve the purpose, the invention is realized by the following technical scheme: the invention provides a method for converting S adenosylmethionine into ACC by using magnetic nano immobilized enzyme, which is characterized by comprising the following steps: the method comprises the following steps: cultivating at least one of the seeds of towel gourd, tomato, soybean and corn under a certain condition to obtain etiolated seedling; step two: extracting 1-aminocyclopropanecarboxylate synthetase (ACS) solution from etiolated seedlings; step three: obtaining high-purity 1-aminocyclopropanecarboxylate synthetase (ACS) by using the extracting solution in the step two through an ammonium persulfate salting-out method and an ultrafiltration method; step four: obtaining magnetic nano immobilized 1-aminocyclopropanecarboxylic acid synthetase by a glutaraldehyde crosslinking method from high-purity 1-aminocyclopropanecarboxylic acid synthetase (ACS); step five: the magnetic nano immobilized 1-aminocyclopropanecarboxylate synthetase is utilized to carry out continuous cyclic biotransformation on S-adenosylmethionine in a coenzyme solution system to synthesize the 1-aminocyclopropanecarboxylic acid.
In the above method for synthesizing ACC by converting S-adenosylmethionine using magnetic nano-immobilized enzyme, as a preferred embodiment, the first step is specifically: cleaning one of 1kg of towel gourd, tomato, soybean or corn seeds with distilled water, soaking for 24h, placing on a wet gauze, and germinating in an incubator at 25 deg.C; washing the seedlings with distilled water once a day, germinating for 6d, selecting the seedlings with good growth condition, grinding to slightly damage the tail part of hypocotyl, and refrigerating for 15-26h to obtain enzyme extraction material.
In the above method for synthesizing ACC by converting S-adenosylmethionine using a magnetic nano-immobilized enzyme, as a preferred embodiment, the second step is specifically: adding 0.01-0.05% of dithiothreitol and 0.0001-0.0005% of pyridoxal phosphate into a phosphate buffer solution with the pH of 8.0 to serve as an extracting solution, wherein the feed-liquid ratio is 1:5-15, crushing the seedlings by a beater for 1-3min, and then putting the crushed seedlings into an ultrasonic extractor for extraction for 2-5min; filtering the extractive solution with 5-6 layers of gauze to obtain extractive solution.
In the above method for synthesizing ACC by converting S-adenosylmethionine using a magnetic nano-immobilized enzyme, as a preferred embodiment, the third step is specifically: adding 20-30% ammonium sulfate by mass into the extractive solution in the second step, fully dissolving, preserving at 4 deg.C for 50-90min, centrifuging at 5000-8000 rpm for 15-20min with a centrifugal extractor, and collecting supernatant; ultrafiltering the supernatant with ultrafiltration membrane with molecular weight cutoff of 3KDa, ultrafiltering the cut-off solution with ultrafiltration membrane of 10KDa, and collecting the cut-off solution as 1-aminocyclopropanecarboxylate synthetase enzyme solution.
In the above method for synthesizing ACC by converting S-adenosylmethionine using a magnetic nano-immobilized enzyme, as a preferred embodiment, the step four is specifically: taking 50-100g of APTS-Fe 4 O 3 Dispersing magnetic nanoparticles in 1-5L mass fraction 10-20% Glutaraldehyde (GA) water solution, stirring for 2-5h, and adding magnet to absorb the magnetic nanoparticles; the prepared APTS-Fe 4 O 3 Uniformly dispersing GA nano particles in 1-5L enzyme liquid, stirring at 20-30 ℃ and 100-200 rpm for 3-6 h to obtain the immobilized enzyme, washing the immobilized enzyme with phosphate buffer solution for several times, wherein the pH of the phosphate buffer solution is =8.0, and storing the phosphate buffer solution in the buffer solution to obtain the 1-aminocyclopropane carboxylic acid synthesized magnetic nano immobilized enzyme.
In the method for synthesizing ACC by converting S-adenosylmethionine using a magnetic nano-immobilized enzyme, the phosphate buffer is preferably a sodium phosphate buffer or a potassium phosphate buffer.
In the above method for synthesizing ACC by converting S-adenosylmethionine using a magnetic nano-immobilized enzyme, as a preferred embodiment, the step five is specifically: preparing an S-adenosylmethionine solution 100-500L with the mass fraction of 1% -10% by using a phosphate buffer solution with the pH value of 8.0, adding 20-60 g dithiothreitol, 1-5g pyridoxal phosphate and 10-50g of magnetic nanoparticle immobilized enzyme, fully sealing and mixing, and then placing into a constant-temperature reaction kettle at the temperature of 20-30 ℃ for stirring and reacting for 3-6 h; then, adding a magnet-adsorbed magnetic nanoparticle immobilized enzyme to the bottom of the reaction kettle, taking out a reaction liquid, and adding a new reaction liquid; continuously and circularly reacting for 5-20 times, and combining reaction liquid to obtain the crude product of the 1-aminocyclopropane carboxylic acid.
The invention provides a method for converting S adenosylmethionine into ACC by using magnetic nano immobilized enzyme, which has the following beneficial effects:
the invention extracts and prepares 1-aminocyclopropane-1-carboxylic acid synthetase from plant etiolated seedlings by a specific method, and prepares magnetic nano immobilized enzyme by a cross-linking method. The immobilized enzyme has strong stability and high catalytic activity. And the reaction solution is easily and quickly separated from the reaction solution through an external magnetic field, and the reaction solution is recycled, so that the cost is saved.
The method for continuously and circularly synthesizing the 1-aminocyclopropane-1-carboxylic acid by utilizing the immobilized 1-aminocyclopropane-1-carboxylic acid synthetase is convenient to amplify and can be used for producing and preparing the 1-aminocyclopropane-1-carboxylic acid on a large scale.
The method for continuously and circularly synthesizing the 1-aminocyclopropane-1-carboxylic acid by using the immobilized 1-aminocyclopropane-1-carboxylic acid synthetase reacts under mild conditions, has low energy consumption, does not use organic reagents and heavy metals, and is environment-friendly.
Drawings
FIG. 1 is a scanning electron microscope image of a magnetic nano immobilized enzyme synthesized from 1-aminocyclopropane carboxylic acid in the invention;
FIG. 2 is an HPLC chromatogram of an ACC standard of the present invention;
FIG. 3 is an HPLC chromatogram of a purified product of the present invention;
FIG. 4 shows the pure product of ACC of the present invention 1 H-NMR spectrum;
FIG. 5 shows the pure ACC of the present invention 13 C-NMR spectrum.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Exemplary embodiments of the present invention are described below in conjunction with specific cases:
example 1
As shown in fig. 1 to 5, the present invention provides a method for synthesizing ACC by converting S adenosylmethionine using magnetic nano-immobilized enzyme, comprising: step one, cultivating at least one of towel gourd, tomato, soybean and corn seeds under a certain condition to obtain etiolated seedlings; the method specifically comprises the following steps: cleaning one of 1kg of towel gourd, tomato, soybean or corn seeds with distilled water, soaking for 24h, placing on a wet gauze, and germinating in an incubator at 25 deg.C; washing the seedlings with distilled water once a day, germinating for 6d, selecting the seedlings with good growth condition, grinding to slightly damage the tail part of hypocotyl, and refrigerating for 15-26h to obtain enzyme extraction material.
Step two: extracting 1-aminocyclopropanecarboxylate synthetase (ACS) solution from etiolated seedlings; the method comprises the following specific steps: taking a phosphate buffer solution with the pH of 8.0, adding 0.01-0.05% of dithiothreitol and 0.0001-0.0005% of pyridoxal phosphate as extracting solutions, wherein the material-liquid ratio is 1:5-15, crushing seedlings by a beater for 1-3min, and then putting the crushed seedlings into an ultrasonic extractor for extraction for 2-5min; filtering the extractive solution with 5-6 layers of gauze to obtain extractive solution.
Step three: using the extracting solution in the second step to obtain high-purity 1-aminocyclopropanecarboxylate synthetase (ACS) by an ammonium persulfate salting-out method and an ultrafiltration method; the method specifically comprises the following steps: adding ammonium sulfate with the mass fraction of 20% -30% into the extracting solution in the second step, fully dissolving, then placing into a storage tank at 4 ℃ for 50min, centrifuging by a centrifugal extractor at 6000 r/min for 15-20min, and taking supernatant; ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 3KDa, ultrafiltering the cut-off solution with ultrafiltration membrane of 10KDa, and collecting the cut-off solution, which is the 1-aminocyclopropanecarboxylate synthetase enzyme solution.
Step four: obtaining magnetic nano immobilized 1-aminocyclopropanecarboxylate synthetase by a glutaraldehyde crosslinking method from high-purity 1-aminocyclopropanecarboxylate synthetase (ACS); the method specifically comprises the following steps: taking 50-100g of APTS-Fe 4 O 3 Dispersing magnetic nanoparticles in 1-5L aqueous solution of Glutaraldehyde (GA) with mass fraction of 10-20%, stirring for 2-5h, and adding magnet to absorb the magnetic nanoparticles; the prepared APTS-Fe 4 O 3 Uniformly dispersing GA nano particles in 1-5L enzyme liquid, stirring at 20-30 ℃ and 100-200 rpm for 3-6 h to obtain the immobilized enzyme, washing the immobilized enzyme with phosphate buffer solution for several times, wherein the pH of the phosphate buffer solution is =8.0, and storing the phosphate buffer solution in the buffer solution to obtain the 1-aminocyclopropane carboxylic acid synthesized magnetic nano immobilized enzyme. The phosphate buffer is a sodium phosphate buffer or a potassium phosphate buffer.
Step five: the magnetic nano immobilized 1-aminocyclopropanecarboxylate synthetase is utilized to carry out continuous cyclic biotransformation on S-adenosylmethionine in a coenzyme solution system to synthesize the 1-aminocyclopropanecarboxylic acid. The method comprises the following specific steps: preparing an S-adenosylmethionine solution 100-500L with the mass fraction of 1% -10% by using a phosphate buffer solution with the pH value of 8.0, adding 20-60 g dithiothreitol, 1-5g pyridoxal phosphate and 10-50g of magnetic nanoparticle immobilized enzyme, fully sealing and mixing, and then placing into a constant-temperature reaction kettle at the temperature of 20-30 ℃ for stirring and reacting for 3-6 h; then, adding a magnet-adsorbed magnetic nanoparticle immobilized enzyme to the bottom of the reaction kettle, taking out a reaction liquid, and adding a new reaction liquid; continuously and circularly reacting for 5-20 times, and combining reaction liquid to obtain the crude product of the 1-aminocyclopropane carboxylic acid.
Example 2
1. Culture of etiolated seedling
1kg of soybeans are cleaned by distilled water, soaked for 24 hours, placed on a wet gauze and germinated in an incubator at 25 ℃. Washing the seedlings with distilled water once a day, after germinating for 6d, selecting the seedlings with better growth condition, grinding to slightly damage the tail part of hypocotyl, and refrigerating for 15h to obtain the enzyme extraction material.
2. 1-aminocyclopropanecarboxylate synthetase extraction
Adding 0.01 wt% of dithiothreitol and 0.0001 wt% of pyridoxal phosphate into pH 8.0 phosphate buffer solution as extractive solution, wherein the ratio of the material to the solution is 1:5, pulverizing the seedling with beater for 1min, and extracting with ultrasonic extractor for 2min. Filtering the extractive solution with 5 layers of gauze to obtain extractive solution.
3. Purification of 1-aminocyclopropanecarboxylate synthetase
Adding ammonium sulfate 20 wt% into the above extractive solution, dissolving completely, preserving at 4 deg.C for 50min, centrifuging at 5000 rpm for 15 min, and collecting supernatant. Ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 3KDa, ultrafiltering the cut-off solution with ultrafiltration membrane of 60KDa, and collecting the cut-off solution, which is the 1-aminocyclopropanecarboxylate synthetase enzyme solution.
4. Immobilization of 1-aminocyclopropane carboxylic acid synthetase
50g of APTS-Fe was taken 4 O 3 Dispersing the magnetic nanoparticles in 1L mass fraction 10% Glutaraldehyde (GA) water solution, stirring for 2h, and adding a magnet to absorb the magnetic nanoparticles. The prepared APTS-Fe 4 O 3 Uniformly dispersing GA nano particles in 1L enzyme solution, stirring 3 h at 20 ℃ and 100-200 rpm to obtain the immobilized enzyme, washing the immobilized enzyme with phosphate buffer (pH = 8.0) for several times, and storing the washed immobilized enzyme in the buffer to obtain the 1-aminocyclopropane carboxylic acid synthesized magnetic nano immobilized enzyme.
5. Process for synthesizing 1-aminocyclopropane carboxylic acid by circulating biotransformation of immobilized enzyme
S-adenosylmethionine solution 100L with mass fraction of 1% is prepared by phosphate buffer solution with pH 8.0, 20 g dithiothreitol, 1g pyridoxal phosphate and 10g magnetic nanoparticle immobilized enzyme are added, and after being sufficiently sealed and mixed, the mixture is placed in a constant temperature reaction kettle at 20 ℃ to be stirred and reacted for 3 h. Then, adding a magnet to adsorb the magnetic nanoparticle immobilized enzyme at the bottom of the reaction kettle, taking out the reaction liquid, and adding a new reaction liquid. Thus, the reaction was continuously circulated 10 times. And combining the reaction liquid to obtain the crude product of the 1-aminocyclopropane carboxylic acid.
Example 3
1. Culture of etiolated seedling
Cleaning 1kg of towel gourd seeds with distilled water, soaking for 24h, placing on a moist gauze, and germinating in an incubator at 25 ℃. Washing the seedlings once every day with distilled water, after germinating for 6d, selecting the seedlings with better growth condition, grinding to slightly damage the tail parts of hypocotyls of the seedlings, and refrigerating for 9h to be used as an enzyme extraction material.
2.1-Aminocyclopropanecarboxylic acid synthetase extraction
Adding 0.01 wt% of dithiothreitol and 0.0001 wt% of pyridoxal phosphate into pH 8.0 phosphate buffer solution as extractive solution, wherein the ratio of the material to the solution is 1:8, pulverizing the seedling with beater for 1-3min, and extracting with ultrasonic extractor for 2min. Filtering the extractive solution with 5 layers of gauze to obtain extractive solution.
3.1-Aminocyclopropanecarboxylic acid synthetase purification
Adding 20-30% ammonium sulfate, dissolving, preserving at 4 deg.C for 50min, centrifuging at 5000 rpm for 15-20min, and collecting supernatant. Ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 3KDa, ultrafiltering the cut-off solution with ultrafiltration membrane of 10KDa, and collecting the cut-off solution, which is the 1-aminocyclopropanecarboxylate synthetase enzyme solution.
4.1-Aminocyclopropanecarboxylic acid synthetase immobilization
Taking 50-100g of APTS-Fe 4 O 3 Dispersing magnetic nanoparticles in 1-5L mass fraction 10-20% Glutaraldehyde (GA) water solution, stirring for 2-5h, and adding magnet to absorb magnetic nanoparticles. The prepared APTS-Fe 4 O 3 Uniformly dispersing GA nano particles in 1-5L enzyme liquid, stirring at 20-30 ℃ and 100-200 rpm for 3-6 h to obtain immobilized enzyme, washing the immobilized enzyme with phosphate buffer (pH = 8.0) for several times, and storing the washed immobilized enzyme in the buffer to obtain the 1-aminocyclopropane carboxylic acid synthesized magnetic nano immobilized enzyme.
5. Process for synthesizing 1-aminocyclopropane carboxylic acid by immobilized enzyme cyclic biotransformation
S-adenosylmethionine solution 100L with mass fraction of 1% is prepared by phosphate buffer solution with pH 8.0, 20 g dithiothreitol, 1g pyridoxal phosphate and 10g magnetic nanoparticle immobilized enzyme are added, and after being sufficiently sealed and mixed, the mixture is placed in a constant temperature reaction kettle at 20 ℃ to be stirred and reacted for 6 h. Then, adding a magnet to adsorb the magnetic nanoparticle immobilized enzyme at the bottom of the reaction kettle, taking out the reaction liquid, and adding a new reaction liquid. Thus, the reaction was continuously circulated 10 times. And combining the reaction liquid to obtain the crude product of the 1-aminocyclopropane carboxylic acid.
Example 4
1. Culture of etiolated seedling
Cleaning 1kg of tomato seeds with distilled water, soaking for 24h, placing on a wet gauze, and germinating in an incubator at 25 ℃. Washing the seedlings with distilled water once a day, germinating for 6d, selecting the seedlings with good growth condition, grinding to slightly damage the tail part of hypocotyl, and refrigerating for 15-26h to obtain enzyme extraction material.
2.1-Aminocyclopropanecarboxylic acid synthetase extraction
Adding 0.01-0.05% of dithiothreitol and 0.0001-0.0005% of pyridoxal phosphate as extracting solutions in a mass fraction of 1:5-15 into phosphate buffer solution with pH of 8.0, pulverizing the seedlings with a beater for 1-3min, and extracting with an ultrasonic extractor for 2-5min. Filtering the extractive solution with 5-6 layers of gauze to obtain extractive solution.
3.1-Aminocyclopropanecarboxylic acid synthetase purification
Adding ammonium sulfate 20-30 wt% into the extractive solution, dissolving completely, preserving at 4 deg.C for 50-90min, centrifuging at 5000-8000r/min for 15-20min, and collecting supernatant. Ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 3KDa, ultrafiltering the cut-off solution with ultrafiltration membrane of 10KDa, and collecting the cut-off solution, which is the 1-aminocyclopropanecarboxylate synthetase enzyme solution.
4.1-Aminocyclopropanecarboxylic acid synthetase immobilization
Taking 50-100g of APTS-Fe 4 O 3 Dispersing the magnetic nanoparticles in 5L mass fraction 10% Glutaraldehyde (GA) water solution, stirring for 5h, and adding a magnet to absorb the magnetic nanoparticles. Uniformly dispersing the prepared APTS-Fe2O3-GA nano particles in 1-5L enzyme liquid, and stirring at 30 ℃ and 200 rpmAnd (3) mixing with 3-6 h to obtain the immobilized enzyme, washing the immobilized enzyme with a phosphate buffer (pH = 8.0) for several times, and storing the washed immobilized enzyme in the buffer to obtain the 1-aminocyclopropane carboxylic acid synthesized magnetic nano immobilized enzyme.
5. Process for synthesizing 1-aminocyclopropane carboxylic acid by immobilized enzyme cyclic biotransformation
Preparing an S-adenosylmethionine solution 100L with the mass fraction of 1% by using a phosphate buffer solution with the pH of 8.0, adding 20 g dithiothreitol, 1g pyridoxal phosphate and 10g of magnetic nanoparticle immobilized enzyme, fully sealing and mixing, and then placing in a constant-temperature reaction kettle with the temperature of 20 ℃ to stir and react for 6 h. Then, adding a magnet to adsorb the magnetic nanoparticle immobilized enzyme at the bottom of the reaction kettle, taking out the reaction liquid, and adding a new reaction liquid. Thus, the reaction was continuously circulated 5 times. And combining the reaction liquid to obtain the crude product of the 1-aminocyclopropane carboxylic acid.
Experimental example 1
Separating and purifying the product
Taking 100ml of the reaction solution in the example 2, concentrating the reaction solution at 50 ℃ and-0.09 MPa under reduced pressure by 200 times, filtering the reaction solution by using a 0.45-um microporous filter membrane, and separating and purifying the reaction solution by using a preparative high performance liquid phase, wherein a chromatographic Column is Atlantis T3 OBD Prep Column, 100A, 10 mu m, 30 mm X150 mm, a mobile phase is 1% methanol-water (containing 0.1% formic acid, v/v), the flow rate is 0.5 ml/min, the detection wavelength is 260nm, and the sample introduction amount is 100 uml. The target off-peak time 13.676 min, fractions were collected and freeze dried to yield 199mg of a white powder material.
Product detection
(1) Detection by HPLC method
Dissolving the separated and purified product 1 mg in 1ml of ultrapure water, filtering with a 0.45um microporous filter membrane, and detecting by an HPLC method. The chromatographic Column was Wotter Atlantis dC18 Column, 100, 5 μm, 4.6 mm X150 mm, mobile phase 1% methanol-water (containing 0.1% formic acid, v/v), flow rate 0.5 ml/min, detection wavelength 260nm, sample introduction 10 uml.
(2) Detection by nuclear magnetic resonance
Taking the separated and purified product 15 mg, and using 0.5 ml DMSO-d 6 Dissolving, carrying out 1 H-NMR、 13 C-NMR detection.
Description of the analysis: the detection and analysis of the product are shown in figure-2, figure-3, figure-4 and figure-5, the data are consistent with the literature reports, and the product obtained by the invention is the 1-aminocyclopropane carboxylic acid.
Conversion rate of reaction
The reaction yield was calculated by detecting the product 1-aminocyclopropanecarboxylic acid in the reaction solution by the above-mentioned HPLC method.
(see the following Table)
| Serial number | Conversion rate |
| Example 2 | 79.2% |
| Example 3 | 79.3% |
| Example 4 | 79.2% |
| Example 5 | 79.1% |
Finally, it should also be noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, 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. Without further limitation, an element defined by the phrase "comprising a … …" does not exclude the presence of another identical element in a process, method, article, or apparatus that comprises the element.
The principle and the implementation of the present invention are explained above by applying specific examples, and the above description of the embodiments is only used to help understanding the method and the core idea of the present invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (7)
1. A method for converting S adenosylmethionine into ACC by using magnetic nano immobilized enzyme is characterized by comprising the following steps:
the method comprises the following steps: cultivating at least one of the seeds of towel gourd, tomato, soybean and corn under a certain condition to obtain etiolated seedling;
step two: extracting 1-aminocyclopropanecarboxylate synthetase (ACS) solution from etiolated seedlings;
step three: obtaining high-purity 1-aminocyclopropanecarboxylate synthetase (ACS) by using the extracting solution in the step two through an ammonium persulfate salting-out method and an ultrafiltration method;
step four: obtaining magnetic nano immobilized 1-aminocyclopropanecarboxylic acid synthetase by a glutaraldehyde crosslinking method from high-purity 1-aminocyclopropanecarboxylic acid synthetase (ACS);
step five: the magnetic nano immobilized 1-aminocyclopropanecarboxylate synthetase is utilized to carry out continuous cyclic biotransformation on S-adenosylmethionine in a coenzyme solution system to synthesize the 1-aminocyclopropanecarboxylate.
2. The method for synthesizing ACC by converting S-adenosylmethionine with magnetic nano-immobilized enzyme according to claim 1, wherein the first step is specifically: cleaning one of 1kg of towel gourd, tomato, soybean or corn seeds with distilled water, soaking for 24h, placing on a moist gauze, and germinating in an incubator at 25 deg.C; washing the seedlings with distilled water once a day, germinating for 6d, selecting the seedlings with good growth condition, grinding to slightly damage the tail part of hypocotyl, and refrigerating for 15-26h to obtain enzyme extraction material.
3. The method for synthesizing ACC by converting S adenosylmethionine with magnetic nano immobilized enzyme according to claim 1, wherein the second step is specifically: adding 0.01-0.05% of dithiothreitol and 0.0001-0.0005% of pyridoxal phosphate into a phosphate buffer solution with the pH of 8.0 to serve as an extracting solution, wherein the feed-liquid ratio is 1:5-15, crushing the seedlings by a beater for 1-3min, and then putting the crushed seedlings into an ultrasonic extractor for extraction for 2-5min; filtering the extractive solution with 5-6 layers of gauze to obtain extractive solution.
4. The method for synthesizing ACC by converting S adenosylmethionine with magnetic nano immobilized enzyme according to claim 1, wherein the third step is specifically: adding ammonium sulfate with the mass fraction of 20% -30% into the extracting solution in the second step, fully dissolving, placing into a storage container at 4 ℃ for 50-90min, centrifuging by a centrifugal extractor at 5000-8000r/min for 15-20min, and taking supernatant; ultrafiltering the supernatant with ultrafiltration membrane with cut-off molecular weight of 3-10KDa, ultrafiltering the cut-off solution with ultrafiltration membrane of 10-20KDa, and collecting the cut-off solution as 1-aminocyclopropanecarboxylate synthetase enzyme solution.
5. The method for synthesizing ACC by converting S adenosylmethionine with magnetic nano immobilized enzyme according to claim 1, wherein the fourth step is specifically: taking 50-100g of APTS-Fe 4 O 3 Dispersing magnetic nanoparticles in 1-5L aqueous solution of Glutaraldehyde (GA) with mass fraction of 10-20%, stirring for 2-5h, and adding magnet to absorb the magnetic nanoparticles; the prepared APTS-Fe 4 O 3 Uniformly dispersing GA nano particles in 1-5L enzyme solution, stirring at 20-30 ℃ and 100-200 rpm for 3-6 h to obtain immobilized enzyme, washing the immobilized enzyme with phosphate buffer solution for several times, wherein the pH of the phosphate buffer solution is =8.0,storing the immobilized product in the buffer solution to obtain the magnetic nano immobilized enzyme synthesized by 1-aminocyclopropane carboxylic acid.
6. The method for converting S-adenosylmethionine into ACC by using magnetic nano-immobilized enzyme according to claim 5, wherein the phosphate buffer is sodium phosphate buffer or potassium phosphate buffer.
7. The method for synthesizing ACC by converting S adenosylmethionine with magnetic nano immobilized enzyme according to claim 1, wherein the step five is specifically as follows: preparing 100-500L of an S-adenosylmethionine solution with the mass fraction of 1% -10% by using a phosphate buffer solution with the pH of 8.0, adding 20-60 g dithiothreitol, 1-5g pyridoxal phosphate and 10-50g of magnetic nanoparticle immobilized enzyme, fully sealing and mixing, and then placing in a constant-temperature reaction kettle with the temperature of 20-30 ℃ to stir and react for 3-6 h; then, adding a magnet-adsorbed magnetic nanoparticle immobilized enzyme to the bottom of the reaction kettle, taking out a reaction liquid, and adding a new reaction liquid; continuously and circularly reacting for 5-20 times, and combining reaction liquid to obtain the crude product of the 1-aminocyclopropane carboxylic acid.
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