CN110904161A - Method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting enzyme method - Google Patents
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Abstract
The invention discloses a method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting an enzyme method. Compared with a chemical method and a whole-cell fermentation method, the method has the advantages of mild reaction conditions, less waste liquid generation, high chemical purity of (R) - (-) -3-hydroxybutyric acid, single optical activity and the like. Because the components of the culture medium for producing (R) - (-) -3-hydroxybutyric acid by the enzyme method are far simpler than those of the culture medium produced by the whole-cell fermentation method, the cost for separating the product is greatly reduced, and the purity is higher. The method adopts Diaphorobacter polyhydroxybutyrate ivorans to generate PHB depolymerizing enzyme in a specific culture environment, and simultaneously uses PHB as a carbon source and an inducer for enzymolysis, so that the catalytic efficiency of the enzyme is extremely high.
Description
Technical Field
The invention belongs to the field of bioengineering, relates to a method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting an enzyme method, and particularly relates to a method for producing poly-3-hydroxybutyrate depolymerase by microbial fermentation and producing a high-purity (R) - (-) -3-hydroxybutyric acid monomer by degrading poly-3-hydroxybutyrate by utilizing the depolymerase.
Background
(R) - (-) -3-hydroxybutyric acid ((R) - (-) -3-Hydroxybutanoic acid) is an R-configuration isomer in the 3-hydroxybutyric acid racemate, and is an optically active chiral compound with CAS number 625-72-9.
In the human body, (R) - (-) -3-hydroxybutyric acid is usually synthesized in the liver from acetyl-CoA, a reaction catalyzed by (R) - (-) -3-hydroxybutyric acid dehydrogenase. (R) - (-) -3-hydroxybutyric acid may be used as a backup energy source for the brain during hypoglycemia, a compound that plays an important role in the physiological response to hypoglycemia during hungry states in humans. In addition, (R) - (-) -3-hydroxybutyric acid has certain therapeutic functions for certain diseases, such as: (1) can be used for treating symptoms related to ketone body level, such as relieving nervous disorder diseases or neurodegenerative diseases. (2) Can be used for treating diseases related to carbohydrate metabolism, such as type I diabetes and type II diabetes with low ketosis. (3) (R) - (-) -3-hydroxybutyric acid is a blocker of endogenous histone deacetylase, protecting cells from "oxidative stress" at lower concentrations. Based on the nutritive value and the medicinal value of (R) - (-) -3-hydroxybutyric acid, the derivative and the compound have great potential for producing health-care products and medicines.
At present, the production method of 3-hydroxybutyric acid is mainly a chemical method, and the (S/R) -3-hydroxybutyric acid is obtained by catalytic oxidation of 3-hydroxybutyraldehyde, the reaction needs to be carried out under an acidic condition, and under the condition, the 3-hydroxybutyric acid is easy to generate intramolecular dehydration to form butenal/acid, so that the final conversion rate and yield of the reaction are influenced; or 3-hydroxybutyric acid with higher purity is obtained by hydrolysis reaction of 3-hydroxybutyric acid lactone, but virulent cyanide is added, so that the waste liquid pollutes the environment. In the production method of (R) -3-hydroxybutyric acid and salts thereof (patent No. (CN 107162893A)), a method of alcoholysis of PHB to obtain ethyl trihydroxybutyrate and then reacting with strong base to obtain 3-hydroxybutyric acid salt is adopted, a large amount of alcohol substances are consumed during the alcoholysis reaction, sulfuric acid is required to be added to maintain an acidic condition, and 3-hydroxybutyric acid or salts cannot be directly obtained in one step.
The method for synthesizing 3-hydroxybutyric acid by the biological method mainly comprises the steps of converting a carbon source into acetyl coenzyme A by the self metabolic cycle of microorganisms, and further converting the carbon source into acetoacetyl coenzyme A, 3-hydroxybutyryl coenzyme A and (R) -3-hydroxybutyric acid. For example, in patents (CN2017103961413 and CN2016800351607), two methods for producing 3-hydroxybutyric acid by using different strains can directly extract sodium 3-hydroxybutyrate from fermentation broth, and then obtain (R) -3-hydroxybutyric acid by direct decolorization and acidification. The patent application No. 200910106659.4 discloses a method for preparing ethyl (R) -3-hydroxybutyrate by fermentation of genetically engineered bacteria, wherein the method for directly synthesizing () -3-hydroxybutyrate by using starch as a substrate carbon source by using the genetically engineered bacteria is involved, but the method for separating and purifying (R) -3-hydroxybutyrate and the discussion of the yield of related products are not shown in the examples. Compared with the chemical method, the biological method has low energy consumption, simple process and shorter reaction time, but the substrate conversion rate is very low (the concentration of a glucose substrate is 650 g/L, the concentration of a product, namely 3-hydroxybutyric acid is 11.5 g/L, and the conversion rate of saccharic acid is only 1.77 percent) as seen in the patent examples, a plurality of microorganisms, namely Escherichia (Escherichia), Pseudomonas (Pseudomonas) and Aeromonas (Aeromonas) need to be used in the biological fermentation process, the process is complicated, and Escherichia coli has antibacterial property as a biological engineering bacterium and contains endotoxin, and has potential harm to production personnel and environment. Therefore, its industrial feasibility is low or the production cost will be very high. In addition, the production strain is used for over-expressing related enzymes in metabolic pathways by means of genetic engineering, but the genetic engineering bacteria still have some inevitable problems, such as activity reduction caused by plasmid loss, and various antibiotic resistances left in the genetic manipulation process still have threats to the environment. Meanwhile, products produced by the whole-cell fermentation method are inevitably accompanied by certain byproducts, which have certain influence on the quality of the end product (R) -3-hydroxybutyric acid.
Disclosure of Invention
The invention aims to solve the technical problems that the prior art cannot realize high-efficiency production of high-purity (R) - (-) -3-hydroxybutyric acid monomers and has simple process, and provides a method for producing high-purity (R) - (-) -3-hydroxybutyric acid by adopting an enzyme method.
The invention relates to a method for producing a high-purity (R) - (-) -3-hydroxybutyric acid monomer by adopting an enzyme method, which comprises the following steps:
step (1), amplification culture:
the Diaphorobacter polyhydroxybutyrate ivorans of Diaphorobacter are subjected to amplification culture in an LB culture medium to obtain a large number of strains.
Diaphorobacter polyhybutyrativorans of the genus Diaphorobacter are currently mature strains and can be obtained by purchase.
The culture conditions are as follows: the temperature was controlled at 37 ℃, the pH at 7.2, 180rpm, and the incubation time 16 h.
Step (2), fermenting to produce enzyme:
putting the strain obtained by amplification culture in the step (1) into a fermentation culture medium for fermentation culture to obtain fermentation liquor containing PHB depolymerizing enzyme; and centrifuging and filtering the fermentation liquor to obtain separated and purified PHB depolymerizing enzyme crude enzyme liquid.
Of the chosen fermentation mediumThe components of the composition are as follows: 0.1-10g/L, KNO PHB32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H20.01-1 g/L, NaCl 0.2.2 g/L of O and 0.15g/L, CaCl g of yeast powder20.02-1 g/L, 0.01g/L ferric ammonium citrate and 0-0.1g/L defoaming agent.
The defoaming agent is a polyether compound.
The culture conditions are as follows: the dissolved oxygen constant is controlled to be 15-30%, and more preferably 20-25%; the temperature is controlled to be 30-40 ℃, and is more preferably controlled to be 35 ℃; the pH is controlled to be 6.5-8.0, and the pH is preferably controlled to be 7.2.
In the process of producing the enzyme by fermentation, a fed-batch mode is adopted, and the PHB concentration in the whole process of producing the enzyme by fermentation is controlled to be 0.5 g/L.
The supernatant from which the cells were removed was obtained by centrifugation as described above, and the filtrate was concentrated as necessary to obtain a crude PHB-depolymerizing enzyme solution. Removing residual thalli and macromolecular substances from the PHB depolymerizing enzyme crude enzyme liquid by a microfiltration membrane filtration method to obtain sterile filtrate; concentrating the filtrate if necessary to obtain crude PHB depolymerizing enzyme liquid.
Step (3), enzymolysis:
and (3) degrading PHB by using PHB as a substrate and the PHB depolymerizing enzyme crude enzyme liquid in the step (2), and controlling the pH value to be 6.5-8.0 by alkali to finally obtain the enzymatic hydrolysate containing (R) - (-) -3-hydroxybutyrate.
Depolymerase buffer solution composition: KNO32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H20.01-1 g/L, NaCl 0.2.2 g/L of O and 0.15g/L, CaCl g of yeast powder20.02-1 g/L, and ferric ammonium citrate 0.01 g/L.
And (3) enzymolysis conditions: the temperature is controlled to be 30-45 ℃, and more preferably controlled to be 37 ℃; the pH is controlled to be 6.5-8.5, and the pH is preferably controlled to be 7.5.
In the enzymolysis process, a substrate PHB adopts a fed-batch mode, and the concentration of the substrate PHB is controlled to be 0.1-10g/L, preferably 1 g/L.
Step (4), monomer purification:
extracting high-concentration (R) - (-) -3-hydroxybutyrate from the enzymolysis liquid in the step (3) by adopting absolute ethyl alcohol, and obtaining a high-purity (R) - (-) -3-hydroxybutyrate monomer through acidification and distillation.
In the process of producing PHB depolymerizing enzyme by fermentation, PHB is used as a carbon source and an inducer.
The invention has the beneficial effects that:
compared with a chemical method and a whole-cell fermentation method, the method has the advantages of mild reaction conditions, less waste liquid generation, high chemical purity of (R) - (-) -3-hydroxybutyric acid, single optical activity and the like. Because the components of the culture medium for producing (R) - (-) -3-hydroxybutyric acid by the enzyme method are far simpler than those of the culture medium produced by the whole-cell fermentation method, the cost for separating the product is greatly reduced, and the purity is higher. The invention adopts Diaphorobacter polyhydroxybutyrate novorans to generate PHB depolymerase under a specific culture environment, and simultaneously uses PHB as a carbon source and an inducer for enzymolysis, the catalytic efficiency of the enzyme is extremely high (more than 250g of poly-3-hydroxybutyrate to (R) - (-) -3-hydroxybutyrate monomer can be catalytically depolymerized by crude enzyme per gram). Therefore, the process for producing (R) - (-) -3-hydroxybutyric acid by the enzyme method has a prospect of large-scale industrial application.
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FIG. 1 is a graph showing the analysis of PHB depolymerase degradation efficiency.
Detailed Description
The technical solutions and effects of the present invention are further described below with reference to examples, but the present invention is not limited to the materials, the ratios and the methods shown in the examples, and any variations that can be easily imagined by a person of ordinary skill based on the materials combinations and methods shown in the examples belong to the scope of the present invention.
The invention relates to the addition amount, content and concentration of various substances, wherein the percentage content refers to the mass percentage content except for special description.
In the embodiment of the present invention, if no particular description is made on the operation temperature, the temperature is generally worth to be room temperature (15 to 30 ℃).
The enzyme activity detection method adopted in the following examples is as follows:
preparing PHB suspension with concentration of 3g/L, treating with ultrasonic wave for 5min before use, and shaking up. 400 mu L of suspension is mixed with a sample to be tested to 4mL, and the final concentration of PHB is 0.3 g/L. In A650nmThe absorbance of the mixture was measured by using distilled water to zero and then measuring the value of A650The absorbance value is recorded as A0. Placing the mixed solution in 35 deg.C constant temperature water bath for reaction for 30min, and measuring absorbance at 650nm wavelength and recording as A1. One unit of enzyme activity is defined as: causing a reduction of light absorption of 0.001 per minute(A650)The amount of enzyme required. Calculating the formula: enzyme activity per mL (U/mL) ((A)1-A0)/30min)/3.6mL。
Example 1: activating seed liquid
The strain Diaphorobacter polyhydroxybutyrate vorans stored in the freeze-dried tube in example 1 was inoculated into LB slant medium (5 g/L yeast powder, 10g/L peptone, 10g/L sodium chloride, 20g/L agar) and subjected to static culture at 35 ℃ for 12 hours. A single colony of EMD bacteria is selected, inoculated in an LB liquid culture medium (5 g/L of yeast powder and 10g/L, NaCl 10g/L of peptone), and subjected to shaking culture at 35 ℃ and 180rpm for 12 hours to prepare a primary seed solution. Inoculating the primary seed liquid into enzyme production medium (PHB 1g/L, KNO) according to the inoculation amount of 10%32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H2O0.2 g/L, NaCl 0.2.2 g/L, yeast powder 0.15g/L, CaCl g/L20.036g/L and 0.01g/L ferric ammonium citrate), and culturing at 35 deg.C and 180rpm for 12 hr under shaking to obtain secondary seed solution.
Comparative example 1: enzyme production activity with glucose as sole carbon source
The secondary seed liquid in example 1 is cultured and fermented by a strain culture in a 250mL shake flask, the liquid loading capacity is 100mL, and the components of a culture medium are as follows: glucose 1g/L, KNO32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H2O0.01 g/L, NaCl 0.2.2 g/L, yeast powder 0.15g/L, CaCl g/L20.02g/L、0.01g/L of ferric ammonium citrate. The culture temperature is 35 ℃, the pH value is 7.2, the rotation speed is about 180rpm, and the culture time is 18 h. The fermentation time is 18h, the thalli are removed by centrifugation, the supernatant is taken, and the enzyme activity is measured, and the total enzyme activity is 68U.
Comparative example 2: enzyme production activity of glucose as sole carbon source added with PHB as inducer
The secondary seed liquid in example 1 is cultured and fermented by a strain culture in a 250mL shake flask, the liquid loading capacity is 100mL, and the components of a culture medium are as follows: glucose 1g/L, PHB 0.01.01 g/L, KNO32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H2O0.01 g/L, NaCl 0.2.2 g/L, yeast powder 0.15g/L, CaCl g/L20.02g/L and ferric ammonium citrate 0.01 g/L. The culture temperature is 35 ℃, the pH value is 7.2, the rotation speed is about 180rpm, and the culture time is 18 h. The fermentation time is 18h, the thalli are removed by centrifugation, and the supernatant is taken to measure the enzyme activity, and the total enzyme activity is 129U.
Example 2-1: and (3) fermenting to produce enzyme: enzyme production activity of PHB as sole carbon source and inducer
The secondary seed liquid in example 1 is cultured and fermented by a strain culture in a 250mL shake flask, the liquid loading capacity is 100mL, and the components of a culture medium are as follows: PHB 1g/L, KNO32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H2O0.01 g/L, NaCl 0.2.2 g/L, yeast powder 0.15g/L, CaCl g/L20.02g/L and ferric ammonium citrate 0.01 g/L. The culture temperature is 35 ℃, the pH value is 7.2, the rotation speed is about 180rpm, and the culture time is 18 h. The fermentation time is 18h, the thalli are removed by centrifugation, the supernatant is taken, and the enzyme activity is measured, wherein the total enzyme activity is 210U.
Examples 2-2 to 2-4: enzyme production by fermentation
500mL of the secondary seed solution of example 1 was inoculated into a 7L fermenter, and the liquid loading was 5L. The fermentation medium components are shown in Table 1. The culture temperature is 35 ℃, the pH value is about 7.2, the dissolved oxygen constant is controlled to be 20-25%, and the ventilation quantity and the stirring speed are flexibly adjusted according to the dissolved oxygen constant. Feeding PHB in a fed-batch manner, and controlling the concentration of PHB in the whole fermentation enzyme production process to be 0.5 g/L. The total fermentation time was 18 h.
TABLE 1 examples 2-2 to 2-4 Total enzyme Activity Using fermentation Medium without Contents of ingredients
Examples 2-5 to 2-7: enzyme production by fermentation
500mL of the secondary seed solution of example 1 was inoculated into a 7L fermenter, and the liquid loading was 5L. The fermentation medium comprises the following components: PHB 1g/L, KNO32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H2O0.2 g/L, NaCl 0.2.2 g/L, yeast powder 0.15g/L, CaCl g/L20.036g/L and 0.01g/L of ferric ammonium citrate. The dosage of the defoaming agent is 0.05 g/L. The culture temperature is 35 ℃, the pH value is about 7.2, the dissolved oxygen constant is shown in table 2, and the ventilation quantity and the stirring speed are flexibly adjusted according to the dissolved oxygen constant. Feeding PHB in a fed-batch manner, and controlling the concentration of PHB in the whole fermentation enzyme production process to be 0.5 g/L. The total fermentation time was 18 h.
TABLE 2 examples 2-5 to 2-7 Total enzyme Activity in culture environments without dissolved oxygen constant
| Dissolved oxygen constant | Total enzyme activity | |
| Examples 2 to 5 | 15～20％ | 7.8×104U |
| Examples 2 to 6 | 20～25％ | 9.5×104U |
| Examples 2 to 7 | 25～30％ | 8.5×104U |
Examples 2-8 to 2-9: enzyme production by fermentation
The culture temperatures of examples 2 to 6 were changed to those in Table 3, and the other conditions were not changed.
TABLE 3 examples 2-6, 2-8, 2-9 use of total enzyme activity without incubation temperature
| Temperature of culture | Total enzyme activity | |
| Examples 2 to 6 | 35℃ | 9.5×104U |
| Examples 2 to 8 | 30℃ | 6.7×104U |
| Examples 2 to 9 | 40℃ | 5.4×104U |
Examples 2-10 to 2-11: ferment to produce enzyme (ferment pH 6.5)
The pH of the fermentations of examples 2-6 was changed to Table 4, with the other conditions being unchanged.
TABLE 4 examples 2-6, 2-10, 2-11 use of total enzyme activity without fermentation pH
| pH of fermentation | Total enzyme activity | |
| Examples 2 to 6 | 7.2 | 9.5×104U |
| Examples 2 to 10 | 6.5 | 4.9×104U |
| Examples 2 to 11 | 8.0 | 7.8×104U |
Example 3: preparation of crude enzyme solution
5L of the fermentation liquid obtained in examples 2 to 6 was centrifuged at 4 ℃ and 6000rcf for 10min to remove the microbial cells, and the supernatant was collected. The supernatant was filtered through a sterile 0.22 μm PES microfiltration membrane to remove the residual bacteria that were not centrifuged off, and a clear and transparent filtrate was collected. And (5) performing biological flat plate coating detection, and sterilizing the filtrate.
PHB depolymerase degradation efficiency: 120mL of sterilized PHB depolymerizing enzyme crude enzyme liquid is taken, and the mass of total protein is 4.32 g; 1.2g of PHB powder was added to a final concentration of 10 g/L. Reacting at 35 deg.C, maintaining pH at about 7.5, and sampling at regular time to determine PHB residue. And (4) preparing a standard curve according to the relation between the OD value and the concentration of the PHB powder suspension, and calculating the PHB residual quantity. The data results are shown in table 1:
TABLE 1
| Time (h) | Residual quantity (g) |
| 0 | 1.189 |
| 1 | 1.044 |
| 2 | 0.576 |
| 3 | 0.516 |
| 4 | 0.456 |
| 5 | 0.396 |
| 6 | 0.348 |
| 7 | 0.336 |
As can be seen from the analysis of Table 1 and FIG. 1, the degradation rate of PHB is fast around the first 2h, the degradation rate becomes slower and slower after 2h, and the degradation rate approaches zero after 6h, but the enzyme activity still plays a role, but the rate becomes slower and slower.
From the above data it is known that 0.853g PHB requires 4.32mg crude protease to function within 7h for complete degradation, plus error and continued extension of reaction time, it is estimated that 4mg crude protease is required for 1g PHB degradation, i.e. 1g crude protease is able to catalyze the degradation of 250g PHB.
Examples 4-1 to 4-3: method for producing (R) - (-) -3-hydroxybutyric acid by enzymatic hydrolysis of PHB
5L of the sterile filtrate from example 3 was charged to a sterile fermenter. The temperature of the enzymatic hydrolysis is shown in Table 5, and the pH is maintained at about 7.5. Feeding PHB in a flowing mode, and controlling the concentration of PHB in the whole enzymolysis process to be 1 g/L. When the alkali liquor maintaining the pH is not supplemented any more, the (R) - (-) -3-hydroxybutyric acid is not produced any more, and the enzymolysis process is finished. And (3) determining the purity of the 3-hydroxybutyric acid by adopting high performance liquid chromatography. The chromatographic column is ZORBAX SB-C18 column (4.6X 250mm), the mobile phase is acetonitrile and HCl-water (pH3.0) (gradient elution conditions are 0-5min, 90% HCl-water, 10% acetonitrile, 5-20min, 90 → 10% HCl-water, 10 → 90%, 20-25min, 10% HCl-water, 90% acetonitrile, 25-27min, 10 → 90% HCl-water, 90 → 10%, 27-35min, 90% HCl-water, 10%) ultraviolet detection wavelength is 210nm, the sample injection amount is 10 muL, the flow rate is 1mL/min, and the column temperature is 30 ℃.
TABLE 5 examples 4-1 to 4-3 production of (R) - (-) -3-hydroxybutyric acid without enzymatic hydrolysis temperature
| Temperature of enzymolysis | (R) - (-) -3-hydroxybutyric acid g/L | |
| Example 4-1 | 30℃ | 12.1 |
| Example 4 to 2 | 37℃ | 15.8 |
| Examples 4 to 3 | 45℃ | 7.2 |
Examples 4-4 to 4-5: method for producing (R) - (-) -3-hydroxybutyric acid by enzymatic hydrolysis of PHB
The fermentation pH of example 4-2 was changed to Table 6, and other conditions were not changed.
TABLE 6 examples 4-2, 4-4, 4-5 production of (R) - (-) -3-hydroxybutyric acid using pH without enzymatic hydrolysis
| pH of enzymolysis | (R) - (-) -3-hydroxybutyric acid g/L | |
| Example 4 to 2 | 7.5 | 15.8 |
| Examples 4 to 4 | 6.5 | 11.8 |
| Examples 4 to 5 | 8.5 | 8.4 |
Example 5: extracting and purifying (R) - (-) -3-hydroxybutyric acid (enzymolysis temperature 37 deg.C, enzymolysis pH7.5)
5L of the enzymatic hydrolysate 6000rcf obtained in example 4-2 was centrifuged for 10min to remove the PHB solids which were not completely degraded in the solution, and the supernatant of the enzymatic hydrolysate after centrifugation was collected. The supernatant was filtered through a 0.1 μm hollow fiber membrane to remove PHB depolymerase from the supernatant to prevent interference with subsequent purification. Then adding 1% active carbon, stirring and decolorizing for 30min, and filtering to obtain light yellow or colorless liquid. After concentrating 5L of the filtrate to 250mL by using a forward osmosis device, the concentrate was dried in an oven at 80 ℃ to obtain 100g of solid powder.
100g of the powder was mixed with absolute ethanol at a ratio of 1:5(m/v), and stirred for 2 hours to be sufficiently dissolved in the absolute ethanol. Filtering to remove other inorganic salts (derived from the culture medium) insoluble in absolute ethyl alcohol, and distilling to remove absolute ethyl alcohol to obtain 3-hydroxybutyrate with high purity. And dissolving the obtained 3-hydroxybutyrate in an excessive HCl solution, and stirring uniformly and fully acidifying. Then according to the different boiling points of water and 3-hydroxybutyric acid, firstly removing water by means of reduced pressure distillation, and then collecting high-purity (R) - (-) -3-hydroxybutyric acid.
The purity of (R) - (-) -3-hydroxybutyric acid was found to be 99%, and the specific optical rotation was-25 ° (C-6% in H)2O)。
The sequence map of the PHB depolymerizing enzyme gene is shown in SIQ ID NO. 1:
ATGCCATTCCATCGTTTTCTGCTCGCTGCTGCGTTGGCATCCGCCGGCATGGCGCAGGCGGCGGCGCCGCTGGGCCAGTACAACATCGACACGGGCAAGATCAGCGTTTCAGGGCTGTCCTCAGGCGGCTTCATGGCCAACCAGCTTGGCAATGCGTACTCGTCCACCTTCATGGGCGTGGGCATCTTTGCCGCGGGGCCGTACATGTGCGCGGGCCACAACAACTACACGGCCTGCATGTACAACGCCAGCATCAGCGCCAGCCAGCAGAGCGCGATGCAGGGCAGCATCGACAGCTACAGCGCCAACGGCACCATCGATGGCAAGTCCGGCATTGCTGCTCAGAAGATCTACATCTTCACGGGCACCAGCGACTACACCGTGGGGCCCAACCTGACCGATGCGCTGCAGACCCAGTACCTCAACAATGGGGTGCCCGCGGGCAACATCACGTACGTCAAGCGCAGCGGGACCGCGCATGTGCTGCCCACCGACTTTGACAGCACGGGCAACAACGCATGCAGCAGCAGCACCTCGCCCTTCATCAGCAACTGCGGTTATGACGGCGCGGGAGCGGTGCTCTCACATTTCTATGGCGCGCTGAATGCGCGCAACAACGCCCCTGCGGCAGCCAACTACATCGAGTTCGATCAAAGCGCCTATACCGCAGGCAACCCGGGCATGGCCGCCAACGCCTGGCTGTACGTGCCCGCAAGCTGCGCCAACGGCGCGCAGTGCAAGCTGCACGTCGTGCTGCACGGTTGCCAGCAGAGCACCGACAAGATCGGCGACAAGTTCGTCAAGAACACCGGCTATAGCCGCTGGGCCGATACCAACCAGATCATCCTGCTGTTCCCGCAGACCCGGATCGACAACACCAGCCGCAGCACCGCCAAGAGCGGATCGCTGGCCAACCCCAACGCCTGCTGGGACTGGATCGGCTGGTACGGCGGCAATTTCGCGCAAAAGAGCGGCGCGCAGATGGCCGCGATCAAGGCCATGGTGGATCGCATCGCTTCCGGTGCAGGTTCGGGCGACGGCGGCGGCACCCCACCGCCCACCGATCCGGTCCTGCCAGCGCCCACCGGTCTCAGCGCGTCGGGCGCCACCGCCACAAGCATGAACCTGGCCTGGAATGCCGTGGCTGGCGCCAACGGCTACAACGTCTACCGCAACGGCAACAAAGCCAACGCCCTTACCGTCTACGCCACCAGCTATACCGACTCGGCGCTGACCGCTTCCACCACCTACAGCTGGGCGGTGCGTGCGGTGGATGCCAACGGCGCAGAGGGTGATGCGTCGAACGCCGTGAACGGCACCACGCTCGCGCCCACGCAGTCCCCTGGCACCTGCACCACAGCCAGCAACTATGCGCACACACAAGCGGGGCGCGCCTACCAGCAGGGCGGCTACACCTACGCCAATGGCTCCGGCCAGAGCATGGGGCTGTGGAACGTGTTCGTGACCACCACGCTCAAGCAGACAGCCACCAATTACTACGTGATCGGCACCTGCCCCTGA
Claims (10)
1. a method for producing high-purity (R) - (-) -3-hydroxybutyric acid by an enzymatic method is characterized by comprising the following steps:
step (1), amplification culture:
carrying out amplification culture on Diaphorobacter polyhydroxybutyrate ivorans of Diaphorobacter in an LB culture medium to obtain a large number of strains;
step (2), fermenting to produce enzyme:
putting the strain obtained by amplification culture in the step (1) into a fermentation culture medium for fermentation culture to obtain fermentation liquor containing PHB depolymerizing enzyme; centrifuging and filtering the fermentation liquor to obtain separated and purified PHB depolymerizing enzyme crude enzyme liquid;
the composition of the selected fermentation medium included the following: 0.1-10g/L, KNO PHB32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H2O 0.01～1gL, NaCl 0.2.2 g/L, yeast powder 0.15g/L, CaCl g20.02-1 g/L, and 0.01g/L ferric ammonium citrate;
the culture conditions are as follows: controlling the dissolved oxygen constant at 15-30%; controlling the temperature to be 30-40 ℃; controlling the pH value to be 6.5-8.0;
the PHB concentration in the fermentation enzyme production process is controlled to be 0.5 g/L;
step (3), enzymolysis:
degrading PHB by using PHB depolymerizing enzyme crude enzyme liquid in the step (2) by using PHB as a substrate, and controlling the pH value to be 6.5-8.0 by using alkali to finally obtain enzymatic hydrolysate containing (R) - (-) -3-hydroxybutyrate;
step (4), monomer purification:
extracting high-concentration (R) - (-) -3-hydroxybutyrate from the enzymolysis liquid in the step (3) by adopting absolute ethyl alcohol, and obtaining a high-purity (R) - (-) -3-hydroxybutyrate monomer through acidification and distillation.
2. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by enzymatic method according to claim 1, wherein the culture conditions in step (1): the temperature was controlled at 37 ℃, the pH at 7.2, 180rpm, and the incubation time 16 h.
3. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to any of claims 1-2, wherein the fermentation medium of step (2) further comprises antifoaming agent 0-0.1 g/L.
4. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to claim 3, wherein the defoaming agent is selected from polyether compounds.
5. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to any of claims 1-4, wherein the culture conditions in step (2): controlling the dissolved oxygen constant to be 20-25%; controlling the temperature at 35 ℃; the pH was controlled at 7.2.
6. The process for producing (R) - (-) -3-hydroxy of high purity by enzymatic method according to any one of claims 1 to 5A method of butyric acid, characterized in that step (3) depolymerase buffer solution composition: KNO32g/L、KH2PO41g/L、Na2HPO43g/L、(NH4)2SO41g/L、MgCl2·6H20.01-1 g/L, NaCl 0.2.2 g/L of O and 0.15g/L, CaCl g of yeast powder20.02-1 g/L, and ferric ammonium citrate 0.01 g/L.
7. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to any one of claims 1 to 6, wherein the enzymatic hydrolysis conditions of step (3): the temperature is controlled to be 30-45 ℃, and more preferably controlled to be 37 ℃; the pH is controlled to be 6.5-8.5, and the pH is preferably controlled to be 7.5.
8. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to any one of claims 1 to 6, wherein the enzymatic hydrolysis conditions of step (3): controlling the temperature at 37 ℃; the pH was controlled at 7.5.
9. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to any of claims 1-8, wherein the concentration of PHB as the substrate during the enzymatic hydrolysis in step (3) is controlled to be 0.1-10 g/L.
10. The process for producing high-purity (R) - (-) -3-hydroxybutyric acid by the enzymatic method according to any of claims 1-8, wherein the concentration of PHB substrate during the enzymatic hydrolysis in step (3) is controlled to 1 g/L.
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| CN114436807A (en) * | 2022-01-24 | 2022-05-06 | 珠海麦得发生物科技股份有限公司 | Preparation method of (R) -3-hydroxybutyrate |
| CN114436807B (en) * | 2022-01-24 | 2023-12-22 | 珠海麦得发生物科技股份有限公司 | Preparation method of (R) -3-hydroxybutyrate |
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| CN114591499B (en) * | 2022-03-17 | 2023-06-13 | 珠海麦得发生物科技股份有限公司 | Preparation method and application of poly (R) -3-hydroxybutyrate |
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