CN114381490A - Crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol - Google Patents
Crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol Download PDFInfo
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Abstract
The invention provides a crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol. The crystallization method of the (beta, S) configuration hydroxypropyl tetrahydropyrane triol specifically comprises the following steps: (1) in the presence of carbonyl reductase and coenzyme regeneration enzyme, carrying out asymmetric reduction reaction on D-xylopyranoside-2- (S) acetone to obtain a crude product of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol; (2) and (2) sequentially extracting, concentrating and crystallizing the reaction system obtained in the step (1) to obtain crystals of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol. The invention provides a high-purity (beta, S) configuration hydroxypropyl tetrahydropyrane triol synthesized by utilizing high stereoselectivity and carbonyl reductase aiming at a specific substrate D-xylopyranoside-2- (S) acetone, and a single configuration solid is obtained by crystallization from a specific mixed solvent, so that the purity is high, the quality is good, and the de value can reach 100%.
Description
Technical Field
The invention belongs to the field of chemistry and biology, and particularly relates to a crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol.
Background
Hydroxypropyl tetrahydropyran triol, also known as boscalid, is a glycoprotein mixture derived from xylose. Since xylose is present in large amounts in beech trees and has the ability to promote the production of glycosaminoglycans, i.e. mucopolysaccharides. The hydroxypropyl tetrahydropyrane triol can improve the water content simulation content among extracellular matrixes by stimulating the generation of mucopolysaccharide. Thereby fully filling the clearance of ECM, reducing wrinkles of the skin and showing more delicate. Meanwhile, the collagen peptide plays a role in the DEJ position, promotes the synthesis of collagen VII and collagen IV, enables the epidermis and the dermis of people to be linked more tightly, and enables the whole skin to be plump, more compact and elastic.
Hydroxypropyl tetrahydropyran triol, as a β -xylose derivative, was originally an anti-aging active substance developed by the company Irelaya, and has the following structural formula:
hydroxypropyl tetrahydropyrane triol is added into skin care products in Eurya 2006, and a plurality of brands under the Eurya flag are promoted in a large scale in Eurya 2018, so that the hydroxypropyl tetrahydropyrane triol has an obvious anti-aging effect, and is popular with the beauty industry. The biological activity and safety of a compound is related to its steric structure, and different steric structures may have different activities. According to the degree of influence of the chemical structure of the active substance on the biological activity, the active substance is generally divided into a structure non-specific compound and a structure specific compound, and the hydroxypropyl tetrahydropyrane triol is a structure specific compound, and the stereochemical characteristics of the compound have great influence on the biological activity.
CN200510071732.0 reports that the (β, S) configuration is a dominant configuration, and the bioactivity thereof is much higher than that of a mixture of (β, S) and (β, R) configurations, and the higher the proportion of the (β, S) configuration in the mixture, the higher the bioactivity.
CN200510071732.0 reports that a C-beta-xylopyranoside-2- (S) hydroxypropane compound is selectively obtained by taking D-xylopyranoside-2- (S) acetone as a raw material and adding sodium borohydride and acetic acid into an isopropanol solution, wherein dr is more than or equal to 90%. However, the method needs to add sodium borohydride which is an easily explosive compound, a large amount of hydrogen is released in the reaction, so that the method has certain danger, and the final product needs to be purified by a silica gel column, so that the method is not beneficial to industrialization.
CN201910785216.6 discloses a method for synthesizing hydroxypropyl tetrahydropyrane triol by using rare earth metal complexes, which uses the rare earth metal complexes as catalysts to react D-xylose with ethyl acetoacetate, and then further reduces the D-xylose to hydroxypropyl tetrahydropyrane triol. The yield of the method is only 80 percent, and the C-beta-xylopyranoside-2- (S) hydroxypropane compound cannot be selectively obtained.
CN202010629023.4 discloses a method for preparing hydroxypropyl tetrahydropyrane triol by using a biological enzyme one-pot method. The method is simple and environment-friendly. However, the yield was only 80%, but the C-. beta. -xylopyranoside-2- (S) hydroxypropane compound was also not selectively obtained.
Therefore, the development of a crystallization method for obtaining a single-configuration solid through crystallization, and obtaining the (beta, S) -configuration hydroxypropyl tetrahydropyrane triol with high purity and good quality is urgently needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol, selects high stereoselectivity and carbonyl reductase aiming at specific substrate D-xylopyranoside-2- (S) acetone, synthesizes and obtains high-purity (beta, S) -configuration hydroxypropyl tetrahydropyrane triol, and crystallizes from a specific solvent to obtain a single-configuration solid with high purity and good quality.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a crystallization method of (β, S) -configuration hydroxypropyl tetrahydropyrane triol, specifically comprising the following steps:
(1) in the presence of carbonyl reductase and coenzyme regeneration enzyme, carrying out asymmetric reduction reaction on D-xylopyranoside-2- (S) acetone to obtain a crude product of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol;
(2) and (2) sequentially extracting, concentrating and crystallizing the reaction system obtained in the step (1) to obtain crystals of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol.
In the prior art, most of hydroxypropyl tetrahydropyrane triol exists in aqueous solution or freeze-dried powder because high-purity (beta, S) configuration hydroxypropyl tetrahydropyrane triol cannot be obtained, and the defects of low content, more impurities and poor using effect exist. The invention particularly screens out carbonyl reductase with high stereoselectivity, and uses mixed solvent for crystallization to obtain the high-purity (beta, S) configuration hydroxypropyl tetrahydropyrane triol, which has high purity and good quality.
Preferably, in the step (1), the coenzyme-regenerating enzyme includes any one of formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase or a combination of at least two thereof.
Preferably, the specific operation of step (1) is: mixing D-xylopyranoside-2- (S) acetone, cosubstrate and liquid containing carbonyl reductase and coenzyme regeneration enzyme, and carrying out heat preservation reaction to obtain a reaction liquid containing a (beta, S) -configuration hydroxypropyl tetrahydropyrane triol crude product.
Preferably, the concentration of the D-xylopyranoside-2- (S) propanone in the reaction system is 20-1000g/L, such as 20g/L, 50g/L, 100g/L, 200g/L, 400g/L, 600g/L, 800g/L, 1000g/L, etc., preferably 100-200g/L, such as 100g/L, 120g/L, 140g/L, 160g/L, 180g/L, 200g/L, etc.
Preferably, the volume ratio of the co-substrate to the carbonyl reductase-and coenzyme-regenerating enzyme-containing liquid is (0.5-2):1, and may be, for example, 0.5:1, 0.6:1, 0.8:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, etc., preferably 1: 1.
Preferably, the co-substrate comprises any one or a combination of at least two of an aqueous isopropanol solution, an aqueous glucose solution, or an aqueous formic acid solution, preferably an aqueous isopropanol solution.
Preferably, the co-substrate is present in an amount of 30-50% by mass, for example 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50% etc.
Preferably, the temperature of the incubation reaction is 25-30 ℃, for example, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃ and the like.
Preferably, the incubation time is 8-24h, for example, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc., preferably 10-12 h.
Preferably, the carbonyl reductase-containing liquid and coenzyme regenerating enzyme are prepared by the following preparation method:
(a) after recombining carbonyl reductase and coenzyme regenerative enzyme, inoculating the carbonyl reductase and engineering escherichia coli together into a liquid culture medium for culture;
(b) centrifuging the system obtained by culturing in the step (a) and collecting thalli; and (3) putting the thalli into a buffer solution, crushing, and centrifuging to collect supernatant to obtain liquid containing carbonyl reductase and coenzyme regenerative enzyme.
Preferably, in step (a), the recombination specifically comprises the steps of: extracting genome, cloning carbonyl reductase gene, and constructing and expressing E.coli/pET-28a (+) -KR01 and other recombinant engineering bacteria.
Preferably, the extracted genome is the Streptomyces cyaneogyreius and Rhodococcus ruber DSM 44541 genome.
Preferably, the carbonyl reductase gene cloning comprises the steps of: and (3) sequentially carrying out codon optimization and chemical synthesis on the extracted genome I to obtain carbonyl reductase genes, cloning the carbonyl reductase genes from the microbial genome, and cloning the carbonyl reductase genes by site-directed mutagenesis to obtain plasmids pET-28a (+) -LK02 and pET-28a (+) -LK 03.
Preferably, the construction and expression of the recombinant engineering bacteria such as E.coli/pET-28a (+) -KR01 and the like comprises the following steps: the cloned plasmids are respectively transformed into BL21(DE3) competent cells to obtain recombinant engineering bacteria for respectively expressing carbonyl reductases such as KR01, KR02 and the like.
Preferably, in the step (2), the solvent for extraction is any one or a combination of at least two of ethyl acetate, methyl acetate or methyl formate, and preferably ethyl acetate.
Preferably, in the step (2), the crystallization comprises the following specific steps: and mixing the concentrated product with a crystallization solvent, and then sequentially stirring for crystallization, cooling for crystal growing, filtering, washing and drying to obtain the crystals of (beta, S) configuration hydroxypropyl tetrahydropyrane triol.
Preferably, the crystallization solvent comprises a first solvent and a second solvent, the concentrated product is dissolved by the first solvent, and the second solvent is added dropwise under stirring to precipitate crystals.
Preferably, the first solvent comprises any one or a combination of at least two of ethanol, isopropanol, ethyl acetate or methyl acetate, preferably ethanol.
Preferably, the second solvent comprises any one or a combination of at least two of acetone, methyl isobutyl ketone, methyl tert-ether or isopropyl ether, preferably acetone.
Preferably, the volume ratio of the second solvent to the first solvent is (1-10):1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc., preferably (1-5): 1.
Preferably, the rotation speed of the stirring crystallization is 50-300rpm, such as 50rpm, 100rpm, 150rpm, 200rpm, 250rpm, 300rpm, etc., the temperature of the stirring crystallization is-10-25 ℃, such as-10 ℃, 5 ℃, 0 ℃, 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, etc., and the time of the stirring crystallization is 1-2h, such as 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.
Preferably, the temperature for cooling and crystal growth is 0-5 ℃, for example, 0 ℃, 1 ℃, 2 ℃,3 ℃,4 ℃, 5 ℃ and the like, and the time for cooling and crystal growth is 0.5-2h, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h and the like.
Preferably, the solvent used for washing comprises any one or a combination of at least two of acetone, methyl isobutyl ketone, methyl tert-ether or isopropyl ether, preferably acetone.
Preferably, the crystallization method of the (beta, S) configuration hydroxypropyl tetrahydropyrane triol specifically comprises the following steps:
(1) mixing D-xylopyranoside-2- (S) acetone, cosubstrate and liquid containing carbonyl reductase and coenzyme regeneration enzyme, and carrying out heat preservation reaction at 25-30 ℃ for 8-24h to obtain reaction liquid containing (beta, S) configuration hydroxypropyl tetrahydropyrane triol crude product;
(2) extracting the reaction liquid of the crude product containing (beta, S) configuration hydroxypropyl tetrahydropyrane triol obtained in the step (1) and concentrating; dissolving the concentrated product by using a first solvent, stirring at 50-300rpm for 1-2h, and simultaneously dropwise adding a second solvent to separate out crystals; and cooling the system to 0-5 ℃ for crystal growth for 0.5-2h, washing and drying to obtain the crystal of (beta, S) configuration hydroxypropyl tetrahydropyrane triol.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention selects carbonyl reductase with high stereoselectivity and specific substrate D-xylopyranoside-2- (S) acetone, synthesizes and obtains high-purity (beta, S) configuration hydroxypropyl tetrahydropyrane triol, and crystallizes from specific solvent to obtain single configuration solid with high purity and good quality;
(2) the purity of the (beta, S) -configuration hydroxypropyl tetrahydropyrane triol prepared by the method is over 99 percent through detection, the yield is over 85 percent, and the de value is 100 percent.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Preparation example
The preparation example constructs carbonyl reductase, and the carbonyl reductase is prepared by the following method:
s1 genome extraction
The cells were collected when Streptomyces cyanomericus glycerol and Rhodococcus ruber DSM 44541 glycerol were cultured in a medium to the logarithmic growth phase. According to the method in the molecular cloning guidelines, the genome is extracted by lysing the bacteria with lysozyme, dissolving the polysaccharides, SDS and denatured proteins with organic solvents in high concentrations of salt, and finally precipitating the DNA with precooled ethanol. The extracted Streptomyces cyaneogyreius and Rhodococcus ruber DSM 44541 genomes were stored in a-20 ℃ freezer for later use.
Cloning of S2 carbonyl reductase Gene
S21 codon optimization and chemical synthesis of carbonyl reductase gene
The gene sequences of carbonyl reductase KR01-KR11 (except KR09 and KR10), LkCR, LK01, LK04 and LK05 are subjected to codon optimization and chemical synthesis aiming at the codon preference of Escherichia coli (the optimized gene sequences are connected in a pUC57 vector, and the middle part of the optimized gene sequences has no NdeI and HindIII enzyme cutting sites). NdeI and HindIII enzyme cutting sites are selected, and the carbonyl reductase target genes such as KR01, LkCR and the like are constructed into an expression vector pET-28a (+) by a molecular cloning method. The specific operation is as follows: the PUC57 plasmid and pET-28a (+) vector which are connected with the carbonyl reductase target gene are subjected to double enzyme digestion by NdeI and HindIII at the temperature of 37 ℃; recovering pET-28a (+) vector having cohesive end and target gene such as KR01 and LkCR by gel electrophoresis, and purifying them by using kit; the purified products were recovered by T4DNAligase enzyme ligation, and plasmids such as pET-28a (+) -KR01 and pET-28a (+) -LkCR were finally constructed.
S22 cloning of carbonyl reductase Gene from microbial genome
Carbonyl reductases KR09 and KR10 were obtained by amplification from the genomes of Streptomyces cyanoensis and Rhodococcus ruber DSM 44541, respectively, by designed primers. NdeI and HindIII enzyme cutting sites are selected to be connected with the carbonyl reductase target gene and an expression vector pET-28a (+) through a homologous recombination kit. The specific operation is as follows: according to the Clonexpress kit, corresponding amplification primers KR09-F, KR09-R, KR10-F and KR10-R (see 2.1.4 for details) are designed, and the two carbonyl reductases KR09 and KR10 are amplified; selecting NdeI and HindIII enzyme double-enzyme digestion pET-28a (+) vector, and linearizing the vector; and recombining the recovered KR09 and pET-28a (+) vectors and KR10 and pET-28a (+) vectors by using an Exnase homologous recombinase, and finally constructing pET-28a (+) -KR09 and pET-28a (+) -KR10 plasmids.
S23 site-directed mutagenesis cloning of carbonyl reductase gene
In order to change the selectivity of carbonyl reductase LK01, Tyr and Ala mutations were introduced in sequence at amino acid sequences Gly190 and Val 94 of carbonyl reductase LK 01. Primers for introducing mutation sites (LK02-F, LK02-R and LK03-F, LK03-R) were synthesized in this experiment. Using carbonyl reductase pUC57-LK01 plasmid as template, and using GXL enzyme to amplify whole plasmid; purifying and recovering the amplified product by gel electrophoresis and a kit; then connecting the linearized plasmids with T4DNA Ligase to construct mutated pUC57-LK02 and pUC57-LK03 plasmids; then pET-28a (+) -LK02 and pET-28a (+) -LK03 plasmids are constructed according to the molecular cloning method of S1.
Construction and expression of recombinant engineering bacteria such as S3 E.coli/pET-28a (+) -KR01 and the like
Plasmids such as pET-28a (+) -KR01 and pET-28a (+) -KR02 constructed above are transformed into BL21(DE3) competent cells respectively to obtain recombinant engineering bacteria expressing carbonyl reductases such as KR01 and KR02 respectively, and the recombinant engineering bacteria are named as E.coli/pET-28a (+) -KR01 and E.coli/pET-28a (+) -KR02 respectively.
coli/pET-28a (+) -KR01 was inoculated into 25mL seed medium containing Kana at a final concentration of 50. mu.g/mL and cultured overnight at 37 ℃. The next day, seed solutions of recombinant engineering bacteria such as E.coli/pET-28a (+) -KR01 were transferred to 100mL fermentation media containing Kana at a final concentration of 50. mu.g/mL, respectively, at an inoculum size of 1.5%. Culturing at 37 deg.C for 3h (OD600 value greater than 2), cooling to 25 deg.C, adding 0.1mL 100mmol IPTG (final concentration of 0.1mmol) for inducing culture overnight, collecting thallus of recombinant engineering bacteria such as E.coli/pET-28a (+) -KR01 (4000g,30min,4 deg.C centrifugation), and storing in refrigerator at-20 deg.C for use.
Example 1
The embodiment provides a crystallization method of (beta, S) configuration hydroxypropyl tetrahydropyrane triol, which comprises the following steps:
(1) after carbonyl reductase and coenzyme regenerating enzyme were recombined (recombinant enzyme provided in preparation example), they were inoculated in a liquid medium (peptone 10g, yeast extract 5g, sodium chloride 10g, 1L of purified water) together with 0.5% engineered E.coli for culture; culturing at 37 deg.C for 16 hr, centrifuging, collecting thallus, and adding phosphate buffer solution (K)2HPO4·3H2O 1.392g，KH2PO40.53g, adding 1L of purified water to prepare phosphate buffer solution with pH of 7.0), and crushing with a wall breaking machine with crushing power of 60W and crushing time of 1 min. Then centrifugally collectingSupernatant fluid;
taking 63g of D-xylopyranoside-2- (S) acetone, adding 100mL of water and 100mL of isopropanol, controlling the temperature to be 25 ℃, adding 200mL of the crude enzyme solution collected in the previous step, keeping the temperature for reaction for 12 hours, monitoring the reaction by HPLC, and stopping the reaction when the substrate is less than or equal to 1%.
(2) Adding 200mL of ethyl acetate for extraction, standing for layering, collecting an upper ethyl acetate phase for later use, adding 100mL of ethyl acetate into a lower water phase, stirring for extraction, standing for layering, collecting an upper ethyl acetate phase for later use, adding 50mL of ethyl acetate into a lower water phase, stirring for extraction, standing for layering, mixing organic phases extracted for 3 times, concentrating under reduced pressure at a temperature of less than 25 ℃, removing solvent to dryness, adding 100mL of ethanol, stirring for dissolution, dropwise adding 400mL of methyl tert-ether, slowly precipitating crystals, after dropwise addition, cooling to 0-5 ℃ for crystal growth for 1h, filtering, washing with acetone, and vacuum drying to obtain 54.12g of a product. The purity is 99.1 percent through detection, the yield is 85 percent, and the de value is 100 percent
Example 2
The embodiment provides a crystallization method of (beta, S) configuration hydroxypropyl tetrahydropyrane triol, which comprises the following steps:
(1) after carbonyl reductase and coenzyme regeneration enzyme are recombined (recombinase provided by preparation example), the carbonyl reductase and coenzyme regeneration enzyme are inoculated in a liquid culture medium (agar 10g, yeast extract 5g, sodium chloride 10g, liquid culture medium composed of 1L purified water is added) together with 0.5 percent engineering escherichia coli for culture; culturing at 37 deg.C for 16h, centrifuging to collect thallus, and adding phosphate buffer (K) with pH of 7.02HPO4·3H2O 1.392g，KH2PO40.53g, adding 1L of purified water to prepare phosphate buffer solution with pH of 7.0), crushing for 0.5min by using a wall breaking machine of 100W, and then centrifuging to collect supernatant;
taking 63g of D-xylopyranoside-2- (S) acetone, adding 100mL of water and 100mL of isopropanol, controlling the temperature to be 30 ℃, adding 200mL of the crude enzyme solution collected in the previous step, keeping the temperature for reaction for 12 hours, monitoring the reaction by HPLC, and stopping the reaction when the substrate is less than or equal to 1%;
(2) adding 200mL of ethyl acetate for extraction, standing for layering, collecting an upper ethyl acetate phase for later use, adding 100mL of ethyl acetate into a lower water phase, stirring for extraction, standing for layering, collecting an upper ethyl acetate phase for later use, adding 50mL of ethyl acetate into a lower water phase, stirring for extraction, standing for layering, mixing organic phases extracted for 3 times, concentrating under reduced pressure at a temperature of less than 25 ℃, desolventizing until dry, adding 100mL of ethanol, stirring for dissolution, dropwise adding 400mL of acetone, slowly precipitating crystals, after dropwise addition, cooling to 0-5 ℃ for crystal growth for 1h, filtering, washing with acetone, and vacuum drying to obtain 60.5g of a product. The purity is 99.5 percent, the yield is 95 percent and the de value is 100 percent.
Example 3
The embodiment provides a crystallization method of (beta, S) configuration hydroxypropyl tetrahydropyrane triol, which comprises the following steps:
(1) after carbonyl reductase and coenzyme regenerating enzyme were recombined (recombinant enzyme provided in preparation example), they were inoculated in a liquid medium (12 g of soybean protein, 5g of yeast extract, 10g of sodium chloride, 1L of purified water was added) together with 0.5% engineered escherichia coli for culture; culturing at 37 deg.C for 16h, centrifuging to collect thallus, and adding phosphate buffer (K) with pH of 7.02HPO4·3H2O 1.392g，KH2PO40.53g, adding 1L of purified water to prepare phosphate buffer solution with pH of 7.0), crushing for 1min by a 60W wall breaking machine, and then centrifuging to collect supernatant;
taking 63g of D-xylopyranoside-2- (S) acetone, adding 100mL of water and 100mL of isopropanol, controlling the temperature to be 25 ℃, adding 200mL of the crude enzyme solution collected in the previous step, keeping the temperature for reaction for 12 hours, monitoring the reaction by HPLC, and stopping the reaction when the substrate is less than or equal to 1%;
(2) adding 200mL of ethyl acetate for extraction, standing for layering, collecting an upper ethyl acetate phase for later use, adding 100mL of ethyl acetate into a lower water phase, stirring for extraction, standing for layering, collecting an upper ethyl acetate phase for later use, adding 50mL of ethyl acetate into a lower water phase, stirring for extraction, standing for layering, mixing organic phases extracted for 3 times, concentrating under reduced pressure at a temperature of less than 25 ℃, desolventizing to dryness, adding 100mL of ethanol, stirring for dissolution, dropwise adding 200mL of acetone, slowly separating out crystals, after dropwise addition is finished, cooling to 0-5 ℃ for crystal growth for 1h, filtering, washing with acetone, and drying in vacuum to obtain 59.3g of a product. The purity is 99.65%, the yield is 93.1% and the de value is 100% through detection.
Example 4
This example provides a method for crystallizing hydroxypropyl tetrahydropyrane triol having (β, S) configuration, which is different from example 1 only in that isopropanol is replaced with glucose having an equal mass in step (1).
Example 5
This example provides a method for crystallizing hydroxypropyl tetrahydropyrane triol having (β, S) configuration, which is different from example 1 only in that isopropanol is replaced with glucose having an equal mass in step (1).
Example 6
This example provides a method for crystallizing (β, S) -configuration hydroxypropyl tetrahydropyrane triol, which is different from example 1 only in that the concentration of D-xylopyranoside-2- (S) acetone in the reaction system of the step (1) is 20 g/L.
Example 7
This example provides a method for crystallizing (β, S) -configuration hydroxypropyl tetrahydropyrane triol, which is different from example 1 only in that the concentration of D-xylopyranoside-2- (S) acetone in the reaction system of the step (1) is 300 g/L.
Example 8
This example provides a crystallization method of hydroxypropyl tetrahydropyrane triol with (beta, S) configuration, which is different from example 1 only in that in the step (1), the reaction is kept for 8 h.
Example 9
This example provides a crystallization method of hydroxypropyl tetrahydropyrane triol with (beta, S) configuration, which is different from example 1 only in that in the step (1), the reaction is kept for 8 h.
Example 10
This example provides a method for crystallizing (β, S) -configuration hydroxypropyl tetrahydropyrane triol, which is different from example 1 only in that methyl tert-ether is not added dropwise in step (2), and after vacuum concentration and desolventization to dryness, a mixture of 100mL of ethanol and 400mL of methyl tert-ether is directly added to the mixture and stirred for crystallization.
Comparative example 1
This comparative example provides a method for crystallizing hydroxypropyl tetrahydropyrane triol having (β, S) configuration, which is different from example 1 only in that alcohol dehydrogenase is not added.
Comparative example 2
This comparative example provides a method for crystallizing hydroxypropyl tetrahydropyrane triol having (β, S) configuration, which is different from example 1 only in that carbonyl reductase is not added.
Performance testing
Test samples: crystals of hydroxypropyl tetrahydropyrane triol of (β, S) configuration provided in examples 1 to 10 and comparative examples 1 to 2;
the test method comprises the following steps: and (4) HPLC detection: a chromatographic column: sinochrom ODS-BP C18, 5 μm,4.6mm × 250 mm; mobile phase: 70% acetonitrile water; detection wavelength: 240 nm.
The specific test results are shown in table 1 below:
TABLE 1
The test results in table 1 show that the purity of the (beta, S) -configuration hydroxypropyl tetrahydropyran triol prepared by the method is over 99 percent, the yield is over 85 percent, and the de value is 100 percent. The invention selects carbonyl reductase with high stereoselectivity and specific substrate D-xylopyranoside-2- (S) acetone, synthesizes and obtains high-purity (beta, S) configuration hydroxypropyl tetrahydropyrane triol, and crystallizes from specific solvent to obtain single configuration solid with high purity and good quality.
The applicant states that the present invention is illustrated by the above examples to the crystallization method of (β, S) -configuration hydroxypropyl tetrahydropyrane triol of the present invention, but the present invention is not limited to the above examples, i.e., it does not mean that the present invention must be implemented by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol is characterized by comprising the following steps:
(1) in the presence of carbonyl reductase and coenzyme regeneration enzyme, carrying out asymmetric reduction reaction on D-xylopyranoside-2- (S) acetone to obtain a crude product of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol;
(2) and (2) sequentially extracting, concentrating and crystallizing the reaction system obtained in the step (1) to obtain crystals of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol.
2. The method for crystallizing (β, S) -configured hydroxypropyl tetrahydropyrane triol according to claim 1, wherein in the step (1), the coenzyme-regenerating enzyme comprises any one of formate dehydrogenase, glucose dehydrogenase or alcohol dehydrogenase or a combination of at least two thereof.
3. The crystallization method of (β, S) -configuration hydroxypropyl tetrahydropyrane triol as claimed in claim 1, characterized in that the specific operation of the step (1) is as follows: mixing D-xylopyranoside-2- (S) acetone, cosubstrate and liquid containing carbonyl reductase and coenzyme regeneration enzyme, and carrying out heat preservation reaction to obtain a reaction liquid containing a (beta, S) -configuration hydroxypropyl tetrahydropyrane triol crude product.
4. The crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol as claimed in claim 3, characterized in that the concentration of the D-xylopyranoside-2- (S) acetone in the reaction system is 20-1000g/L, preferably 100-200 g/L;
preferably, the volume ratio of the cosubstrate to the carbonyl reductase-and coenzyme regenerating enzyme-containing liquid is (0.5-2) to 1, preferably 1 to 1;
preferably, the co-substrate comprises any one or a combination of at least two of an aqueous isopropanol solution, an aqueous glucose solution, or an aqueous formic acid solution, preferably an aqueous isopropanol solution;
preferably, the mass percentage content of the cosubstrate is 30-50%;
preferably, the temperature of the heat preservation reaction is 25-30 ℃;
preferably, the time for the incubation reaction is 8-24h, preferably 10-12 h.
5. The crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol according to claim 3 or 4, characterized in that the carbonyl reductase-containing liquid and coenzyme regeneration enzyme are prepared by the following preparation method:
(a) after recombining carbonyl reductase and coenzyme regenerative enzyme, inoculating the carbonyl reductase and engineering escherichia coli together into a liquid culture medium for culture;
(b) centrifuging the system obtained by culturing in the step (a) and collecting thalli; putting the thalli into a buffer solution, crushing, and centrifuging to collect supernatant to obtain liquid containing carbonyl reductase and coenzyme regenerative enzyme;
preferably, in step (a), the recombination specifically comprises the steps of: extracting genome, cloning carbonyl reductase gene, and constructing and expressing E.coli/pET-28a (+) -KR01 recombinant engineering bacteria;
preferably, the extracted genome is the Streptomyces cyaneogrieus and Rhodococcus ruber DSM 44541 genome;
preferably, the carbonyl reductase gene cloning comprises the steps of: sequentially carrying out codon optimization and chemical synthesis on the extracted genome to obtain a carbonyl reductase gene, cloning the carbonyl reductase gene from a microbial genome, and cloning the carbonyl reductase gene by site-directed mutagenesis to obtain plasmids pET-28a (+) -LK02 and pET-28a (+) -LK 03;
preferably, the construction and expression of the E.coli/pET-28a (+) -KR01 recombinant engineering bacterium comprises the following steps: the plasmids obtained by cloning are respectively transformed into BL21(DE3) competent cells to obtain recombinant engineering bacteria for respectively expressing KR01 and KR02 carbonyl reductase.
6. The crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol according to any one of claims 3 to 5, characterized in that in the step (2), the solvent for extraction is any one or a combination of at least two of ethyl acetate, methyl acetate or methyl formate, preferably ethyl acetate.
7. The crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol according to any one of claims 3 to 6, characterized in that in the step (2), the crystallization is specifically performed by the following steps: and mixing the concentrated product with a crystallization solvent, and then sequentially stirring for crystallization, cooling for crystal growing, filtering, washing and drying to obtain the crystals of (beta, S) configuration hydroxypropyl tetrahydropyrane triol.
8. The crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol according to claim 7, characterized in that the crystallization solvent comprises a first solvent and a second solvent, the concentrated product is dissolved by the first solvent, and the second solvent is added dropwise under stirring to separate out crystals;
preferably, the first solvent comprises any one or a combination of at least two of ethanol, isopropanol, ethyl acetate or methyl acetate, preferably ethanol;
preferably, the second solvent comprises any one or a combination of at least two of acetone, methyl isobutyl ketone, methyl tert-ether or isopropyl ether, preferably acetone;
preferably, the volume ratio of the second solvent to the first solvent is (1-10):1, preferably (1-5): 1.
9. The crystallization method of (beta, S) -configuration hydroxypropyl tetrahydropyrane triol as claimed in claim 7 or 8, characterized in that the rotation speed of the stirring crystallization is 50-300rpm, the temperature of the stirring crystallization is-10-25 ℃, and the time of the stirring crystallization is 1-2 h;
preferably, the temperature for cooling and crystal growing is 0-5 ℃, and the time for cooling and crystal growing is 0.5-2 h;
preferably, the solvent used for washing comprises any one or a combination of at least two of acetone, methyl isobutyl ketone, methyl tert-ether or isopropyl ether, preferably acetone.
10. The crystallization method of (β, S) configuration hydroxypropyl tetrahydropyrane triol according to claims 1 to 9, characterized in that the crystallization method of (β, S) configuration hydroxypropyl tetrahydropyrane triol specifically comprises the following steps:
(1) mixing D-xylopyranoside-2- (S) acetone, cosubstrate and liquid containing carbonyl reductase and coenzyme regeneration enzyme, and carrying out heat preservation reaction at 25-30 ℃ for 8-24h to obtain reaction liquid containing (beta, S) configuration hydroxypropyl tetrahydropyrane triol crude product;
(2) extracting the reaction liquid of the crude product containing (beta, S) configuration hydroxypropyl tetrahydropyrane triol obtained in the step (1) and concentrating; dissolving the concentrated product by using a first solvent, stirring at 50-300rpm for 1-2h, and simultaneously dropwise adding a second solvent to separate out crystals; and cooling the system to 0-5 ℃ for crystal growth for 0.5-2h, washing and drying to obtain the crystal of (beta, S) configuration hydroxypropyl tetrahydropyrane triol.
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| CN111876452A (en) * | 2020-07-01 | 2020-11-03 | 江苏瑞蓓丽生物科技有限公司 | Method for preparing vitronectin by biological enzyme one-pot method |
| CN113416756A (en) * | 2021-07-12 | 2021-09-21 | 上海克琴科技有限公司 | Method for synthesizing hydroxypropyl tetrahydropyrane triol catalyzed by biological enzyme |
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| CN111876452A (en) * | 2020-07-01 | 2020-11-03 | 江苏瑞蓓丽生物科技有限公司 | Method for preparing vitronectin by biological enzyme one-pot method |
| CN113416756A (en) * | 2021-07-12 | 2021-09-21 | 上海克琴科技有限公司 | Method for synthesizing hydroxypropyl tetrahydropyrane triol catalyzed by biological enzyme |
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