CN118064513A - Application of biological enzyme mutant in acetyl hydroxyproline synthesis - Google Patents
Application of biological enzyme mutant in acetyl hydroxyproline synthesis Download PDFInfo
- Publication number
- CN118064513A CN118064513A CN202410284215.4A CN202410284215A CN118064513A CN 118064513 A CN118064513 A CN 118064513A CN 202410284215 A CN202410284215 A CN 202410284215A CN 118064513 A CN118064513 A CN 118064513A
- Authority
- CN
- China
- Prior art keywords
- mutant
- biological enzyme
- hydroxyproline
- synthesis
- acetyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 57
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 57
- BAPRUDZDYCKSOQ-RITPCOANSA-N (2s,4r)-1-acetyl-4-hydroxypyrrolidine-2-carboxylic acid Chemical compound CC(=O)N1C[C@H](O)C[C@H]1C(O)=O BAPRUDZDYCKSOQ-RITPCOANSA-N 0.000 title claims abstract description 35
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 25
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 41
- 239000000047 product Substances 0.000 claims abstract description 18
- 239000001963 growth medium Substances 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims abstract description 9
- 239000013612 plasmid Substances 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims abstract description 6
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 4
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 4
- 239000002244 precipitate Substances 0.000 claims abstract description 3
- 230000008569 process Effects 0.000 claims abstract description 3
- 230000001131 transforming effect Effects 0.000 claims abstract description 3
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 claims description 29
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 claims description 29
- 229960002591 hydroxyproline Drugs 0.000 claims description 29
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 claims description 29
- 238000003756 stirring Methods 0.000 claims description 17
- 239000007853 buffer solution Substances 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 4
- 125000003342 alkenyl group Chemical group 0.000 claims description 3
- 125000000217 alkyl group Chemical group 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 150000002148 esters Chemical group 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 2
- 230000001681 protective effect Effects 0.000 claims description 2
- 239000002904 solvent Substances 0.000 claims description 2
- 238000005809 transesterification reaction Methods 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 239000000758 substrate Substances 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 42
- 229940088598 enzyme Drugs 0.000 description 37
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 14
- 239000000203 mixture Substances 0.000 description 14
- 229910052757 nitrogen Inorganic materials 0.000 description 14
- 238000010926 purge Methods 0.000 description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
- 238000002390 rotary evaporation Methods 0.000 description 10
- 238000001514 detection method Methods 0.000 description 9
- 238000000605 extraction Methods 0.000 description 6
- 239000002537 cosmetic Substances 0.000 description 4
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 3
- XTXRWKRVRITETP-UHFFFAOYSA-N Vinyl acetate Chemical compound CC(=O)OC=C XTXRWKRVRITETP-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 3
- HHDQVBSXYDRQGD-DMTCNVIQSA-N (2s,4r)-4-hydroxypyrrolidine-1,2-dicarboxylic acid Chemical compound O[C@@H]1C[C@@H](C(O)=O)N(C(O)=O)C1 HHDQVBSXYDRQGD-DMTCNVIQSA-N 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- YKYONYBAUNKHLG-UHFFFAOYSA-N n-Propyl acetate Natural products CCCOC(C)=O YKYONYBAUNKHLG-UHFFFAOYSA-N 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 229940090181 propyl acetate Drugs 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 2
- 230000037303 wrinkles Effects 0.000 description 2
- 125000004973 1-butenyl group Chemical group C(=CCC)* 0.000 description 1
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- -1 acetate ester Chemical class 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 150000003862 amino acid derivatives Chemical class 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000000337 buffer salt Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940106189 ceramide Drugs 0.000 description 1
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 125000004368 propenyl group Chemical group C(=CC)* 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 231100000245 skin permeability Toxicity 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides an application of a biological enzyme mutant in acetyl hydroxyproline synthesis, which is characterized in that: the biological enzyme mutant: transforming mutant plasmid into colibacillus to obtain positive clone strain, inoculating recombinant single colony carrying target gene mutant plasmid into culture medium for culture to obtain bacterial cell precipitate, ultrasonic crushing and centrifuging to obtain coarse enzyme solution; wherein the mutant primer comprises an upstream primer shown as a sequence SEQ ID NO.1 and a downstream primer shown as a sequence SEQ ID NO. 2. In the process of synthesizing acetyl hydroxyproline by adopting the biological enzyme mutant, the substrate stability is good, the enzyme conversion rate is high, the operation is simple and convenient, the subsequent product has no protein residue, the purity of the product is high, and the method has good industrial application prospect.
Description
Technical Field
The invention belongs to the field of organic synthesis, and particularly relates to an organic synthesis reaction catalyzed by biological enzymes, in particular to a synthesis method of acetyl hydroxyproline catalyzed by biological enzyme mutants.
Background
Acetyl hydroxyproline, chemical name N-acetyl-L-4-hydroxyproline, molecular formula C 7H11NO4, CAS registry number 33996-33-7, its structural formula is as follows:
collagen contains about 10% of hydroxyproline, and acetyl hydroxyproline is an acetylated modified amino acid derivative, and is a cosmetic/quasi drug raw material with barrier protection effect. The acetylhydroxyproline has the effects of long-acting moisturizing, promoting ceramide synthesis, relieving itching of patients with atopic dermatitis, repairing skin fine wrinkles, promoting collagen synthesis, enhancing skin permeability and the like. In the cosmetic industry, the cosmetic is mainly used for resisting aging, resisting wrinkle and preserving moisture.
For the synthesis of acetyl hydroxyproline, the currently published literature basically adopts a chemical synthesis method, and needs to protect carboxyl and hydroxyl of the proline, remove the protecting group after removing the acetyl. Long route, high cost, extremely poor atom economy, and more waste water and waste materials, which relate to some environment-unfriendly reagents. And toxic chemical residues may exist, which is unfavorable for downstream fields such as cosmetics.
Disclosure of Invention
The invention aims to overcome the defects, and discloses a method for synthesizing N-acetyl-L-4-hydroxyproline by taking hydroxyproline and acetate as raw materials and taking a specific biological enzyme mutant as a catalyst through one-step reaction.
The application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
the biological enzyme mutant: transforming mutant plasmid into colibacillus to obtain positive clone strain, inoculating recombinant single colony carrying target gene mutant plasmid into culture medium for culture to obtain bacterial cell precipitate, ultrasonic crushing and centrifuging to obtain coarse enzyme solution;
Wherein the mutant primer comprises an upstream primer shown as a sequence SEQ ID NO.1 and a downstream primer shown as a sequence SEQ ID NO. 2.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
the culture medium is LB culture medium containing 10-150 mug/mL kanamycin.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
When the cell concentration OD600 reaches 0.7-0.8, isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.01-0.1mM is added in the culture process in the culture medium.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
The biological enzyme mutant synthesizes acetyl hydroxyproline by catalyzing the ester exchange reaction of hydroxyproline and acetate.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
The acetate is selected from compounds shown in the following structures:
r is selected from alkyl and alkenyl.
The acetate is preferably selected from ethyl acetate, vinyl acetate, propyl acetate, butyl acetate, etc.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
the hydroxyproline: the molar ratio of acetate is 1:1-2;
The hydroxyproline: the mass ratio of the biological enzyme mutant is 1:0.001-0.1. The preferred amount of enzyme is 1-25mg/10g hydroxyproline.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
the reaction is carried out in a system having a pH of 7.0 to 10.0.
Further, the application of the biological enzyme mutant provided by the invention in the synthesis of acetyl hydroxyproline is characterized in that:
the specific method for synthesizing acetyl hydroxyproline is as follows: under the protection of protective gas, sequentially adding a buffer solution with the pH of 7.0-10.0, hydroxyproline, acetate and biological enzyme mutant into a reaction container, uniformly stirring, reacting at 25-45 ℃ until the conversion rate is not lower than 90%, adjusting the pH of a reaction system to 3-5, filtering, extracting, and removing the solvent to obtain the target product.
In addition, the invention also provides a preparation method of acetyl hydroxyproline, which is characterized in that: the biological enzyme mutant is used as a catalyst to catalyze the transesterification reaction of hydroxyproline and acetate to synthesize acetyl hydroxyproline.
The invention has the following functions and effects:
The invention synthesizes acetyl hydroxyproline by catalyzing ester exchange reaction of hydroxyproline and acetate by using acyltransferase, and has the advantages of good substrate stability, high enzyme conversion rate, simple and convenient operation, no protein residue of subsequent products and high purity of the products in the whole preparation process.
Drawings
FIG. 1, nuclear magnetic pattern of the compound of example 2;
* The product patterns of other examples using the method of the present invention are similar to the peak-emitting results of example 2, and can be both indicated as the same target product.
Detailed Description
This example proceeds based on the following equation:
hydroxyproline: acetate ester: the dosage and mass ratio of the biological enzyme mutant is 1:1-2:0.02-0.1;
In the selection of the acetate, a selectivity test is performed on the case of an alkyl group (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, etc.) and an alkenyl group (e.g., vinyl, propenyl, allyl, 1-butenyl, etc.) having not more than 10 carbon atoms as R, wherein the reaction results performed by ethyl acetate, vinyl acetate, propyl acetate, butyl acetate are the best;
Regarding the biological enzyme mutant (MsAcT-T93G), the specific preparation method is as follows:
(1) Primer design
PCR primers were designed using molecular biology software Snap Gene and MsAcT original coding genes as templates. MsAcT-T93G related primer design primer base sequence
The upstream primer SEQ ID NO.1: TGGTTCCGGGACCTGGGGCTGGGTCCCCGTAACG A
The downstream primer SEQ ID NO.2: TTAACAGACAAAGCCACCAAGGGGGTGGACCCC A
(2) PCR reaction system
* Purchase https:// www.takarabio.com// purchase http:// www.miaolingbio.com
(3) PCR amplification procedure
(4) Expression of MsAcT-T93G in E.coli
The mutant plasmid was transformed into E.coli BL21 (DE 3) to obtain a positive clone strain, and the recombinant single colony carrying the mutant plasmid of the target gene was inoculated into LB medium (1.0 g/L pancreatin, 0.5g/L yeast extract, and 1.0 g/LNaCl) containing 50. Mu.g/mL kanamycin, and cultured at 37℃and 200 rpm. When the concentration of the bacterial cells OD600 reaches 0.7-0.8, isopropyl-beta-D-thiopyran galactoside (final concentration is 0.1 mM) is added, and after continuous culture for 12 hours at 25 ℃, bacterial liquid is collected, and centrifugation is carried out for 3min at 6793 Xg at 4 ℃ to obtain bacterial cell sediment. The collected cell pellet was then resuspended in sodium phosphate buffer (100 mM, pH 8.0) at 4℃and, after sonication, centrifuged at 10012 Xg for 30min at 4℃to remove cell debris and uncrushed cells, the supernatant was MsAcT-T93G crude enzyme (i.e.enzyme and buffer salt mixture, wherein the enzyme content was about 1 mg/mL).
The following are preferred examples of experiments conducted using the most preferred conditions, and the implementation conditions used in the examples may be further adjusted according to the specific requirements of the application, and the implementation conditions not noted are those in conventional experiments.
Example 1
200ML of 0.1M PBS buffer solution with pH of 8.0, 5mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 9G of ethyl acetate are sequentially added into a 500mL three-neck flask, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is detected by HPLC (high performance liquid chromatography). Hydrochloric acid is added to adjust the pH to 3-5, diatomite is filtered, equal volume of dichloromethane is added to extract twice, and rotary evaporation is carried out to obtain 14.1 g of product with the purity of 93%.
Example 2
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 9G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 97% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is filtered, equal volume of dichloromethane is added to extract twice, and 16.9 g of product is obtained by rotary evaporation, and the purity is 98%.
Example 3
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 25mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 9G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 95% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is added for filtration, equal volume of dichloromethane is added for extraction twice, 15.2 g of product is obtained by rotary evaporation, and the purity is 98%.
Example 4
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 100mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 9G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 94% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is filtered, equal volume of dichloromethane is added to extract twice, and rotary evaporation is carried out to obtain 14.1 g of product with the purity of 95%.
Example 5
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 11G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 97% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is added for filtration, equal volume of dichloromethane is added for extraction twice, 16.2 g of product is obtained by rotary evaporation, and the purity is 97%
Example 6
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 15G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 97% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is added for filtration, equal volume of dichloromethane is added for extraction twice, 15.4 g of product is obtained by rotary evaporation, and the purity is 98 percent
Example 7
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 20G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 94% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is added for filtration, equal volume of dichloromethane is added for extraction twice, 15.6 g of product is obtained by rotary evaporation, and the purity is 95%
Example 8
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 5.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 11G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate detected by HPLC is less than 5%. (the same is true, i.e., almost no reaction occurs at pH 11)
Example 9
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 7.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 11G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 80% by HPLC detection.
Example 10
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 10.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 11G of ethyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is detected by HPLC (high Performance liquid chromatography). Hydrochloric acid is added to adjust the pH to 3-5, diatomite is added for filtration, equal volume of dichloromethane is added for extraction twice, and rotary evaporation is carried out to obtain 15.4 g of product with the purity of 93%.
Example 11
In a 500mL three-necked flask, 200mL of a 0.1M PBS buffer solution with pH of 8.0, DMF, 13 g of hydroxyproline and 11g of ethyl acetate were sequentially added, and the mixture was stirred at 25℃and 200rpm with stirring paddle, and reacted for 24 hours under a nitrogen purge of 0.01MPa, without result.
Example 12
200ML of 0.1M PBS buffer solution with pH of 8.0, concentrated sulfuric acid, 13 g of hydroxyproline, 11g of ethyl acetate and stirring with a 200rpm stirring paddle at 25 ℃ are sequentially added into a 500mL three-neck flask, and the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and no result is obtained.
Example 13
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 10G of vinyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 97% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is added for filtration, equal volume of dichloromethane is added for extraction twice, 15.7 g of product is obtained by rotary evaporation, and the purity is 97%
Example 14
In a 500mL three-neck flask, 200mL of 0.1M PBS buffer solution with pH of 8.0, 10mL of MsACT-T93G crude enzyme solution, 13G of hydroxyproline and 13G of butyl acetate are sequentially added, the mixture is stirred by a stirring paddle at 25 ℃ and 200rpm, the reaction is carried out for 24 hours under the condition of nitrogen purging at 0.01MPa, and the conversion rate is 96% by HPLC detection. Hydrochloric acid is added to adjust the pH to 3-5, diatomite is filtered, equal volume of dichloromethane is added to extract twice, and 16.1 g of product with the purity of 97% is obtained by rotary evaporation.
Claims (9)
1. The application of the biological enzyme mutant in the synthesis of acetyl hydroxyproline is characterized in that:
the biological enzyme mutant: transforming mutant plasmid into colibacillus to obtain positive clone strain, inoculating recombinant single colony carrying target gene mutant plasmid into culture medium for culture to obtain bacterial cell precipitate, ultrasonic crushing and centrifuging to obtain coarse enzyme solution;
Wherein the mutant primer comprises an upstream primer shown as a sequence SEQ ID NO.1 and a downstream primer shown as a sequence SEQ ID NO. 2.
2. The application of the biological enzyme mutant in the synthesis of acetyl hydroxyproline is characterized in that:
the culture medium is LB culture medium containing 10-150 mug/mL kanamycin.
3. The application of the biological enzyme mutant in the synthesis of acetyl hydroxyproline is characterized in that:
When the cell concentration OD600 reaches 0.7-0.8, isopropyl-beta-D-thiogalactopyranoside with the final concentration of 0.01-0.1mM is added in the culture process in the culture medium.
4. Use of a mutant biological enzyme according to claim 1 for the synthesis of acetylhydroxyproline, characterized in that:
The biological enzyme mutant synthesizes acetyl hydroxyproline by catalyzing the ester exchange reaction of hydroxyproline and acetate.
5. Use of a mutant biological enzyme according to claim 1 for the synthesis of acetylhydroxyproline, characterized in that:
The acetate is selected from compounds shown in the following structures:
r is selected from alkyl and alkenyl.
6. Use of a mutant biological enzyme according to claim 1 for the synthesis of acetylhydroxyproline, characterized in that:
the hydroxyproline: the molar ratio of acetate is 1:1-2;
The hydroxyproline: the mass ratio of the biological enzyme mutant is 1:0.001-0.1.
7. Use of a mutant biological enzyme according to claim 1 for the synthesis of acetylhydroxyproline, characterized in that:
the reaction is carried out in a system having a pH of 7.0 to 10.0.
8. Use of a mutant biological enzyme according to claim 1 for the synthesis of acetylhydroxyproline, characterized in that:
the specific method for synthesizing acetyl hydroxyproline is as follows: under the protection of protective gas, sequentially adding a buffer solution with the pH of 7.0-10.0, hydroxyproline, acetate and biological enzyme mutant into a reaction container, uniformly stirring, reacting at 25-45 ℃ until the conversion rate is not lower than 90%, adjusting the pH of a reaction system to 3-5, filtering, extracting, and removing the solvent to obtain the target product.
9. A preparation method of acetyl hydroxyproline is characterized in that: the use of the biological enzyme mutant according to any one of claims 1-3 as a catalyst for catalyzing transesterification of hydroxyproline with acetate to synthesize acetyl hydroxyproline.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410284215.4A CN118064513A (en) | 2024-03-13 | 2024-03-13 | Application of biological enzyme mutant in acetyl hydroxyproline synthesis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410284215.4A CN118064513A (en) | 2024-03-13 | 2024-03-13 | Application of biological enzyme mutant in acetyl hydroxyproline synthesis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118064513A true CN118064513A (en) | 2024-05-24 |
Family
ID=91098852
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410284215.4A Pending CN118064513A (en) | 2024-03-13 | 2024-03-13 | Application of biological enzyme mutant in acetyl hydroxyproline synthesis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118064513A (en) |
-
2024
- 2024-03-13 CN CN202410284215.4A patent/CN118064513A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210071216A1 (en) | Lov-d acyltransferase mediated acylation | |
| CN108690854B (en) | Method for producing L-glufosinate-ammonium by using chemical-enzymatic method | |
| CN112175918B (en) | 7 alpha-hydroxysteroid dehydrogenase mutant St-2-2 delta C10 and application thereof | |
| CN111690585B (en) | recombinant serratia marcescens with rcsB gene deletion and application thereof | |
| CN110791488A (en) | Lipase for splitting chiral compound and preparation method and application thereof | |
| CN112322596B (en) | 7 alpha-hydroxysteroid dehydrogenase mutant J-1-1 delta C6 and application thereof | |
| CN118064513A (en) | Application of biological enzyme mutant in acetyl hydroxyproline synthesis | |
| EP2448912A2 (en) | Enzymes and methods for resolving amino vinyl cyclopropane carboxylic acid derivatives | |
| CN111471662B (en) | SlEH1 mutant and application thereof in enantiotropic hydrolyzed epoxides | |
| CN113502305B (en) | Method for synthesizing (R) -isobutyl glutarate monoamide by utilizing recombinant imidinase | |
| Kobayashi et al. | Nitrilase-catalyzed production of p-aminobenzoic acid from p-aminobenzonitrile with Rhodococcus rhodochrous J1 | |
| CN111944774B (en) | Alcohol dehydrogenase, encoding gene thereof and application of alcohol dehydrogenase in catalytic synthesis of (R) -styrene glycol | |
| CN110804602B (en) | L-aspartic acid beta-decarboxylase mutant and application thereof | |
| CN111321177B (en) | Method for synthesizing cinacalcet intermediate (R) -1- (1-naphthyl) ethylamine by enzyme method | |
| CN115433721A (en) | Carbonyl reductase mutant and application thereof | |
| CN114507652A (en) | Purification and crystal preparation method of Burkholderia ester synthetase Bur01 | |
| CN116410940A (en) | Asymmetric reduction method for potential chiral carbon-carbon double bond by using alkene reductase and mutant thereof | |
| CN115433747A (en) | Enzymatic synthesis method of puerarin 6'' -O-acetate | |
| US4198481A (en) | Process for the preparation of 2-hydroxymethyl-3,4,5-trihydroxy piperidine | |
| CN114480315B (en) | Baeyer-Villiger monooxygenase and application thereof in brivaracetam synthesis | |
| Jost et al. | Downstream processing of serinol from a glycerol‐based fermentation broth and transfer to other amine containing molecules | |
| CN110643650A (en) | Preparation method of (S) -1-benzyl-1, 2,3,4,5,6,7, 8-octahydroisoquinoline compound | |
| CN109536468A (en) | Dicarbapentaborane reductase and its application in statins drug midbody synthesis | |
| CN117363667B (en) | Use of imine reductase in preparation of dapoxetine intermediate and/or dapoxetine | |
| CN117778371B (en) | Co-immobilized enzyme of phenylpyruvate decarboxylase and alcohol dehydrogenase, preparation and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |