CN116904543A - Application of dehydrogenase in synthesis of R-configuration vitronectin and synthesis method - Google Patents
Application of dehydrogenase in synthesis of R-configuration vitronectin and synthesis method Download PDFInfo
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- CN116904543A CN116904543A CN202311146033.2A CN202311146033A CN116904543A CN 116904543 A CN116904543 A CN 116904543A CN 202311146033 A CN202311146033 A CN 202311146033A CN 116904543 A CN116904543 A CN 116904543A
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- 101710088194 Dehydrogenase Proteins 0.000 title claims abstract description 28
- 102100035140 Vitronectin Human genes 0.000 title claims abstract description 18
- 108010031318 Vitronectin Proteins 0.000 title claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 title claims description 10
- 238000003786 synthesis reaction Methods 0.000 title claims description 10
- 238000001308 synthesis method Methods 0.000 title abstract description 7
- 239000011521 glass Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 21
- 239000013067 intermediate product Substances 0.000 claims abstract description 18
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 claims abstract description 14
- 239000005515 coenzyme Substances 0.000 claims abstract description 11
- 239000000126 substance Substances 0.000 claims abstract description 10
- 230000002194 synthesizing effect Effects 0.000 claims abstract description 10
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 7
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- 150000004729 acetoacetic acid derivatives Chemical class 0.000 claims abstract description 3
- 238000010189 synthetic method Methods 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 21
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- YRKCREAYFQTBPV-UHFFFAOYSA-N acetylacetone Chemical compound CC(=O)CC(C)=O YRKCREAYFQTBPV-UHFFFAOYSA-N 0.000 claims description 6
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical class CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 3
- REIYHFWZISXFKU-UHFFFAOYSA-N Butyl acetoacetate Chemical compound CCCCOC(=O)CC(C)=O REIYHFWZISXFKU-UHFFFAOYSA-N 0.000 claims description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 235000015497 potassium bicarbonate Nutrition 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 235000011181 potassium carbonates Nutrition 0.000 claims description 2
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 235000011118 potassium hydroxide Nutrition 0.000 claims description 2
- GVIIRWAJDFKJMJ-UHFFFAOYSA-N propan-2-yl 3-oxobutanoate Chemical compound CC(C)OC(=O)CC(C)=O GVIIRWAJDFKJMJ-UHFFFAOYSA-N 0.000 claims description 2
- DHGFMVMDBNLMKT-UHFFFAOYSA-N propyl 3-oxobutanoate Chemical compound CCCOC(=O)CC(C)=O DHGFMVMDBNLMKT-UHFFFAOYSA-N 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-O 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 description 24
- 108090000790 Enzymes Proteins 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 5
- 229960001031 glucose Drugs 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 238000000703 high-speed centrifugation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000012137 tryptone Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 108010029645 galactitol 2-dehydrogenase Proteins 0.000 description 3
- -1 lithium aluminum hydrogen Chemical class 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000003321 amplification Effects 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 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 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 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 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000010802 sludge Substances 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102000005751 Alcohol Oxidoreductases Human genes 0.000 description 1
- 108010031132 Alcohol Oxidoreductases Proteins 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 241000122973 Stenotrophomonas maltophilia Species 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000009903 catalytic hydrogenation reaction Methods 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000019353 potassium silicate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
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Abstract
The invention provides an application of dehydrogenase in synthesizing R-configuration vitronectin and a synthetic method thereof, wherein the step 1 of the method is that D-xylose, an acetoacetate analogue and an alkaline substance are mixed and heated in aqueous solution, and a vitronectin intermediate product is obtained after reaction; step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol. The synthesis method of the R-configuration glass color factor adopts dehydrogenase SMADH2 and glucose dehydrogenase GDH to specifically synthesize the glass color factor with a single R configuration, and simultaneously avoids the use of harmful chemicals, so that the whole reaction process is more environment-friendly.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of dehydrogenase in synthesis of R-configuration vitronectin and a synthesis method.
Background
Most of the synthesis routes of the existing chemical method for the vitrein are two-step reactions, the first step is to prepare xyloside from D-xylose, the conversion is smooth, and the difficulty is that the second step is to selectively reduce xyloside. The separation of the pair of diastereomers obtained is difficult due to the poor stereoselectivity of the carbonyl groups of the chemically reduced ketones. Moreover, the existing route often needs to use dangerous reagents and processes such as sodium borohydride, lithium aluminum hydrogen, catalytic hydrogenation and the like, and a large amount of pollutants are introduced.
There are few patents for synthesizing vitrein by enzyme catalysis and no examples for synthesizing a single R configuration.
The Chinese patent publication No. CN111876452A (publication No. 2020, 11/3) discloses a method for preparing vitronectin by an enzyme one-pot method, but the sources of isopropyl alcohol dehydrogenase, vitronectin synthase, carbonyl reductase and the like used in the method are unknown and difficult to be trusted.
Chinese patent publication No. CN 113717997A (publication No. 2022, 2, 8) discloses a method for preparing vitriol factor by using combined enzyme, but the method can only synthesize S vitriol factor.
Disclosure of Invention
In view of the above, the present invention aims to overcome the defects in the prior art, and provides an application of dehydrogenase in synthesizing R-configuration vitronectin and a synthesis method thereof.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
the invention provides an application of dehydrogenase SMADH2 in synthesis of R-configuration vitronectin.
The invention also provides a dehydrogenase composition, which comprises dehydrogenase SMADH2 and glucose dehydrogenase GDH.
Further, the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1.
The invention also provides application of the dehydrogenase composition in synthesis of R-configuration vitronectin.
The invention also provides a synthesis method of the R-configuration glass color factor, which comprises the following steps:
step 1, mixing D-xylose, an acetoacetic ester analogue and an alkaline substance in an aqueous solution, heating, and reacting to obtain a glassy factor intermediate product;
step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol.
Further, the temperature of the heating step in the step 1 is 50-56 ℃ and the time is 3-5 hours; the molar ratio of D-xylose, the acetoacetate analogue and the alkaline substance in the step 1 is 1:1-1.3:1.3-1.6; the ethyl acetoacetate analogue is at least one of acetylacetone, ethyl acetoacetate, propyl acetoacetate, isopropyl acetoacetate or butyl acetoacetate; the alkaline substance is at least one of potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate.
Further, after the reaction step in the step 1 is finished, the intermediate product of the vitriol factor is obtained after the pH value of the solution is regulated, decolorized and filtered; the pH value of the step of adjusting the pH value of the solution is 7.0-8.0; the decoloring step is performed by using activated carbon.
Further, the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.075-0.15; the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1. Preferably, the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.1.
Further, the molar ratio of the glass-color factor intermediate product to glucose in the step 2 is 1:1-2; the molar ratio of the glass color factor intermediate product to the coenzyme in the step 2 is 198-794:1, a step of; the coenzyme in the step 2 is NADP.
Preferably, the molar ratio of the glass-color factor intermediate product to glucose in the step 2 is 1:1.5; the molar ratio of the vitriol intermediate product to the coenzyme in the step 2 is 396-794:1, a step of; more preferably, the molar ratio of the glass-color factor intermediate product to the coenzyme in the step 2 is 594:1.
further, the buffer solution in the step 2 is potassium phosphate buffer solution; the pH value of the buffer solution in the step 2 is 6.25-7.0; the concentration of the potassium phosphate buffer solution is 10-100mM; the reaction temperature in the step 2 is 20-40 ℃ and the reaction time is 6-12 hours.
Preferably, the pH value of the buffer solution in the step 2 is 6.75; the concentration of the potassium phosphate buffer solution is 10-50mM; the reaction temperature in the step 2 is 25-35 ℃ and the reaction time is 8-10 hours.
More preferably, the concentration of the potassium phosphate buffer is 25mM; the reaction temperature in the step 2 is 30 ℃ and the time is 10 hours.
Compared with the prior art, the invention has the following advantages:
the synthesis method of the R-configuration glass color factor adopts dehydrogenase SMADH2 and glucose dehydrogenase GDH to specifically synthesize the glass color factor with a single R configuration, and simultaneously avoids the use of harmful chemicals, so that the whole reaction process is more environment-friendly.
Drawings
FIG. 1 is a gel electrophoresis diagram of SMADH2 according to example 1 of the present invention;
FIG. 2 is a gel electrophoresis diagram of GDH according to example 2 of the present invention;
FIG. 3 is a synthetic route diagram of a glassy factor intermediate according to example 3 of the present invention;
FIG. 4 is a liquid chromatogram of a glassy chromogenic intermediate according to example 3 of the present invention;
FIG. 5 is a synthetic route diagram of the R-configuration glass color factor according to example 3 of the present invention;
FIG. 6 is a nuclear magnetic resonance spectrum of R-configuration vitronectin according to example 3 of the present invention;
FIG. 7 is a chiral liquid chromatogram of a commercial vitronectin product according to example 3 of the present invention (in the racemic state (S: R=42:58));
FIG. 8 is a chiral liquid chromatogram of a single R-configuration glass color factor according to example 3 of the present invention.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The amount of enzyme required for converting 1. Mu. Mol of the substrate at room temperature for 1 minute was U, and the amount of enzyme U/g per gram of cells was calculated based on the cell concentration of the bacterium.
The present invention will be described in detail with reference to examples.
EXAMPLE 1 preparation of dehydrogenase composition
1. The dehydrogenase is SMADH2 derived from Stenotrophomonas maltophilia LH and has NCBI accession number WP_088028380.1. The construction mode of the recombinant enzyme vector and the synthesis mode of the recombinant enzyme are as follows: after sequence optimization, the genes corresponding to the enzymes are synthesized by the company from the order to the department of Optimago (Beijing), and NdeI/BamHI restriction sites are introduced and subcloned into a pET 28a expression vector. Plasmid with correct sequence is transferred by heat shock methodE.coli(BL 21) competent cells were subjected to plate culture (Optimago) and monoclonal miniculture, and the bacteria with correct protein expression were finally subjected to stepwise amplification liquid culture. Specifically, the single colony was transferred to 5ml LB medium (37 ℃) containing 50. Mu.M kanamycin for culture, and when the cells were grown to the logarithmic phase, they were inoculated to 250 ml TB medium containing the same antibiotics, and when the cells were grown to the logarithmic phase, they were transferred to a 5L culture fermenter for culture and final protein expression was performed. In 5L fermenter culture, 0.25. 0.25 mM isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added at 16℃to induce protein expression for 18 hours when the cell OD was about 20, and finally the cells were collected by high-speed centrifugation (6500 rpm,5 min) to obtain 450g of wet cells over-expressed with the enzyme. A small amount of cells were mixed with potassium phosphate buffer (50 mM, pH 7.0) and then disrupted by an ultrasonic disrupter, and the supernatant after cell wall removal by high-speed centrifugation was subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to detect protein expression, and the results are shown in FIG. 1. Cells with correct protein expression were used for the next catalytic experiment. LB medium consisted of: 1% of tryptone, 0.5% of yeast powder and 1% of NaCl; the TB medium consisted of: 1.2% tryptone, 2.4% yeast powder, 0.232% dipotassium hydrogen phosphate, 1.665% dipotassium hydrogen phosphate and 0.5% glycerol.
2. Glucose dehydrogenase GDH, derived from Priestia megaterium NBRC 15308, accession number CP035094.1. Recombinant enzyme vector construction mode and recombinationThe enzyme synthesis mode is as follows: after sequence optimization, the genes corresponding to the enzymes are synthesized by the company from the order to the department of Optimago (Beijing), and NdeI/BamHI restriction sites are introduced and subcloned into a pET 28a expression vector. Plasmid with correct sequence is transferred by heat shock methodE.coli(BL 21) competent cells were subjected to plate culture (Optimago) and monoclonal miniculture, and the bacteria with correct protein expression were finally subjected to stepwise amplification liquid culture. Specifically, the single colony was transferred to 5ml LB medium (37 ℃) containing 50. Mu.M kanamycin for culture, and when the cells were grown to the logarithmic phase, they were inoculated to 250 ml TB medium containing the same antibiotics, and when the cells were grown to the logarithmic phase, they were transferred to a 5L culture fermenter for culture and final protein expression was performed. In 5L fermenter culture, 0.25. 0.25 mM isopropyl-. Beta. -D-thiogalactopyranoside (IPTG) was added at 16℃to induce protein expression for 18 hours when the cell OD was about 20, and finally the cells were collected by high-speed centrifugation (6500 rpm,5 min) to obtain 450g of wet cells over-expressed with the enzyme. A small amount of cells were mixed with potassium phosphate buffer (50 mM, pH 7.0) and then disrupted by an ultrasonic disrupter, and the supernatant after cell wall removal by high-speed centrifugation was subjected to SDS-PAGE gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to detect protein expression, and the results are shown in FIG. 2. Cells with correct protein expression were used for the next catalytic experiment. LB medium consisted of: 1% tryptone, 0.5% yeast powder and 1% NaCl. The TB medium consisted of: 1.2% tryptone, 2.4% yeast powder, 0.232% dipotassium hydrogen phosphate, 1.665% dipotassium hydrogen phosphate and 0.5% glycerol.
The amount of enzyme required for converting 1. Mu. Mol of the substrate at room temperature for one minute was U, and the amount of enzyme contained per g of cell was U/g.
Example 2 enzyme Activity detection
1. Enzyme activity detection system of SMADH 2: 1g of bacterial sludge is taken and mixed with 20ml of potassium phosphate buffer solution with pH7.0 and molar concentration of 50mM, and the cell suspension is broken by an ultrasonic breaker for 400w, 15min and 2s for 4s. Subsequently, the mixture was centrifuged at 10000rpm for 20 minutes at a low temperature (4 ℃) to obtain 100ul of a supernatant, which was diluted to 900ul of pH7.0 and prepared into a crude enzyme solution in 50mM potassium phosphate buffer. The total reaction system was 3ml, including: 50mM,2910ul of potassium phosphate buffer pH7.0, 30ul of 25% of the vitronectin intermediate, 30ul of 20mM of coenzyme NADPH in water and 30ul of crude enzyme solution. After the components are evenly mixed, an ultraviolet spectrophotometer is used for detecting the change of the absorbance at 340 nm. The calculation formula is [ Δax3000/(6.22×30) ] ×200, where Δa is the difference in absorbance range. The enzyme activity obtained in the invention is generally 200U/g-250U/g.
2. Enzymatic activity detection system for GDH: 1g of bacterial sludge is taken and mixed with 20ml of potassium phosphate buffer solution with pH7.0 and molar concentration of 50mM, and the cell suspension is broken by an ultrasonic breaker for 400w, 15min and 2s for 4s. Subsequently, the mixture was centrifuged at 10000rpm for 20 minutes at a low temperature (4 ℃) to obtain 100ul of a supernatant, which was diluted to 900ul of pH7.0 and prepared into a crude enzyme solution in 50mM potassium phosphate buffer. The total reaction system was 3ml, including: 50mM,2910ul of potassium phosphate buffer pH7.0, 30ul of 25% glucose solution, 30ul of 20mM coenzyme NADP aqueous solution and 30ul of crude enzyme solution. After the components are evenly mixed, an ultraviolet spectrophotometer is used for detecting the change of the absorbance at 340 nm. The calculation formula is [ Δax3000/(6.22×30) ] ×200, where Δa is the difference in absorbance range. The enzyme activity obtained in the invention is generally 200U/g-250U/g.
Example 3 Synthesis of a vitronectin of R configuration
1. Firstly, putting 36L of water and 2.4kg of sodium hydroxide into a 100L reaction kettle, controlling the temperature to 25 ℃ and controlling the error to be not more than 3 ℃; then adding 6kg of D-xylose and 4.8kg of acetylacetone to react for 3 hours at the temperature of 53 ℃ and the error of not more than 3 ℃; adding 35kg of 2.5N dilute hydrochloric acid to adjust the pH to 7.0-8.0; adding 1.2kg of active carbon, keeping the temperature at 53 ℃ for decoloring for 1h, filtering the active carbon, and obtaining a filtrate which is a vitronectin intermediate solution required by the second enzyme reaction, wherein the molecular formula of the vitronectin intermediate is C 8 H 14 O 5 The synthetic route of the intermediate of the glass color factor is shown in figure 3, and the liquid chromatogram is shown in figure 4.
2. To 833g of the vitriol intermediate solution (containing 100g of the vitriol intermediate) were added 156g of glucose monohydrate, 25mL of 1M potassium phosphate buffer pH7.0, 0.66g of NADP, the temperature was raised to 25℃and the pH was adjusted to 7.0 with 4M sodium hydroxide, and finally 100ml of 100g/L of SMADH2 enzyme solution and 50ml of 100g/L of GDH enzyme solution were added. And inserting a PH electrode, heating the system to 30 ℃, supplementing 4M sodium hydroxide solution by a peristaltic pump according to the pH falling speed displayed by the electrode to maintain the pH of the system to 6.75, stirring at constant temperature for 10 hours, ending the reaction, and detecting the reaction result by a liquid phase. After the reaction, hydrochloric acid is added to adjust the pH to 2, and sodium hydroxide is added to adjust the pH to neutrality. The synthetic route of the R-configuration glass color factor is shown in fig. 5, the nuclear magnetic spectrum is shown in fig. 6, the chiral liquid chromatography is shown in fig. 7-8, and the configuration ratio of fig. 7 to the production of Shanghai Yun Luo is (S: R) 42:58, wherein a peak with retention time of 11.256min in the figure is an S-configuration glass color factor, a peak with retention time of 12.717min in the figure is an R-configuration glass color factor, and S and R configurations exist in the liquid glass color factor at the same time; FIG. 8 shows that the product of this example was subjected to liquid chromatography and only peaked at a retention time of 12.307min, indicating that there was only a single R-configuration of the glass color factor in the product.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. An application of dehydrogenase SMADH2 in synthesizing R-configuration vitronectin.
2. A dehydrogenase composition, characterized in that: the composition comprises dehydrogenase SMADH2 and glucose dehydrogenase GDH.
3. The dehydrogenase composition of claim 2, wherein: the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1.
4. Use of a dehydrogenase composition according to claim 2 or 3 for the synthesis of R-configuration vitronectin.
5. A synthetic method of R-configuration glass color factor is characterized in that: the method comprises the following steps:
step 1, mixing D-xylose, an acetoacetic ester analogue and an alkaline substance in an aqueous solution, heating, and reacting to obtain a glassy factor intermediate product;
step 2, mixing the vitriol intermediate product, dehydrogenase SMADH2, glucose dehydrogenase GDH, coenzyme and glucose in a buffer solution, and reacting to obtain the R-configuration vitriol.
6. The method for synthesizing R-configuration glass color factor according to claim 5, wherein the method comprises the following steps: the temperature of the heating step in the step 1 is 50-56 ℃ and the time is 3-5 hours; the molar ratio of D-xylose, the acetoacetate analogue and the alkaline substance in the step 1 is 1:1-1.3:1.3-1.6; the ethyl acetoacetate analogue is at least one of acetylacetone, ethyl acetoacetate, propyl acetoacetate, isopropyl acetoacetate or butyl acetoacetate; the alkaline substance is at least one of potassium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate or potassium carbonate.
7. The method for synthesizing R-configuration glass color factor according to claim 5, wherein the method comprises the following steps: after the reaction step in the step 1 is finished, the intermediate product of the vitreous color factor is obtained after the pH value of the solution is regulated, decolored and filtered; the pH value of the step of adjusting the pH value of the solution is 7.0-8.0; the decoloring step is performed by using activated carbon.
8. The method for synthesizing R-configuration glass color factor according to claim 5, wherein the method comprises the following steps: the mass ratio of the vitriol intermediate product to the dehydrogenase SMADH2 in the step 2 is 1:0.075-0.15; the mass ratio of the dehydrogenase SMADH2 to the glucose dehydrogenase GDH is 1-3:1.
9. The method for synthesizing R-configuration glass color factor according to claim 5, wherein the method comprises the following steps: the molar ratio of the glass color factor intermediate product to glucose in the step 2 is 1:1-2; the molar ratio of the glass color factor intermediate product to the coenzyme in the step 2 is 198-794:1, a step of; the coenzyme in the step 2 is NADP.
10. The method for synthesizing R-configuration glass color factor according to claim 5, wherein the method comprises the following steps: the buffer solution in the step 2 is potassium phosphate buffer solution; the pH value of the buffer solution in the step 2 is 6.25-7.0; the concentration of the potassium phosphate buffer solution is 10-100mM; the reaction temperature in the step 2 is 20-40 ℃ and the reaction time is 6-12 hours.
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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 |
| CN113717997A (en) * | 2021-11-04 | 2021-11-30 | 深圳瑞德林生物技术有限公司 | Enzyme composition and method for synthesizing vitronectin by chemical enzyme method |
| CN114540380A (en) * | 2022-03-17 | 2022-05-27 | 成都格纯生物医药有限公司 | Sorbitol dehydrogenase sorDHGo gene, encoding protein and application in preparation of vitreous chromogen |
| CN115747272A (en) * | 2022-11-30 | 2023-03-07 | 闽江学院 | Synthetic method of vitreous chromogen |
| CN115896199A (en) * | 2022-12-30 | 2023-04-04 | 杭州海普沃辉生物医药有限公司 | Method for synthesizing high-concentration (S) -configuration vitronectin by double-enzyme coupling |
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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 |
| CN113717997A (en) * | 2021-11-04 | 2021-11-30 | 深圳瑞德林生物技术有限公司 | Enzyme composition and method for synthesizing vitronectin by chemical enzyme method |
| CN114540380A (en) * | 2022-03-17 | 2022-05-27 | 成都格纯生物医药有限公司 | Sorbitol dehydrogenase sorDHGo gene, encoding protein and application in preparation of vitreous chromogen |
| CN115747272A (en) * | 2022-11-30 | 2023-03-07 | 闽江学院 | Synthetic method of vitreous chromogen |
| CN115896199A (en) * | 2022-12-30 | 2023-04-04 | 杭州海普沃辉生物医药有限公司 | Method for synthesizing high-concentration (S) -configuration vitronectin by double-enzyme coupling |
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