CN104263767A - Method for producing iodophenyl methyl propionate through reduction by virtue of candida utilis - Google Patents
Method for producing iodophenyl methyl propionate through reduction by virtue of candida utilis Download PDFInfo
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- CN104263767A CN104263767A CN201410518174.7A CN201410518174A CN104263767A CN 104263767 A CN104263767 A CN 104263767A CN 201410518174 A CN201410518174 A CN 201410518174A CN 104263767 A CN104263767 A CN 104263767A
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- 229940017219 methyl propionate Drugs 0.000 title claims abstract description 15
- 241000235646 Cyberlindnera jadinii Species 0.000 title claims description 21
- 230000009467 reduction Effects 0.000 title claims description 5
- -1 iodophenyl methyl Chemical group 0.000 title abstract description 3
- 238000004519 manufacturing process Methods 0.000 title abstract description 3
- 239000000758 substrate Substances 0.000 claims abstract description 69
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 19
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 126
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 38
- 210000005253 yeast cell Anatomy 0.000 claims description 19
- 235000015097 nutrients Nutrition 0.000 claims description 17
- 238000009423 ventilation Methods 0.000 claims description 17
- 210000004027 cell Anatomy 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 13
- 229960003487 xylose Drugs 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 12
- 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 description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 208000012839 conversion disease Diseases 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 6
- 238000003810 ethyl acetate extraction Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 238000010531 catalytic reduction reaction Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 4
- 238000006722 reduction reaction Methods 0.000 abstract description 4
- 240000008564 Boehmeria nivea Species 0.000 abstract description 2
- 241000235056 Pichia norvegensis Species 0.000 abstract 2
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 230000001105 regulatory effect Effects 0.000 abstract 1
- 239000007787 solid Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 238000012807 shake-flask culturing Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 230000002210 biocatalytic effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012847 fine chemical Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 241000024287 Areas Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 101710157860 Oxydoreductase Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 150000001728 carbonyl compounds Chemical class 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The invention relates to a method for producing chiral bromophenyl methyl propionate by virtue of a cell process and particularly relates to a method for preparing chiral (-)-(2R, 3S)-3-(4-bromophenyl)-3-hydroxy-2-methyl propionate through direct catalytic reduction by virtue of candida norvegensis cells. Phosphate buffer solution is added into a reaction tank, the concentrations of a substrate and a product are regulated by adding ramie gauze and candida norvegensis cells are used to perform biological reduction reaction. The yield of the product is high, the enantiomeric excess is high and the method has good application value.
Description
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to the reduction of Candida utilis cell biological and prepare chiral medicinal intermediate ()-(2S, 3S)-3-(4-iodophenyl) technology of-3-hydroxy-2-methyl methyl propionate.
Background technology
Hydroxyl chiral ester is the important intermediate of synthesis of chiral medicine, fine chemicals, agricultural chemicals product and other exotic materialss.The asymmetric reduction of biocatalysis carbonyl compound has environmental friendliness, mild condition, selectivity advantages of higher, prepares the first approach of hydroxyl chiral ester as green high-efficient, is applied to the hydroxyl chiral ester producing some high added values more and more.Biocatalysis is prepared hydroxyl chiral ester and is had good application prospect.
Biocatalysis has the outstanding advantages such as catalytic efficiency is high, selectivity strong, mild condition, environmental friendliness, is the important method substituting and expand traditional organic chemical synthesis in process of sustainable development.Wherein chiral separation and asymmetric synthesis are the Application Areass of biocatalysis most magnetism.As in six large fermentoids of biological catalyst, lytic enzyme catalytic kinetics resolution of racemates can obtain quiral products, in industrial biocatalytic, play key player always.In recent years, oxydo-reductase application industrially obtained and increased rapidly.At present, the ratio adopting the industry of lytic enzyme Kinetic Resolution, biological reducing method and biological oxidation process to prepare optical activity chirality compound is 4:2:1.
Biological reducing method is for Kinetic Resolution, and maximum advantage is that theoretical yield can reach 100%, and Atom economy is good.But bioreduction needs the participation of coenzyme or cofactor, limit its application to a certain extent.Due to the dependent cause of coenzyme, in bioreduction, many Bian intact cells are as catalyzer, realize the purification procedures eliminating enzyme in body while coenzyme cyclic regeneration.
()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate is the important chiral building block of synthesis of chiral medicine, fine chemicals, agricultural chemicals product, owing to there being 2 chiral centres, make chemosynthesis comparatively difficult, cost is high.The present invention will adopt biocatalysis to prepare ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate.
Summary of the invention
The present invention adopts Candida utilis cell catalysis to prepare ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction formula is as follows:
Substrate 2-(4-iodobenzene formyl)-2-methyl acrylate (1) through Candida utilis catalytic reduction, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate (2).There is multiple-microorganism can catalytic reduction, through great many of experiments screening, finally determine to adopt Candida utilis as catalyzer, because its catalytic reduction
1effect best, reaction yield, enantiomeric excess rate (ee%) are all very high.
Many Candida utilis can carry out biocatalytic reduction, but its effect is different, differs greatly, and through experiment, the present invention selects Candida utilis bacterial strain to be
aTCC 16321, its catalytic reduction best results.
developing medium:
1, seed culture medium composition is: yeast extract 10 g/L, glucose 20 g/L, peptone 20 g/L, pH6; The agar powder of 15g/L is added during preparation solid medium.
2, nutrient solution composition: glucose 20-22 g/L, malt extract 23-25 g/L, wood sugar 6-7 g/L, (NH
4)
2sO
44.8-5.3 g/L, KH
2pO
49-11 g/L, MgSO
4.7H
2o 0.3-0.4 g/L, CaCl
20.35-0.4 g/L, ammonium sulfate 0.2-0.25g/L, magnesium sulfate 0.3-0.4g/L, pH5.6.
2, solid medium composition: add the agar powder of 2% in liquid medium within.
Prepared by yeast cell.Candida utilis through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, Candida utilis through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, coefficient is 0.6-0.7,121 DEG C of autoclavings 30 minutes, be cooled to 27-28 DEG C, Candida utilis seed liquor is seeded to fermentor tank, inoculative proportion is 6-6.5%, ventilation ratio is 0.5-1V/(V minute), cultivate 48-56 hour, obtain wet yeast cell, as bioreduction catalyzer with filtering centrifuge is centrifugal for 29-31 DEG C.
Because substrate, product all have restraining effect to yeast cell, in order to reduce the suppression of substrate, products upon cell, the present invention adopts diatomite adsorption substrate, in reaction, when substrate is reduced by cell catalysis, when concentration reduces, substrate from diatomite stripping, postreaction consume substrate.Meanwhile, product, by diatomite adsorption, decreases its concentration in water, thus considerably reduces the suppression of substrate, products upon cell.Substrate and diatomaceous ratio, determine the concentration of substrate in reaction solution, product, and concentration is also relevant with temperature of reaction, reaction solution composition.Different cell is different to the susceptibility of substrate, product, so, great many of experiments be carried out, best substrate and diatomaceous ratio could be determined.Experiment shows, for bacterial strain of the present invention, reaction solution composition, temperature of reaction, best substrate and diatomaceous ratio are 0.37-0.4.During concrete absorption, substrate is dissolved in ethyl acetate, the ratio of substrate and ethyl acetate is 0.5:2, obtains the ethyl acetate solution of substrate, absorbs this solution with diatomite, then, dries, reclaims ethyl acetate, namely obtain the diatomite of immunoabsorbent substrate.Regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.37-0.4.Diatomite used is common diatomite, and commercially, sterilizing, Preservation in sterile condition is stand-by.
Phosphate buffered saline buffer is added in bottom ventilation stirred tank, coefficient is 0.6-0.7, pH is 6.2, add the diatomite having adsorbed substrate, making substrate 2-(4-fluorobenzoyl)-2-methyl acrylate addition is 80-90 g/L, malt extract 22-26 g, wood sugar 12-13 g/L, 10 carbon polyoxyethylenated alcohol of straight chain are 16-18g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 27-28 DEG C, add wet yeast cell and make concentration be 29-32g/L, ventilation ratio is 0.17-0.2V/(V minute), carry out bioreduction, the reaction times is 81-86 hour; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid and the diatomaceous ethyl acetate of extraction, steam ethyl acetate, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction conversion ratio 94-96%, product yield 92-94%, enantiomeric excess rate (ee%) 97-98%.
The present invention carries out the work and comprises bacterial strain selection (selecting from more or less a hundred bacterial strain), catalytic reductive conditions optimizes (temperature of reaction, air flow, pH), reaction medium is selected and concentration optimization (concentration of substrate, malt extract content, Xylose Content, kinds of surfactants (more than 20, kind selects 1) and concentration, other multiple components is selected to get rid of), solid absorption is adopted to control substrate product concentration, thus the suppression reduced cell, test carclazyte, diatomite, cotton gauze, ramie gauze, the many kinds of solids materials such as macropore resin, also once carried out the test of water-organic solvent 2 phase system.Because tested number is very large, although have employed response surface optimization design experiment, drastically reduce the area tested number, tested number is still very large, and total Test carries out just completing more than 2 years, reaches current technical scheme.For photolytic activity product, as reaction conversion ratio 94-96%, during product yield 92-94%, enantiomeric excess rate (ee%), still up to 97-98%, is very not easily, and our work achieves marked improvement.
embodiment 1
Seed culture medium composition is: yeast extract 10 g/L, glucose 20 g/L, peptone 20 g/L, pH6; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 20 g/L, malt extract 23 g/L, wood sugar 6 g/L, (NH
4)
2sO
44.8 g/L, KH
2pO
49 g/L, MgSO
4.7H
2o 0.3 g/L, CaCl
20.35 g/L, ammonium sulfate 0.2g/L, magnesium sulfate 0.3g/L, pH6.2.
Wet yeast cell preparation process is as follows: Candida utilis ATCC 16321 through inclined-plane, shake-flask culture, obtain seed liquor.10L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.6,121 DEG C of autoclavings 30 minutes, be cooled to 27 DEG C, Candida utilis seed liquor is seeded to fermentor tank, inoculative proportion is 6%, ventilation ratio is 1.7V/(V minute), cultivate 21 hours, obtain wet yeast cell, as bioreduction catalyzer with filtering centrifuge is centrifugal for 27 DEG C.
The diatomite making method of immunoabsorbent substrate is, substrate is dissolved in ethyl acetate, the ratio of substrate and ethyl acetate is 0.5:2, obtain the ethyl acetate solution of substrate, absorb this solution with diatomite, then, dry, reclaim ethyl acetate, namely obtain the diatomite of immunoabsorbent substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.37.Diatomite used is common diatomite, and commercially, sterilizing, Preservation in sterile condition is stand-by, and other embodiment diatomite process is identical.
Phosphate buffered saline buffer is added in 50L bottom ventilation stirred tank, coefficient is 0.6, pH is 6.2, add the diatomite having adsorbed substrate, making substrate 2-(4-fluorobenzoyl)-2-methyl acrylate addition is 80 g/L, malt extract 22 g, wood sugar 12 g/L, 10 carbon polyoxyethylenated alcohol of straight chain are 16g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 30 DEG C, add wet yeast cell and make concentration be 29g/L, ventilation ratio is 0.17V/(V minute), carry out bioreduction, the reaction times is 81 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid and the diatomaceous ethyl acetate of extraction, steam ethyl acetate, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction conversion ratio 94%, product yield 92%, enantiomeric excess rate (ee%) 97%.
embodiment 2
Seed culture medium composition is: yeast extract 10 g/L, glucose 20 g/L, peptone 20 g/L, pH6; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 22 g/L, malt extract 25 g/L, wood sugar 7 g/L, (NH
4)
2sO
45.3 g/L, KH
2pO
411 g/L, MgSO
4.7H
2o 0.4 g/L, CaCl
20.4 g/L, ammonium sulfate 0.25g/L, magnesium sulfate 0.4g/L, pH6.2.
Wet yeast cell preparation process is as follows: Candida utilis ATCC 16321 through inclined-plane, shake-flask culture, obtain seed liquor.50L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.6-0.7,121 DEG C of autoclavings 30 minutes, be cooled to 28 DEG C, Candida utilis seed liquor is seeded to fermentor tank, inoculative proportion is 6.5%, ventilation ratio is 1.8V/(V minute), cultivate 23 hours, obtain wet yeast cell, as bioreduction catalyzer with filtering centrifuge is centrifugal for 28 DEG C.
The diatomite making method of immunoabsorbent substrate is, substrate is dissolved in ethyl acetate, the ratio of substrate and ethyl acetate is 0.5:2, obtain the ethyl acetate solution of substrate, absorb this solution with diatomite, then, dry, reclaim ethyl acetate, namely obtain the diatomite of immunoabsorbent substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.38.
Phosphate buffered saline buffer is added in 250L bottom ventilation stirred tank, coefficient is 0.7, pH is 6.2, add the diatomite having adsorbed substrate, making substrate 2-(4-fluorobenzoyl)-2-methyl acrylate addition is 90 g/L, malt extract 26 g, wood sugar 13 g/L, 10 carbon polyoxyethylenated alcohol of straight chain are 18g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 31 DEG C, add wet yeast cell and make concentration be 32g/L, ventilation ratio is 0.2V/(V minute), carry out bioreduction, the reaction times is 86 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid and the diatomaceous ethyl acetate of extraction, steam ethyl acetate, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction conversion ratio 96%, product yield 94%, enantiomeric excess rate (ee%) 97%.
embodiment 3
Seed culture medium composition is: yeast extract 10 g/L, glucose 20 g/L, peptone 20 g/L, pH6; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 20-22 g/L, malt extract 23-25 g/L, wood sugar 6-7 g/L, (NH
4)
2sO
44.8-5.3 g/L, KH
2pO
49-11 g/L, MgSO
4.7H
2o 0.3-0.4 g/L, CaCl
20.35-0.4 g/L, ammonium sulfate 0.2-0.25g/L, magnesium sulfate 0.3-0.4g/L, pH6.2.
Wet yeast cell preparation process is as follows: Candida utilis ATCC 16321 through inclined-plane, shake-flask culture, obtain seed liquor.200L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.6-0.7,121 DEG C of autoclavings 30 minutes, be cooled to 27-28 DEG C, Candida utilis seed liquor is seeded to fermentor tank, inoculative proportion is 6-6.5%, ventilation ratio is 1.7-1.8V/(V minute), cultivate 21-23 hour, obtain wet yeast cell, as bioreduction catalyzer with filtering centrifuge is centrifugal for 27-28 DEG C.
The diatomite making method of immunoabsorbent substrate is, substrate is dissolved in ethyl acetate, the ratio of substrate and ethyl acetate is 0.5:2, obtain the ethyl acetate solution of substrate, absorb this solution with diatomite, then, dry, reclaim ethyl acetate, namely obtain the diatomite of immunoabsorbent substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.39.
Phosphate buffered saline buffer is added in 100L bottom ventilation stirred tank, coefficient is 0.65, pH is 6.2, add the diatomite having adsorbed substrate, making substrate 2-(4-fluorobenzoyl)-2-methyl acrylate addition is 805 g/L, malt extract 24 g, wood sugar 12.5 g/L, 10 carbon polyoxyethylenated alcohol of straight chain are 17g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 30.5 DEG C, add wet yeast cell and make concentration be 31g/L, ventilation ratio is 0.18V/(V minute), carry out bioreduction, the reaction times is 84 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid and the diatomaceous ethyl acetate of extraction, steam ethyl acetate, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction conversion ratio 95%, product yield 93%, enantiomeric excess rate (ee%) 97.5%.
embodiment 4
Seed culture medium composition is: yeast extract 10 g/L, glucose 20 g/L, peptone 20 g/L, pH6; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 21 g/L, malt extract 25 g/L, wood sugar 7 g/L, (NH
4)
2sO
45 g/L, KH
2pO
410 g/L, MgSO
4.7H
2o 0.35 g/L, CaCl
20.37 g/L, ammonium sulfate 0.23g/L, magnesium sulfate 0.36g/L, pH6.2.
Wet yeast cell preparation process is as follows: Candida utilis ATCC 16321 through inclined-plane, shake-flask culture, obtain seed liquor.1000L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.7,121 DEG C of autoclavings 30 minutes, be cooled to 28 DEG C, Candida utilis seed liquor is seeded to fermentor tank, inoculative proportion is 6.5%, ventilation ratio is 1.8V/(V minute), cultivate 22 hours, obtain wet yeast cell, as bioreduction catalyzer with filtering centrifuge is centrifugal for 28 DEG C.
The diatomite making method of immunoabsorbent substrate is, substrate is dissolved in ethyl acetate, the ratio of substrate and ethyl acetate is 0.5:2, obtain the ethyl acetate solution of substrate, absorb this solution with diatomite, then, dry, reclaim ethyl acetate, namely obtain the diatomite of immunoabsorbent substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.4.
Phosphate buffered saline buffer is added in 5000L bottom ventilation stirred tank, coefficient is 0.7, pH is 6.2, add the diatomite having adsorbed substrate, making substrate 2-(4-fluorobenzoyl)-2-methyl acrylate addition is 90 g/L, malt extract 26 g, wood sugar 13 g/L, 10 carbon polyoxyethylenated alcohol of straight chain are 18g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 31 DEG C, add wet yeast cell and make concentration be 32g/L, ventilation ratio is 0.2V/(V minute), carry out bioreduction, the reaction times is 81-86 hour; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid and the diatomaceous ethyl acetate of extraction, steam ethyl acetate, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction conversion ratio 95%, product yield 934%, enantiomeric excess rate (ee%) 98%.
Claims (4)
1. Candida utilis cell biological reduction preparation ()-(2S, 3S)-3-(4-iodophenyl) method of-3-hydroxy-2-methyl methyl propionate, it is characterized in that adding phosphate buffered saline buffer in retort, coefficient is 0.6-0.7, pH is 6.2, add the diatomite having adsorbed substrate, making substrate 2-(4-fluorobenzoyl)-2-methyl acrylate addition is 80-90 g/L, malt extract 22-26 g, wood sugar 12-13 g/L, 10 carbon polyoxyethylenated alcohol of straight chain are 16-18g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 30-31 DEG C, add wet yeast cell and make concentration be 29-32g/L, ventilation ratio is 0.17-0.2V/(V minute), carry out bioreduction, the reaction times is 81-86 hour; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid and the diatomaceous ethyl acetate of extraction, steam ethyl acetate, obtain product ()-(2S, 3S)-3-(4-iodophenyl)-3-hydroxy-2-methyl methyl propionate, reaction conversion ratio 94-96%, product yield 92-94%, enantiomeric excess rate 97-98%.
2. method according to claim 1, is characterized in that wet yeast cell preparation process is as follows: Candida utilis through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, coefficient is 0.6-0.7,121 DEG C of autoclavings 30 minutes, be cooled to 27-28 DEG C, Candida utilis seed liquor is seeded to fermentor tank, inoculative proportion is 6-6.5%, ventilation ratio is 1.7-1.8V/(V minute), cultivate 21-23 hour, obtain wet yeast cell, as bioreduction catalyzer with filtering centrifuge is centrifugal for 27-28 DEG C.
3. method according to claim 1, its feature at nutrient solution composition is: glucose 20-22 g/L, malt extract 23-25 g/L, wood sugar 6-7 g/L, (NH
4)
2sO
44.8-5.3 g/L, KH
2pO
49-11 g/L, MgSO
4.7H
2o 0.3-0.4 g/L, CaCl
20.35-0.4 g/L, ammonium sulfate 0.2-0.25g/L, magnesium sulfate 0.3-0.4g/L, pH6.2.
4. method according to claim 1, it is characterized in that described diatomite making method of having adsorbed substrate is, substrate is dissolved in ethyl acetate, and the ratio of substrate and ethyl acetate is 0.5:2, obtains the ethyl acetate solution of substrate, this solution is absorbed with diatomite, then, dry, reclaim ethyl acetate, namely obtain the diatomite of immunoabsorbent substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.37-0.4.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005106006A2 (en) * | 2004-05-05 | 2005-11-10 | Jubilant Organosys Limited | Biotransformation of nicotinic acid to 6-hydroxynicotinic acid |
| WO2014045299A2 (en) * | 2012-08-28 | 2014-03-27 | Privi Biotechnologies Private Limited | Continuous biotransformation of substituted aromatic carboxylic acids to their selective aldehydes and alcohols |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005106006A2 (en) * | 2004-05-05 | 2005-11-10 | Jubilant Organosys Limited | Biotransformation of nicotinic acid to 6-hydroxynicotinic acid |
| WO2014045299A2 (en) * | 2012-08-28 | 2014-03-27 | Privi Biotechnologies Private Limited | Continuous biotransformation of substituted aromatic carboxylic acids to their selective aldehydes and alcohols |
Non-Patent Citations (1)
| Title |
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| MICHEL R.B.: "Regio- and enantioselective bioreduction of methyleneketoesters using both polymeric resin and cellulose matrix.", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
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