CN104313075A - Cell production method for photoactive bromopyridyl ethanol - Google Patents
Cell production method for photoactive bromopyridyl ethanol Download PDFInfo
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- 238000004519 manufacturing process Methods 0.000 title abstract 4
- RHCIUSVSTJVCCN-UHFFFAOYSA-N 1-bromo-1-pyridin-2-ylethanol Chemical compound BrC(C)(O)C1=NC=CC=C1 RHCIUSVSTJVCCN-UHFFFAOYSA-N 0.000 title abstract 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 60
- 239000000758 substrate Substances 0.000 claims abstract description 57
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 21
- RUJTWTUYVOEEFW-UHFFFAOYSA-N 1-(6-bromopyridin-2-yl)ethanone Chemical compound CC(=O)C1=CC=CC(Br)=N1 RUJTWTUYVOEEFW-UHFFFAOYSA-N 0.000 claims description 19
- 239000000243 solution Substances 0.000 claims description 16
- 230000002210 biocatalytic effect Effects 0.000 claims description 12
- 238000009423 ventilation 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 8
- 239000008103 glucose Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 240000008042 Zea mays Species 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 7
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 7
- 208000012839 conversion disease Diseases 0.000 claims description 7
- 235000005822 corn Nutrition 0.000 claims description 7
- 229960003487 xylose Drugs 0.000 claims description 7
- 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 6
- 239000000872 buffer Substances 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
- 238000000605 extraction Methods 0.000 claims description 6
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 6
- 229920000053 polysorbate 80 Polymers 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 abstract description 10
- 230000002349 favourable effect Effects 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 230000008569 process Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 239000005515 coenzyme Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000007787 solid Substances 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
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 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
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000003905 agrochemical Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000012847 fine chemical Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000024287 Areas Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 240000008564 Boehmeria nivea Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101710157860 Oxydoreductase Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 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
- -1 bromopyridine ethanol Chemical compound 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003054 catalyst Substances 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
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 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
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000009467 reduction Effects 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
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a cell production method for photoactive bromopyridyl ethanol. The cell production method comprises the steps of carrying out biological catalysis by using tarlaromyces flavus cells to prepare photoactive (S)-1-(2-(6- bromopyridyl)ethanol; adding a phosphate buffer solution into a reaction tank; and adding diatomite to control the concentrations of a substrate and a product. The tarlaromyces flavus cells are used for biological catalytic reaction, so that the yield of the product is high, the enantiomeric excess rate (ee%) is high, and the cell production method has a favorable application prospect.
Description
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to Tarlaromyces flavus cell biocatalysis and prepare chiral medicinal intermediate (S)-1-(2-(6-bromopyridine base)) technology of ethanol.
Background technology
Chirality (S)-1-(2-(6-bromopyridine base)) ethanol is the important intermediate of synthesis of chiral medicine, fine chemicals, agricultural chemicals product and other exotic materialss.Biocatalysis asymmetric reaction has environmental friendliness, mild condition, selectivity advantages of higher, prepares the first approach of hydroxyl chipal compounds as green high-efficient, is applied to the chipal compounds producing some high added values more and more.Biocatalysis prepares chirality (S)-1-(2-(6-bromopyridine base)) ethanol has 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 catalysis and biological oxidation process to prepare optical activity chirality compound is 4:2:1.Biomass cells reduction 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.
(S)-1-(2-(6-bromopyridine base)) ethanol 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 (S)-1-(2-(6-bromopyridine base)) ethanol.
Summary of the invention
The present invention adopts Tarlaromyces flavus cell catalysis to prepare (S)-1-(2-(6-bromopyridine base)) ethanol, reaction formula is as follows:
Substrate 2-ethanoyl-6-bromopyridine (1), through Tarlaromyces flavus catalyzed reaction, obtains product (S)-1-(2-(6-bromopyridine base)) ethanol (2).Have multiple-microorganism can catalysis this reaction, through great many of experiments screening, finally determine adopt Tarlaromyces flavus as catalyzer because its catalysis 1 reaction effect best, reaction yield, enantiomeric excess rate (ee%) are all very high.
Many Tarlaromyces flavus can carry out this reaction of biocatalysis, but its effect is different, differs greatly, and through experiment, the present invention selects Tarlaromyces flavus bacterial strain to be ATCC
66738, its this reaction effect of catalysis is best.
developing medium:
1, nutrient solution composition: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-4 g/L; yeast extract 8-10 g/L, glucose 18-22 g/L, malt extract 20-25 g; ammonium sulfate 0.5-0.7g/L; magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L, calcium carbonate 0.7 g/L; ferrous sulfate 0.18 g/L, manganous sulfate 0.025 g/L; PH 4.0.
2, solid medium composition: the agar powder adding 1.5-2% in liquid medium within.
Prepared by Tarlaromyces flavus wet cell.Tarlaromyces flavus through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, and coefficient is 0.6-0.7, and 121 DEG C of autoclavings 30 minutes, are cooled to 25-26 DEG C, by Tarlaromyces flavus
aTCC 66738seed liquor is seeded to fermentor tank, and inoculative proportion is 10-15%, and ventilation ratio is 0.5-1V/(V minute), namely per minute air flow is 0.5-1 times of fermentating liquid volume, cultivate 32-40 hour, obtain wet Tarlaromyces flavus cell, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 25-26 DEG C.Wet cell preparation technique is mature technology.
Because substrate, product all have restraining effect to Tarlaromyces flavus cell, in order to reduce the suppression of substrate, products upon cell, the present invention adopts diatomite adsorption substrate, in reaction, when substrate is reacted 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 0.3-0.33.During concrete absorption, substrate 2-ethanoyl-6-bromopyridine diatomite is absorbed, namely obtains the diatomite having adsorbed substrate.Regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.3-0.33.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, pH is 5.5, add the diatomite having adsorbed substrate 2-ethanoyl-6-bromopyridine, substrate 2-ethanoyl-6-bromopyridine addition is made to be 80-90 g/L, corn steep liquor (with dry basis) content is 15-18g/L, and glucose content is 14-16g/L, wood sugar 12-13 g/L, tween 80 content is 13-14g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 29-31 DEG C, add wet Tarlaromyces flavus cell and make concentration be 36-40g/L, ventilation ratio is 0.2-0.26V/(V minute), namely per minute air flow is 0.3-0.34 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 40-44 hour; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-1-(2-(6-bromopyridine base)) ethanol, reaction conversion ratio 96-98%, product yield 94-96%, enantiomeric excess rate (ee%) 98-99%.
The present invention carries out the work and comprises bacterial strain selection (selecting from more or less a hundred bacterial strain), catalytic reaction condition optimizes (temperature of reaction, air flow, pH), reaction medium is selected and concentration optimization (concentration of substrate, glucose 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 96-98%, during product yield 94-96%, enantiomeric excess rate (ee%), still up to 98-99%, is very not easily, and our work achieves marked improvement.
embodiment 1
Wet Tarlaromyces flavus is produced by ordinary method
aTCC 66738cell, as biocatalytic reaction catalyzer.
The diatomite making method of having adsorbed substrate is, is absorbed by the diatomite of substrate 2-ethanoyl-6-bromopyridine, namely obtains the diatomite having adsorbed substrate, regulates substrate and diatomite consumption, makes substrate and diatomaceous quality ratio be 0.3.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 15L bottom ventilation stirred tank, pH is 5.5, add the diatomite having adsorbed substrate 2-ethanoyl-6-bromopyridine, substrate 2-ethanoyl-6-bromopyridine addition is made to be 80 g/L, corn steep liquor (with dry basis) content is 15g/L, and glucose content is 14g/L, wood sugar 12 g/L, tween 80 content is 13g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 29 DEG C, add wet Tarlaromyces flavus cell and make concentration be 36g/L, ventilation ratio is 0.2V/(V minute), namely per minute air flow is 0.3 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 40 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-1-(2-(6-bromopyridine base)) ethanol, reaction conversion ratio 96%, product yield 94%, enantiomeric excess rate (ee%) 99%.
embodiment 2
Wet Tarlaromyces flavus is produced by ordinary method
aTCC 66738cell, as biocatalytic reaction catalyzer.
The diatomite making method of having adsorbed substrate is, is absorbed by substrate 2-ethanoyl-6-bromopyridine diatomite, namely obtains the diatomite having adsorbed substrate, regulates substrate and diatomite consumption, makes substrate and diatomaceous quality ratio be 0.33.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 100L bottom ventilation stirred tank, pH is 5.5, add the diatomite having adsorbed substrate 2-ethanoyl-6-bromopyridine, substrate 2-ethanoyl-6-bromopyridine addition is made to be 90 g/L, corn steep liquor (with dry basis) content is 18g/L, and glucose content is 16g/L, wood sugar 13 g/L, tween 80 content is 14g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 31 DEG C, add wet Tarlaromyces flavus cell and make concentration be 40g/L, ventilation ratio is 0.26V/(V minute), namely per minute air flow is 0.34 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 44 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-1-(2-(6-bromopyridine base)) ethanol, reaction conversion ratio 98%, product yield 96%, enantiomeric excess rate (ee%) 98%.
embodiment 3
Wet Tarlaromyces flavus is produced by ordinary method
aTCC 66738cell, as biocatalytic reaction catalyzer.
The diatomite making method of having adsorbed substrate is; the substrate 2-ethanoyl-6-bromopyridine diatomite being of a size of 0.5cmX0.5cm is absorbed; namely obtain the diatomite having adsorbed substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.32.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 500L bottom ventilation stirred tank, pH is 5.5, add the diatomite having adsorbed substrate 2-ethanoyl-6-bromopyridine, substrate 2-ethanoyl-6-bromopyridine addition is made to be 85 g/L, corn steep liquor (with dry basis) content is 17g/L, and glucose content is 15.5g/L, wood sugar 12.5 g/L, tween 80 content is 13.5g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 30 DEG C, add wet Tarlaromyces flavus cell and make concentration be 38g/L, ventilation ratio is 0.25V/(V minute), namely per minute air flow is 0.32 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 42 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-1-(2-(6-bromopyridine base)) ethanol, reaction conversion ratio 97%, product yield 95%, enantiomeric excess rate (ee%) 98.5%.
embodiment 4
Wet Tarlaromyces flavus is produced by ordinary method
aTCC 66738cell, as biocatalytic reaction catalyzer.
The diatomite making method of having adsorbed substrate is; the substrate 2-ethanoyl-6-bromopyridine diatomite being of a size of 0.5cmX0.5cm is absorbed; namely obtain the diatomite having adsorbed substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.36.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 1000L bottom ventilation stirred tank, pH is 5.5, add the diatomite having adsorbed substrate 2-ethanoyl-6-bromopyridine, substrate 2-ethanoyl-6-bromopyridine addition is made to be 89g/L, corn steep liquor (with dry basis) content is 16.5g/L, and glucose content is 15g/L, wood sugar 13 g/L, tween 80 content is 14g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 31 DEG C, add wet Tarlaromyces flavus cell and make concentration be 40g/L, ventilation ratio is 0.26V/(V minute), namely per minute air flow is 0.34 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 43 hours; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-1-(2-(6-bromopyridine base)) ethanol, reaction conversion ratio 97.5%, product yield 95%, enantiomeric excess rate (ee%) 98.9%.
Claims (2)
1. Tarlaromyces flavus cell biocatalysis preparation (S)-1-(2-(6-bromopyridine base)) method of ethanol, it is characterized in that adding phosphate buffered saline buffer in retort, pH is 5.5, add the diatomite having adsorbed substrate 2-ethanoyl-6-bromopyridine, substrate 2-ethanoyl-6-bromopyridine addition is made to be 80-90 g/L, corn steep liquor (with dry basis) content is 15-18g/L, glucose content is 14-16g/L, wood sugar 12-13 g/L, tween 80 content is 13-14g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 29-31 DEG C, add wet Tarlaromyces flavus cell and make concentration be 36-40g/L, ventilation ratio is 0.2-0.26V/(V minute), carry out biocatalytic reaction, the reaction times is 40-44 hour; After reaction terminates, leach cell, diatomite respectively, be extracted with ethyl acetate reaction solution, extraction diatomite, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-1-(2-(6-bromopyridine base)) ethanol, reaction conversion ratio 96-98%, product yield 94-96%, enantiomeric excess rate (ee%) 98-99%.
2. method according to claim 1; it is characterized in that described diatomite making method of having adsorbed substrate is; substrate 2-ethanoyl-6-bromopyridine diatomite is absorbed; namely the diatomite having adsorbed substrate is obtained; regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.3-0.33.
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Non-Patent Citations (3)
| Title |
|---|
| MOHAN PAL ET AL.: "Bioreduction of methyl heteroaryl and aryl heteroaryl ketones in high enantiomeric excess with newly isolated fungal strains", 《BIORESOURCE TECHNOLOGY》 * |
| 李治林等: "全细胞生物催化制备生物柴油研究", 《林产化学与工业》 * |
| 杨忠华等: "活性酵母细胞不对称催化苯乙酮还原及树脂吸附对反应的促进作用", 《催化学报》 * |
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