CN105177069A - Method for cell production of (S)-hydroxyethyl methyl phenylpropionate - Google Patents
Method for cell production of (S)-hydroxyethyl methyl phenylpropionate Download PDFInfo
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- CN105177069A CN105177069A CN201510560642.1A CN201510560642A CN105177069A CN 105177069 A CN105177069 A CN 105177069A CN 201510560642 A CN201510560642 A CN 201510560642A CN 105177069 A CN105177069 A CN 105177069A
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- ethylbenzene
- methyl propionate
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- 238000000034 method Methods 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title abstract description 4
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 229940017219 methyl propionate Drugs 0.000 claims abstract description 23
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 18
- RJUFJBKOKNCXHH-UHFFFAOYSA-N Methyl propionate Chemical compound CCC(=O)OC RJUFJBKOKNCXHH-UHFFFAOYSA-N 0.000 claims abstract description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- 210000005253 yeast cell Anatomy 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 20
- 230000002210 biocatalytic effect Effects 0.000 claims description 18
- 238000009423 ventilation Methods 0.000 claims description 17
- 235000015097 nutrients Nutrition 0.000 claims description 13
- 239000000203 mixture Substances 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
- 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 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 210000004027 cell Anatomy 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 229960003487 xylose Drugs 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 7
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- 230000035484 reaction time Effects 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 6
- 208000012839 conversion disease Diseases 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
- 238000001914 filtration Methods 0.000 claims description 6
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 241000790917 Dioxys <bee> Species 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 238000006555 catalytic reaction Methods 0.000 abstract description 9
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 3
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 239000000843 powder Substances 0.000 description 11
- 239000000758 substrate Substances 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 239000005515 coenzyme Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 4
- 235000005822 corn Nutrition 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000012807 shake-flask culturing Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 2
- 101710157860 Oxydoreductase Proteins 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
- 230000001419 dependent 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
- 239000007788 liquid Substances 0.000 description 2
- 239000013028 medium composition Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- ALSTYHKOOCGGFT-KTKRTIGZSA-N (9Z)-octadecen-1-ol Chemical compound CCCCCCCC\C=C/CCCCCCCCO ALSTYHKOOCGGFT-KTKRTIGZSA-N 0.000 description 1
- 241000024287 Areas Species 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- BQODPTQLXVVEJG-UHFFFAOYSA-N [O].C=C Chemical compound [O].C=C BQODPTQLXVVEJG-UHFFFAOYSA-N 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 229940055577 oleyl alcohol Drugs 0.000 description 1
- XMLQWXUVTXCDDL-UHFFFAOYSA-N oleyl alcohol Natural products CCCCCCC=CCCCCCCCCCCO XMLQWXUVTXCDDL-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 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
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Abstract
The invention discloses a method for preparing (S)-3-hydroxy-3-(4-ethylbenzene) methyl propionate through biological catalysis of saccharomyces cerevisiae cells, wherein the method comprises steps of adding a phosphate buffer solution to a reaction tank, and applying saccharomyces cerevisiae cells to a biological catalytic reaction. The method is high in product yield and high in enantiomeric excess rate; and problems of an existing method in production are solved.
Description
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to brewing yeast cell biocatalysis and prepare chiral medicinal intermediate (S)-3-hydroxyl-3-(4-ethylbenzene) technology of methyl propionate.
Background technology
Chirality (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate 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 chipal compounds as green high-efficient, is applied to the chipal compounds producing some high added values more and more.Biocatalysis prepares chirality (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate 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 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.
(S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate is the important chiral building block of synthesis of chiral medicine, fine chemicals, agricultural chemicals product.Existing method ubiquity low conversion rate, long reaction time, needs add expensive coenzyme, high in cost of production major defect, are difficult to carry out suitability for industrialized production.The present invention will adopt biomass cells catalysis to prepare (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate.
Summary of the invention
The present invention adopts brewing yeast cell catalysis to prepare (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction formula is as follows:
3-(4-ethylbenzene)-3-methyl propionate (1) through yeast saccharomyces cerevisiae catalyzed reaction, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate (2).Have multiple-microorganism can catalysis this reaction, through great many of experiments screening, finally determine adopt yeast saccharomyces cerevisiae as catalyzer because the effect of its catalyzed reaction is best, reaction yield, enantiomeric excess rate (ee%) are all very high.
Many yeast saccharomyces cerevisiaes can carry out this reaction of biocatalysis, but its effect is different, differs greatly, and through experiment, the present invention selects Wine brewing yeast strain to be ATCC208275, its catalyzed reaction best results.
Because most oxydo-reductase is coenzyme NAD (P) H dependent form, and the coenzyme amount contained by cell itself may be less, therefore, when carrying out redox reaction, for promoting regenerating coenzyme, improving reaction efficiency, in reaction system, also adding cosubstrate form transformation system.Conventional cosubstrate kind is more, and the cosubstrate needed for different cell is different, and effect difference is very large, adopts several cosubstrate to combine, can obtain better effects, need carry out great many of experiments and could determine best cosubstrate.The cosubstrate that the present invention determines through great many of experiments is: wood sugar 42-46g/L, maltose 20-24g/L, Virahol 5-6%V/V (volumetric concentration).
Because substrate solubility is lower, so, add tensio-active agent to increase the solubleness of substrate.
developing medium:
1, seed culture medium composition is: yeast extract 10g/L, glucose 20g/L, peptone 20g/L; The agar powder of 15g/L is added during preparation solid medium.
2, nutrient solution composition: peptone 1.5-2g/L, yeast extract 8-10g/L, glucose 18-22g/L, malt extract 25-30g, ammonium sulfate 0.2-0.25g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; Solid medium composition: add the agar powder of 2% in liquid medium within; PH6.9.
2, solid medium composition: add the agar powder of 2% in liquid medium within.
Prepared by yeast cell.Yeast saccharomyces cerevisiae through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 23-24 DEG C, yeast saccharomyces cerevisiae seed liquor is seeded to fermentor tank, inoculative proportion is 6-6.5%, ventilation ratio is 0.8-1V/(V minute), cultivate 47-50 hour, obtain wet yeast cell, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 23-24 DEG C.
Biocatalytic Conversion.Add phosphate buffered saline buffer in bottom ventilation stirred tank, pH is 7.3, adds substrate 3-(4-ethylbenzene)-3-methyl propionate, make concentration of substrate be 80-90g/L.Other compositions: malt extract 22-26g, paregal O (PerogolO, the oxygen ethene of 15 units and the condenses of oleyl alcohol) 21-22g/L, wood sugar 25-30g/L, maltose 42-46g/L, Virahol 5-6%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 23-24 DEG C, add wet yeast cell and make concentration be 89-92g/L, ventilation ratio is 0.11-0.13V/(V minute), namely per minute air flow is 0.11-0.13 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 70-74 hour; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction conversion ratio 98-99%, product yield 96-97%, enantiomeric excess rate 98.5-99%.
The present invention carries out the work and comprises bacterial strain selection, catalytic reaction condition optimization (temperature of reaction, air flow, pH), reaction medium selection and concentration optimization, comprises concentration of substrate, malt extract content, cosubstrate combination, kinds of surfactants and concentration etc.
embodiment 1
Seed culture medium composition is: yeast extract 10g/L, glucose 20g/L, peptone 20g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 37g/L, wood sugar 78g/L, corn plumule powder 10g/L, yeast extract 4g/L, (NH
4)
2hPO
412g/L, KH
2pO
46.5g/L.MgSO
4.7H
2o 0.7g/L, NaCl 0.08g/L; PH6.9.
Wet yeast cell preparation process is as follows: yeast saccharomyces cerevisiae ATCC208275 through inclined-plane, shake-flask culture, obtain seed liquor.1L fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 23 DEG C, yeast saccharomyces cerevisiae seed liquor is seeded to fermentor tank, inoculative proportion is 6%, and ventilation ratio is 0.8V/(V minute), cultivate 47 hours for 23 DEG C, wet yeast cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal.
Biocatalytic Conversion: add phosphate buffered saline buffer in 0.5L bottom ventilation stirred reactor, pH is 7.3, add substrate 3-(4-ethylbenzene)-3-methyl propionate, concentration is made to be 80g/L, malt extract 22g, paregal O 21-22g/L, wood sugar 25-30g/L, maltose 42-46g/L, Virahol 5-6%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 23 DEG C, add wet yeast cell and make concentration be 89g/L, ventilation ratio is 0.11V/(V minute), carry out biocatalytic reaction, the reaction times is 70 hours; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction conversion ratio 99%, product yield 97%, enantiomeric excess rate 98.5%.
embodiment 2
Seed culture medium composition is: yeast extract 10g/L, glucose 20g/L, peptone 20g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 44g/L, wood sugar 8g/L, corn plumule powder 12g/L, yeast extract 5g/L, (NH
4)
2hPO
415g/L, KH
2pO
47.5g/L.MgSO
4.7H
2o 0.8g/L, NaCl 0.1g/L; PH6.9.
Wet yeast cell preparation process is as follows: yeast saccharomyces cerevisiae ATCC208275 through inclined-plane, shake-flask culture, obtain seed liquor.5L fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 24 DEG C, yeast saccharomyces cerevisiae seed liquor is seeded to fermentor tank, inoculative proportion is 6.5%, and ventilation ratio is 1V/(V minute), cultivate 50 hours for 24 DEG C, wet yeast cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal.
Biocatalytic Conversion: add phosphate buffered saline buffer in 5L bottom ventilation stirred reactor, pH is 7.3, add substrate 3-(4-ethylbenzene)-3-methyl propionate, concentration is made to be 90g/L, malt extract 22g, paregal O 21-22g/L, wood sugar 25-30g/L, maltose 42-46g/L, Virahol 5-6%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 24 DEG C, add wet yeast cell and make concentration be 92g/L, ventilation ratio is 0.12V/(V minute), carry out biocatalytic reaction, the reaction times is 72 hours; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction conversion ratio 98%, product yield 96%, enantiomeric excess rate 99%.
embodiment 3
Seed culture medium composition is: yeast extract 10g/L, glucose 20g/L, peptone 20g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 40g/L, wood sugar 7.5g/L, corn plumule powder 11g/L, yeast extract 4.5g/L, (NH
4)
2hPO
413g/L, KH
2pO
47g/L.MgSO
4.7H
2o 0.75g/L, NaCl 0.09g/L; PH6.9.
Wet yeast cell preparation process is as follows: yeast saccharomyces cerevisiae ATCC208275 through inclined-plane, shake-flask culture, obtain seed liquor.10L fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 23 DEG C, yeast saccharomyces cerevisiae seed liquor is seeded to fermentor tank, inoculative proportion is 6.3%, and ventilation ratio is 0.9V/(V minute), cultivate 48 hours for 23 DEG C, wet yeast cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal.
Biocatalytic Conversion: add phosphate buffered saline buffer in 10L bottom ventilation stirred reactor, pH is 7.3, add substrate 3-(4-ethylbenzene)-3-methyl propionate, concentration is made to be 84g/L, malt extract 22g, paregal O 21-22g/L, wood sugar 25-30g/L, maltose 42-46g/L, Virahol 5-6%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 23 DEG C, add wet yeast cell and make concentration be 90g/L, ventilation ratio is 0.13V/(V minute), carry out biocatalytic reaction, the reaction times is 74 hours; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction conversion ratio 98.5%, product yield 96.5%, enantiomeric excess rate 99%.
embodiment 4
Seed culture medium composition is: yeast extract 10g/L, glucose 20g/L, peptone 20g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: glucose 42g/L, wood sugar 8g/L, corn plumule powder 12g/L, yeast extract 5g/L, (NH
4)
2hPO
415g/L, KH
2pO
47.5g/L.MgSO
4.7H
2o 0.75g/L, NaCl 0.1g/L; PH6.9.
Wet yeast cell preparation process is as follows: yeast saccharomyces cerevisiae ATCC208275 through inclined-plane, shake-flask culture, obtain seed liquor.10L fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 24 DEG C, yeast saccharomyces cerevisiae seed liquor is seeded to fermentor tank, inoculative proportion is 6.5%, and ventilation ratio is 0.85V/(V minute), cultivate 49 hours for 24 DEG C, wet yeast cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal.
Biocatalytic Conversion: add phosphate buffered saline buffer in 20L bottom ventilation stirred reactor, pH is 7.3, add substrate 3-(4-ethylbenzene)-3-methyl propionate, concentration is made to be 86g/L, malt extract 22g, paregal O 21-22g/L, wood sugar 25-30g/L, maltose 42-46g/L, Virahol 5-6%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 24 DEG C, add wet yeast cell and make concentration be 91g/L, ventilation ratio is 0.13V/(V minute), carry out biocatalytic reaction, the reaction times is 73 hours; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction conversion ratio 99%, product yield 96.5%, enantiomeric excess rate 99%.
Claims (2)
1. brewing yeast cell biocatalysis preparation (S)-3-hydroxyl-3-(4-ethylbenzene) method of methyl propionate, it is characterized in that adding phosphate buffered saline buffer in retort, pH is 7.3, add 3-(4-ethylbenzene)-3-methyl propionate, making 3-(4-ethylbenzene)-3-methyl propionate addition is 80-90g/L, malt extract 22-26g, paregal O 21-22g/L, wood sugar 25-30g/L, maltose 42-46g/L, Virahol 5-6%V/V, 121 DEG C of autoclavings 30 minutes; When being cooled to 23-24 DEG C, add wet yeast cell and make concentration be 89-92g/L, ventilation ratio is 0.11-0.13V/(V minute), carry out biocatalytic reaction, the reaction times is 70-74 hour; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-ethylbenzene) methyl propionate, reaction conversion ratio 98-99%, product yield 96-97%, enantiomeric excess rate 98.5-99%.
2. method according to claim 1, is characterized in that wet yeast cell preparation process is as follows: yeast saccharomyces cerevisiae ATCC208275, and through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 23-24 DEG C, yeast saccharomyces cerevisiae seed liquor is seeded to fermentor tank, inoculative proportion is 6-6.5%, and ventilation ratio is 0.8-1.0V/(V minute), cultivate 47-50 hour for 23-24 DEG C, wet yeast cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal; In fermentor tank, nutrient solution composition is: peptone 1.5-2g/L, yeast extract 8-10g/L, glucose 18-22g/L, malt extract 25-30g, ammonium sulfate 0.2-0.25g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; PH6.9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510560642.1A CN105177069A (en) | 2015-09-07 | 2015-09-07 | Method for cell production of (S)-hydroxyethyl methyl phenylpropionate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510560642.1A CN105177069A (en) | 2015-09-07 | 2015-09-07 | Method for cell production of (S)-hydroxyethyl methyl phenylpropionate |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105177069A true CN105177069A (en) | 2015-12-23 |
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|---|---|---|---|
| CN201510560642.1A Withdrawn CN105177069A (en) | 2015-09-07 | 2015-09-07 | Method for cell production of (S)-hydroxyethyl methyl phenylpropionate |
Country Status (1)
| Country | Link |
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2015
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