CN105087682A - Method for biologically producing (S)-hydroxyl bromophenyl methyl propionate - Google Patents
Method for biologically producing (S)-hydroxyl bromophenyl methyl propionate Download PDFInfo
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- 229940017219 methyl propionate Drugs 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 230000002210 biocatalytic effect Effects 0.000 claims abstract description 19
- 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
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 22
- 240000008042 Zea mays Species 0.000 claims description 17
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 17
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 17
- 235000005822 corn Nutrition 0.000 claims description 17
- 238000009423 ventilation Methods 0.000 claims description 17
- 235000015097 nutrients Nutrition 0.000 claims description 16
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 241000790917 Dioxys <bee> Species 0.000 claims description 11
- 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
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 11
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 11
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 11
- 239000011591 potassium Substances 0.000 claims description 11
- 229910052700 potassium Inorganic materials 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000000758 substrate Substances 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 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 7
- 230000035484 reaction time Effects 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 229960003487 xylose Drugs 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 6
- 235000010469 Glycine max Nutrition 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 229910000019 calcium carbonate Inorganic materials 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
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 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
- 238000006555 catalytic reaction Methods 0.000 abstract description 8
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000007796 conventional method Methods 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 239000005515 coenzyme Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 5
- 238000011218 seed culture Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000012807 shake-flask culturing Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 3
- 238000005516 engineering process Methods 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
- 230000001580 bacterial effect Effects 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
- 238000005457 optimization Methods 0.000 description 2
- -1 (S)-hydroxyl bromo-hydrocinnamic acid methyl esters Chemical class 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
- 239000013543 active substance Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000011914 asymmetric synthesis Methods 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
- 239000007788 liquid Substances 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000013028 medium composition Substances 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
- 230000009466 transformation Effects 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing (S)-3-hydroxyl-3-(4-bromophenyl) methyl propionate through black mould cell biological catalysis. According to the method, a phosphate buffer solution is added into a reaction tank, and biocatalytic reaction is carried out through using black mould cells, so that the product is high in yield, the enantiomeric excess rate is high, and the production difficulties in the conventional method is solved.
Description
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to melanomyces cell biocatalysis and prepare chiral medicinal intermediate (S)-3-hydroxyl-3-(4-bromophenyl) technology of methyl propionate.
Background technology
Chirality (S)-3-hydroxyl-3-(4-bromophenyl) 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-bromophenyl) 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-bromophenyl) 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-bromophenyl) methyl propionate.
Summary of the invention
The present invention adopts melanomyces cell catalysis to prepare (S)-3-hydroxyl-3-(4-bromophenyl) methyl propionate, reaction formula is as follows:
3-(4-bromophenyl)-3-propionic acid methyl ester (1) through melanomyces catalyzed reaction, obtain product (S)-3-hydroxyl-3-(4-bromophenyl) methyl propionate (2).Have multiple-microorganism can catalysis this reaction, through great many of experiments screening, finally determine adopt melanomyces as catalyzer because the effect of its catalyzed reaction is best, reaction yield, enantiomeric excess rate (ee%) are all very high.
Many melanomyces can carry out this reaction of biocatalysis, but its effect is different, differs greatly, and through experiment, the present invention selects melanomyces bacterial strain to be ATCC56836, 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 20-24g/L, sucrose 25-28g/L, Virahol 7-9%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: corn steep liquor (with dry basis) content is 35-45g/L, yeast extract 10g/L, glucose 20g/L, ammonium sulfate 0.5-0.7g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; The agar powder of 15g/L is added during preparation solid medium.
2, nutrient solution composition: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-5g/L; yeast extract 6-9g/L, glucose 18-22g/L, malt extract 25-30g; 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 0.7g/L; ferrous sulfate 0.18g/L, manganous sulfate 0.025g/L; PH6.3
2, solid medium composition: add the agar powder of 2% in liquid medium within.
Prepared by melanomyces cell.Melanomyces 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 25-26 DEG C, melanomyces seed liquor is seeded to fermentor tank, inoculative proportion is 10-11%, and ventilation ratio is 0.9-1V/(V minute), cultivate 35-40 hour for 25-26 DEG C, wet melanomyces cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal.
Biocatalytic Conversion.Add phosphate buffered saline buffer in bottom ventilation stirred tank, pH is 6.3, adds substrate 3-(4-bromophenyl)-3-propionic acid methyl ester, make concentration of substrate be 80-90g/L.Other compositions: corn steep liquor (with dry basis) content is 19-20g/L, tween 80 19-20g/L, wood sugar 20-24g/L, sucrose 25-28g/L, Virahol 7-9%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 25-26 DEG C, add wet melanomyces cell and make its concentration be 81-85g/L, ventilation ratio is 0.13-0.14V/(V minute), carry out biocatalytic reaction, the reaction times is 73-76 hour; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-bromophenyl) 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, cosubstrate combination, kinds of surfactants and concentration etc.
embodiment 1
Seed culture medium composition is: corn steep liquor (with dry basis) content is 35-45g/L, yeast extract 10g/L, glucose 20g/L, ammonium sulfate 0.5-0.7g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-5g/L; yeast extract 6-9g/L, glucose 18-22g/L, malt extract 25-30g; 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 0.7g/L; ferrous sulfate 0.18g/L, manganous sulfate 0.025g/L; PH6.3
Wet melanomyces cell preparation process is as follows: melanomyces ATCC56836 through inclined-plane, shake-flask culture, obtain seed liquor.1L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 25-26 DEG C, melanomyces seed liquor is seeded to fermentor tank, inoculative proportion is inoculative proportion is 10-11%, and ventilation ratio is 0.9-1V/(V minute), cultivate 35-40 hour for 25-26 DEG C, wet melanomyces 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 6.3, add substrate 3-(4-bromophenyl)-3-propionic acid methyl ester, its concentration is made to be 80g/L, corn steep liquor (with dry basis) content is 19g/L, tween 80 20g/L, wood sugar 24g/L, sucrose 25g/L, Virahol 9%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 26 DEG C, add wet melanomyces cell and make its concentration be 81g/L, ventilation ratio is 0.13V/(V minute), carry out biocatalytic reaction, the reaction times is 76 hours; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-bromophenyl) methyl propionate, reaction conversion ratio 99%, product yield 97%, enantiomeric excess rate 98.5%.
embodiment 2
Seed culture medium composition is: corn steep liquor (with dry basis) content is 35-45g/L, yeast extract 10g/L, glucose 20g/L, ammonium sulfate 0.5-0.7g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-5g/L; yeast extract 6-9g/L, glucose 18-22g/L, malt extract 25-30g; 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 0.7g/L; ferrous sulfate 0.18g/L, manganous sulfate 0.025g/L; PH6.3
Wet melanomyces cell preparation process is as follows: melanomyces ATCC56836 through inclined-plane, shake-flask culture, obtain seed liquor.5L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 25-26 DEG C, melanomyces seed liquor is seeded to fermentor tank, inoculative proportion is 10-11%, and ventilation ratio is 0.9-1V/(V minute), cultivate 35-40 hour for 25-26 DEG C, wet melanomyces 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 6.3, add substrate 3-(4-bromophenyl)-3-propionic acid methyl ester, its concentration is made to be 90g/L, corn steep liquor (with dry basis) content is 20g/L, tween 80 19g/L, wood sugar 20g/L, sucrose 28g/L, Virahol 7%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 25 DEG C, add wet melanomyces cell and make its concentration be 83g/L, ventilation ratio is 0.14V/(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-bromophenyl) methyl propionate, reaction conversion ratio 98%, product yield 96%, enantiomeric excess rate 99%.
embodiment 3
Seed culture medium composition is: corn steep liquor (with dry basis) content is 35-45g/L, yeast extract 10g/L, glucose 20g/L, ammonium sulfate 0.5-0.7g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-5g/L; yeast extract 6-9g/L, glucose 18-22g/L, malt extract 25-30g; 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 0.7g/L; ferrous sulfate 0.18g/L, manganous sulfate 0.025g/L; PH6.3
Wet melanomyces cell preparation process is as follows: melanomyces ATCC56836 through inclined-plane, shake-flask culture, obtain seed liquor.10L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 25-26 DEG C, melanomyces seed liquor is seeded to fermentor tank, inoculative proportion is 10-11%, and ventilation ratio is 0.9-1V/(V minute), cultivate 35-40 hour for 25-26 DEG C, wet melanomyces 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 6.3, add substrate 3-(4-bromophenyl)-3-propionic acid methyl ester, its concentration is made to be 84g/L, corn steep liquor (with dry basis) content is 19g/L, tween 80 20g/L, wood sugar 22g/L, sucrose 26g/L, Virahol 8%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 26 DEG C, add wet melanomyces cell and make its concentration be 84g/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-bromophenyl) methyl propionate, reaction conversion ratio 98.5%, product yield 96.5%, enantiomeric excess rate 99%.
embodiment 4
Seed culture medium composition is: corn steep liquor (with dry basis) content is 35-45g/L, yeast extract 10g/L, glucose 20g/L, ammonium sulfate 0.5-0.7g/L, magnesium sulfate 0.3-0.4g/L, phosphoric acid dioxy potassium 1.8-2.3g/L; The agar powder of 15g/L is added during preparation solid medium.
Nutrient solution composition is: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-5g/L; yeast extract 6-9g/L, glucose 18-22g/L, malt extract 25-30g; 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 0.7g/L; ferrous sulfate 0.18g/L, manganous sulfate 0.025g/L; PH6.3
Wet melanomyces cell preparation process is as follows: melanomyces ATCC56836 through inclined-plane, shake-flask culture, obtain seed liquor.10L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, 121 DEG C of autoclavings 30 minutes, be cooled to 25-26 DEG C, melanomyces seed liquor is seeded to fermentor tank, inoculative proportion is 10-11%, and ventilation ratio is 0.9-1V/(V minute), cultivate 35-40 hour for 25-26 DEG C, wet melanomyces 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 6.3, add substrate 3-(4-bromophenyl)-3-propionic acid methyl ester, its concentration is made to be 86g/L, corn steep liquor (with dry basis) content is 20g/L, tween 80 19g/L, wood sugar 23g/L, sucrose 27g/L, Virahol 9%V/V (volumetric concentration), 121 DEG C of autoclavings 30 minutes; When being cooled to 25 DEG C, add wet melanomyces cell and make its concentration be 85g/L, ventilation ratio is 0.14V/(V minute), carry out biocatalytic reaction, the reaction times is 75 hours; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-bromophenyl) methyl propionate, reaction conversion ratio 99%, product yield 96.5%, enantiomeric excess rate 99%.
Claims (2)
1. melanomyces cell biocatalysis preparation (S)-3-hydroxyl-3-(4-bromophenyl) method of methyl propionate, it is characterized in that adding phosphate buffered saline buffer in retort, pH is 6.3, add substrate 3-(4-bromophenyl)-3-propionic acid methyl ester, making substrate 3-(4-bromophenyl)-3-propionic acid methyl ester addition is 80-90g/L, corn steep liquor content is 19-20g/L, tween 80 19-20g/L, wood sugar 20-24g/L, sucrose 25-28g/L, Virahol 7-9%V/V, 121 DEG C of autoclavings 30 minutes; When being cooled to 25-26 DEG C, add wet melanomyces cell and make its concentration be 81-85g/L, ventilation ratio is 0.13-0.14V/(V minute), carry out biocatalytic reaction, the reaction times is 73-76 hour; After reaction terminates, leach cell, be extracted with ethyl acetate reaction solution, steam ethyl acetate, obtain product (S)-3-hydroxyl-3-(4-bromophenyl) 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 melanomyces cell preparation process is as follows: melanomyces ATCC56836, 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 25-26 DEG C, melanomyces seed liquor is seeded to fermentor tank, inoculative proportion is 10-11%, and ventilation ratio is 0.9-1V/(V minute), cultivate 35-40 hour for 25-26 DEG C, wet melanomyces cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal; In fermentor tank, nutrient solution composition is: corn steep liquor (with dry basis) content is 35-45g/L; analysis for soybean powder 3-5g/L; yeast extract 6-9g/L, glucose 18-22g/L, malt extract 25-30g; 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 0.7g/L; ferrous sulfate 0.18g/L, manganous sulfate 0.025g/L; PH6.3.
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Application publication date: 20151125 |