CN104313072A - Method for preparing (1R, 3S)dioxo-heptylene alcohol by candida - Google Patents
Method for preparing (1R, 3S)dioxo-heptylene alcohol by candida Download PDFInfo
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- CN104313072A CN104313072A CN201410537231.6A CN201410537231A CN104313072A CN 104313072 A CN104313072 A CN 104313072A CN 201410537231 A CN201410537231 A CN 201410537231A CN 104313072 A CN104313072 A CN 104313072A
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- diatomite
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- glycerose
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- 238000000034 method Methods 0.000 title claims abstract description 23
- 241000222120 Candida <Saccharomycetales> Species 0.000 title abstract 2
- 239000000758 substrate Substances 0.000 claims abstract description 70
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 60
- 238000006243 chemical reaction Methods 0.000 claims abstract description 43
- 210000005253 yeast cell Anatomy 0.000 claims abstract description 20
- 210000004027 cell Anatomy 0.000 claims abstract description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 54
- 239000000243 solution Substances 0.000 claims description 37
- AWAKCWDDISOXOC-QMMMGPOBSA-N (2r)-3-cyclohexylidene-2,3-dihydroxypropanal Chemical compound O=C[C@H](O)C(O)=C1CCCCC1 AWAKCWDDISOXOC-QMMMGPOBSA-N 0.000 claims description 32
- 241000222173 Candida parapsilosis Species 0.000 claims description 20
- 229940055022 candida parapsilosis Drugs 0.000 claims description 20
- 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 17
- 239000008103 glucose Substances 0.000 claims description 17
- 235000015097 nutrients Nutrition 0.000 claims description 17
- 238000009423 ventilation Methods 0.000 claims description 17
- 230000002210 biocatalytic effect Effects 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 229960003487 xylose Drugs 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 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
- 239000000843 powder Substances 0.000 claims description 12
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- 208000012839 conversion disease Diseases 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- XZIIFPSPUDAGJM-UHFFFAOYSA-N 6-chloro-2-n,2-n-diethylpyrimidine-2,4-diamine Chemical compound CCN(CC)C1=NC(N)=CC(Cl)=N1 XZIIFPSPUDAGJM-UHFFFAOYSA-N 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 235000005822 corn Nutrition 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
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 230000035484 reaction time Effects 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
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- 238000006555 catalytic reaction Methods 0.000 abstract description 8
- 230000003197 catalytic effect Effects 0.000 abstract description 3
- 230000003287 optical effect Effects 0.000 abstract description 2
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 239000000126 substance Substances 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
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
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- 230000001580 bacterial effect Effects 0.000 description 4
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- 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
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- HDQDUBPEBVXIBJ-VOTSOKGWSA-N (e)-hept-1-en-1-ol Chemical compound CCCCC\C=C\O HDQDUBPEBVXIBJ-VOTSOKGWSA-N 0.000 description 1
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- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 1
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- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
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Abstract
The invention relates to a method for preparing (1R, 3S)-1, 2-(cyclohexylidene dioxo) heptyl-6-ene-3-alcohol with optical activity in a catalytic way directly by nearly-smooth candida cells. The method comprises the following steps: feeding a phosphate buffer solution into a reaction tank, feeding diatomite to regulate and control the concentrations of the substrate and the product, and carrying out a biological catalysis reaction by the yeast cells. The method is high in product yield, high in enantiomeric excess rate and remarkably superior to a chemical method.
Description
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to Candida parapsilosis cell biocatalysis and prepare chiral medicinal intermediate (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol technology.
Background technology
Chirality cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol be the important intermediate of synthesis of chiral medicine, fine chemicals, agricultural chemicals product and other exotic materialss.Biocatalysis asymmetric synthesis 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 cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol there is 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.
(1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol be 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol.
Summary of the invention
The present invention adopts Candida parapsilosis cell catalysis to prepare (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, reaction formula is as follows:
Substrate R-cyclohexylidene Glycerose (1) and the bromo-1-butylene of 4-(2), through Candida parapsilosis catalyzed reaction, obtain product (1R, 3S)-1,2-(cyclohexylidene dioxy base)-6-alkene-3-alcohol in heptan (3).Have multiple-microorganism can catalysis this reaction, through great many of experiments screening, finally determine adopt Candida parapsilosis as catalyzer because its catalyzed reaction
1 and 2 reactionseffect best, reaction yield, enantiomeric excess rate (ee%) are all very high.
Many Candida parapsilosis can carry out this reaction of biocatalysis, but its effect is different, differs greatly, and through experiment, the present invention selects Candida parapsilosis bacterial strain to be
aTCC 22019D-5, its catalyzed reaction 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 37-44 g/L, wood sugar 7-8g/L, corn plumule powder 10-12 g/L, yeast extract 4-5 g/L, (NH
4)
2h PO
412-15 g/L, KH
2pO
46.5-7.5 g/L. MgSO
4.7H
2o 0.7-0.8 g/L, NaCl 0.08-0.1 g/L; PH value 7.0.
2, solid medium composition: add the agar powder of 2% in liquid medium within.
Prepared by yeast cell.Candida parapsilosis through inclined-plane, shaking flask, seed tank culture obtain seed liquor; Fermentor tank adds nutrient solution, Candida parapsilosis 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 parapsilosis 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 biocatalytic reaction 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 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 0.32-0.35.During concrete absorption, by substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-miscible, R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of 4-are 1, absorb this solution, namely obtain the diatomite having adsorbed substrate with diatomite.Regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.32-0.35.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 6.5, add the diatomite having adsorbed substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-, substrate R-cyclohexylidene Glycerose addition is made to be 80-90 g/L, substrate R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of substrate 4-are 1, and 2 kinds of concentration of substrate all determine thus.Glucose 25-30 g, wood sugar 15-20g/L, sorbitan monolaurate is 19-21g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 26-28 DEG C, add wet yeast cell and make concentration be 29-32g/L, ventilation ratio is 0.15-0.17V/(V minute), namely per minute air flow is 0.15-0.17 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 82-89 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, 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 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 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 37 g/L, wood sugar 78g/L, corn plumule powder 10 g/L, yeast extract 4 g/L, (NH
4)
2h PO
412 g/L, KH
2pO
46.5 g/L. MgSO
4.7H
2o 0.7 g/L, NaCl 0.08 g/L; PH value 7.0.
Wet yeast cell preparation process is as follows: Candida parapsilosis ATCC 22019D-5 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 parapsilosis seed liquor is seeded to fermentor tank, inoculative proportion is 6%, ventilation ratio is 0.5V/(V minute), cultivate 48 hours, obtain wet yeast cell, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 29 DEG C.
The diatomite making method of having adsorbed substrate is, by substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-miscible, R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of 4-are 1, this solution is absorbed with diatomite, namely the diatomite having adsorbed substrate is obtained, 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 50L bottom ventilation stirred tank, coefficient is 0.6, pH is 6.5, add the diatomite having adsorbed substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-, make substrate R-cyclohexylidene Glycerose addition be 80 g/L, substrate R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of substrate 4-are 1, glucose 25 g, wood sugar 15 g/L, sorbitan monolaurate is 19g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 26 DEG C, add wet yeast cell and make concentration be 29g/L, ventilation ratio is 0.15V/(V minute), namely per minute air flow is 0.15 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 82 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, reaction conversion ratio 94%, product yield 92%, enantiomeric excess rate (ee%) 98%.
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 44 g/L, wood sugar 8g/L, corn plumule powder 12 g/L, yeast extract 5 g/L, (NH
4)
2h PO
415 g/L, KH
2pO
47.5 g/L. MgSO
4.7H
2o 0.8 g/L, NaCl 0.1 g/L; PH value 7.0.
Wet yeast cell preparation process is as follows: Candida parapsilosis ATCC 22019D-5 through inclined-plane, shake-flask culture, obtain seed liquor.100L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.7,121 DEG C of autoclavings 30 minutes, be cooled to 31 DEG C, Candida parapsilosis seed liquor is seeded to fermentor tank, inoculative proportion is 6.5%, ventilation ratio is 1V/(V minute), cultivate 56 hours, obtain wet yeast cell, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 31 DEG C.
The diatomite making method of having adsorbed substrate is, by substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-miscible, R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of 4-are 1, this solution is absorbed with diatomite, namely the diatomite having adsorbed substrate is obtained, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.35.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, coefficient is 0.7, pH is 6.5, add the diatomite having adsorbed substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-, make substrate R-cyclohexylidene Glycerose addition be 90 g/L, substrate R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of substrate 4-are 1, glucose 30 g, wood sugar 20g/L, sorbitan monolaurate is 21g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 28 DEG C, add wet yeast cell and make concentration be 32g/L, ventilation ratio is 0.17V/(V minute), namely per minute air flow is 0.12 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 89 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, 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 40 g/L, wood sugar 7.5g/L, corn plumule powder 11 g/L, yeast extract 4.5 g/L, (NH
4)
2h PO
413 g/L, KH
2pO
47 g/L. MgSO
4.7H
2o 0.75 g/L, NaCl 0.09 g/L; PH value 7.0.
Wet yeast cell preparation process is as follows: Candida parapsilosis ATCC 22019D-5 through inclined-plane, shake-flask culture, obtain seed liquor.250L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.65,121 DEG C of autoclavings 30 minutes, be cooled to 30 DEG C, Candida parapsilosis seed liquor is seeded to fermentor tank, inoculative proportion is 6.3%, ventilation ratio is 0.8V/(V minute), cultivate 52 hours, obtain wet yeast cell, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 30 DEG C.
The diatomite making method of having adsorbed substrate is, by substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-miscible, R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of 4-are 1, this solution is absorbed with diatomite, namely the diatomite having adsorbed substrate is obtained, regulate substrate and diatomite consumption, make 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 1000L bottom ventilation stirred tank, coefficient is 0.65, pH is 6.5, add the diatomite having adsorbed substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-, make substrate R-cyclohexylidene Glycerose addition be 85 g/L, substrate R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of substrate 4-are 1, glucose 28 g, wood sugar 18 g/L, sorbitan monolaurate is 20g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 27 DEG C, add wet yeast cell and make concentration be 30g/L, ventilation ratio is 0.16V/(V minute), namely per minute air flow is 0.16 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 87 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, 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 42 g/L, wood sugar 8g/L, corn plumule powder 12 g/L, yeast extract 5 g/L, (NH
4)
2h PO
415 g/L, KH
2pO
47.5 g/L. MgSO
4.7H
2o 0.75 g/L, NaCl 0.1 g/L; PH value 7.0.
Wet yeast cell preparation process is as follows: Candida parapsilosis ATCC 22019D-5 through inclined-plane, shake-flask culture, obtain seed liquor.5000L fermentor tank adds nutrient solution; Fermentor tank adds nutrient solution, coefficient is 0.7,121 DEG C of autoclavings 30 minutes, be cooled to 31 DEG C, Candida parapsilosis seed liquor is seeded to fermentor tank, inoculative proportion is 6.5%, ventilation ratio is 1V/(V minute), cultivate 56 hours, obtain wet yeast cell, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 31 DEG C.
The diatomite making method of having adsorbed substrate is, by substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-miscible, R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of 4-are 1, this solution is absorbed with diatomite, namely the diatomite having adsorbed substrate is obtained, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.35.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 20L bottom ventilation stirred tank, coefficient is 0.7, pH is 6.5, add the diatomite having adsorbed substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-, make substrate R-cyclohexylidene Glycerose addition be 90 g/L, substrate R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of substrate 4-are 1, glucose 30 g, wood sugar 19 g/L, sorbitan monolaurate is 20g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 28 DEG C, add wet yeast cell and make concentration be 32g/L, ventilation ratio is 0.165V/(V minute), namely per minute air flow is 0.165 times of reaction solution volume, carries out biocatalytic reaction, and the reaction times is 89 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, reaction conversion ratio 96%, product yield 93%, enantiomeric excess rate (ee%) 98%.
Claims (4)
1. a Candida parapsilosis cell biocatalysis preparation (1R, 3S)-1, 2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol method, it is characterized in that adding phosphate buffered saline buffer in retort, coefficient is 0.6-0.7, pH is 6.5, add the diatomite having adsorbed substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-, substrate R-cyclohexylidene Glycerose addition is made to be 80-90g/L, substrate R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of substrate 4-are 1, glucose 25-30 g, wood sugar 15-20g/L, sorbitan monolaurate is 19-21g/L, 121 DEG C of autoclavings 30 minutes, when being cooled to 26-28 DEG C, add wet yeast cell and make concentration be 29-32g/L, ventilation ratio is 0.15-0.17V/(V minute), carry out biocatalytic reaction, the reaction times is 82-89 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 (1R, 3S)-1,2-(cyclohexylidene dioxy base) heptan-6-alkene-3-alcohol, 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 parapsilosis 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 29-31 DEG C, Candida parapsilosis 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 biocatalytic reaction catalyzer with filtering centrifuge is centrifugal for 29-31 DEG C.
3. method according to claim 1, is characterized in that nutrient solution composition is: glucose 37-44 g/L, wood sugar 7-8g/L, corn plumule powder 10-12 g/L, yeast extract 4-5 g/L, (NH
4)
2h PO
412-15 g/L, KH
2pO
46.5-7.5 g/L. MgSO
4.7H
2o 0.7-0.8 g/L, NaCl 0.08-0.1 g/L; PH value 7.0.
4. method according to claim 1, it is characterized in that described diatomite making method of having adsorbed substrate is, by substrate R-cyclohexylidene Glycerose and the bromo-1-butylene of 4-miscible, R-cyclohexylidene Glycerose and the bromo-1-butylene mol ratio of 4-are 1, this solution is absorbed with diatomite, namely obtain the diatomite having adsorbed substrate, regulate substrate and diatomite consumption, make substrate and diatomaceous quality ratio be 0.32-0.35.
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| CN1793355A (en) * | 2005-12-01 | 2006-06-28 | 华东理工大学 | Organic solvent slow-releasing system and its preparation and application for catalyzing reaction of enzyme thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793355A (en) * | 2005-12-01 | 2006-06-28 | 华东理工大学 | Organic solvent slow-releasing system and its preparation and application for catalyzing reaction of enzyme thereof |
Non-Patent Citations (1)
| Title |
|---|
| MIYU FURUTA等: "Stereoselective approach to (2R,3S)- and (2R,3R)-1,2-(cyclohexylidenedioxy)hept-6-en-3-ol by microbial reduction", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
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