CN104328154A - Method for producing (S)-phenyl methanol from blue mold - Google Patents

Method for producing (S)-phenyl methanol from blue mold Download PDF

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Publication number
CN104328154A
CN104328154A CN201410594403.3A CN201410594403A CN104328154A CN 104328154 A CN104328154 A CN 104328154A CN 201410594403 A CN201410594403 A CN 201410594403A CN 104328154 A CN104328154 A CN 104328154A
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carclazyte
substrate
reaction
cell
phenyl
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刘均洪
曹延俊
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Qingdao University of Science and Technology
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Qingdao University of Science and Technology
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Abstract

The invention relates to a method for producing (S)-phenyl methanol from blue mold, which comprises the following steps: preparing photoactive (S)-phenyl-(2-pyridyl)methanol by using penicillium cell as a biological catalyst, adding a phosphate buffer solution into the reaction tank, and adding argil to regulate the concentrations of the substrate and product. The penicillium cell is utilized to perform biological catalytic reaction, so the method has the advantages of high product yield and high enantiomeric excess ratio (ee%) and has favorable application prospects.

Description

A kind of mould produces the method for (S)-phenyl methanol
Technical field
The invention belongs to technology of pharmaceutical engineering field, be related specifically to the technology that Penicillium cell biocatalysis prepares chiral medicinal intermediate (S)-phenyl-(2-pyridyl) methyl alcohol.
Background technology
Chirality (S)-phenyl-(2-pyridyl) methyl alcohol 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 is prepared chirality (S)-phenyl-(2-pyridyl) methyl alcohol and is had good application prospect.
Biocatalysis has the outstanding advantages such as catalytic efficiency is high, selectivity strong, mild condition, environmental friendliness, is the important method substituting and expand traditional organic chemical synthesis in process of sustainable development.Wherein chiral separation and asymmetric synthesis are the Application Areass of biocatalysis most magnetism.As in six large fermentoids of biological catalyst, lytic enzyme catalytic kinetics resolution of racemates can obtain quiral products, in industrial biocatalytic, play key player always.In recent years, oxydo-reductase application industrially obtained and increased rapidly.At present, the ratio adopting the industry of lytic enzyme Kinetic Resolution, biological 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)-phenyl-(2-pyridyl) methyl alcohol is the important chiral building block of synthesis of chiral medicine, fine chemicals, agricultural chemicals product, and owing to there being 2 chiral centres, make chemosynthesis comparatively difficult, cost is high.The present invention will adopt biocatalysis to prepare (S)-phenyl-(2-pyridyl) methyl alcohol.
Summary of the invention
The present invention adopts Penicillium cell catalysis to prepare (S)-phenyl-(2-pyridyl) methyl alcohol, and reaction formula is as follows:
Substrate 2-benzoyl pyridine (1), through Penicillium notatum catalyzed reaction, obtains product (S)-phenyl-(2-pyridyl) methyl alcohol (2).Have multiple-microorganism can catalysis this reaction, through great many of experiments screening, finally determine adopt Penicillium notatum as catalyzer because its catalysis 1 reaction effect best, reaction yield, enantiomeric excess rate (ee%) are all very high.
Many Penicillium notatums can carry out this reaction of biocatalysis, but its effect is different, differs greatly, and through experiment, the present invention selects Penicillium strain to be ATCC 204049, and 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; yeast extract 8-10 g/L; glucose 18-22 g/L; malt extract 25-30 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 4.7-4.9.
2, solid medium composition: the agar powder adding 1.5-2% in liquid medium within.
Prepared by Penicillium cell.Penicillium notatum 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 Penicillium notatum aTCC204049 seed liquor are seeded to fermentor tank, inoculative proportion is 10-15%, 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 for 25-26 DEG C, wet Penicillium cell is obtained, as biocatalytic reaction catalyzer with filtering centrifuge is centrifugal.Wet cell preparation technique is mature technology.
Because substrate, product all have restraining effect to Penicillium cell, in order to reduce the suppression of substrate, products upon cell, the present invention adopts bleaching earth adsorption substrate, in reaction, when substrate is reacted by cell catalysis, when concentration reduces, substrate from carclazyte stripping, postreaction consume substrate.Meanwhile, product, by bleaching earth adsorption, decreases its concentration in water, thus considerably reduces the suppression of substrate, products upon cell.The ratio of substrate and carclazyte, determines 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 the ratio of carclazyte could be determined.Experiment shows, for bacterial strain of the present invention, reaction solution composition, temperature of reaction, best substrate and the ratio of carclazyte are 0.27-0.3.During concrete absorption, substrate 2-benzoyl pyridine carclazyte is absorbed, namely obtains the carclazyte having adsorbed substrate.Regulate substrate and carclazyte consumption, make the quality ratio of substrate and carclazyte be 0.27-0.3.Carclazyte used is common carclazyte, and commercially, sterilizing, Preservation in sterile condition is stand-by.
Phosphate buffered saline buffer is added in bottom ventilation stirred tank, pH is 6.6, add the carclazyte having adsorbed substrate 2-benzoyl pyridine, substrate 2-benzoyl pyridine addition is made to be 80-90 g/L, corn steep liquor (with dry basis) content is 12-15g/L, and glucose content is 15-18g/L, wood sugar 13-15g/L, tween 80 content is 12-15g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 26-27 DEG C, add wet Penicillium cell and make concentration be 40-44g/L, ventilation ratio is 0.13-0.14V/(V minute) namely per minute air flow be 0.13-14 times of reaction solution volume, carry out biocatalytic reaction, the reaction times is 60-65 hour; After reaction terminates, leach cell, carclazyte respectively, be extracted with ethyl acetate reaction solution, extraction carclazyte, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-phenyl-(2-pyridyl) methyl alcohol, 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 Penicillium notatum ATCC 204049 cell is produced, as biocatalytic reaction catalyzer by ordinary method.
The carclazyte making method of having adsorbed substrate is, is absorbed by substrate 2-benzoyl pyridine carclazyte, namely obtains the carclazyte having adsorbed substrate, regulates substrate and carclazyte consumption, makes the quality ratio of substrate and carclazyte be 0.27.Carclazyte used is common carclazyte, and commercially, sterilizing, Preservation in sterile condition is stand-by, and other embodiment clay treatment is identical.
Phosphate buffered saline buffer is added in 15L bottom ventilation stirred tank, pH is 6.6, add the carclazyte having adsorbed substrate 2-benzoyl pyridine, substrate 2-benzoyl pyridine addition is made to be 80-90 g/L, corn steep liquor (with dry basis) content is 12g/L, and glucose content is 15g/L, wood sugar 13g/L, tween 80 content is 12g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 26 DEG C, add wet Penicillium cell and make concentration be 40g/L, ventilation ratio is 0.13V/(V minute) namely per minute air flow be 0.13 times of reaction solution volume, carry out biocatalytic reaction, the reaction times is 60 hours; After reaction terminates, leach cell, carclazyte respectively, be extracted with ethyl acetate reaction solution, extraction carclazyte, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-phenyl-(2-pyridyl) methyl alcohol, reaction conversion ratio 96%, product yield 94%, enantiomeric excess rate (ee%) 99%.
embodiment 2
Wet Penicillium notatum ATCC 204049 cell is produced, as biocatalytic reaction catalyzer by ordinary method.
The carclazyte making method of having adsorbed substrate is, is absorbed by substrate 2-benzoyl pyridine carclazyte, namely obtains the carclazyte having adsorbed substrate, regulates substrate and carclazyte consumption, makes the quality ratio of substrate and carclazyte be 0.3.
Phosphate buffered saline buffer is added in 100L bottom ventilation stirred tank, pH is 6.6, add the carclazyte having adsorbed substrate 2-benzoyl pyridine, substrate 2-benzoyl pyridine addition is made to be 90 g/L, corn steep liquor (with dry basis) content is 15g/L, and glucose content is 18g/L, wood sugar 15g/L, tween 80 content is 15g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 27 DEG C, add wet Penicillium cell and make concentration be 44g/L, ventilation ratio is 14V/(V minute) namely per minute air flow be 14 times of reaction solution volumes, carry out biocatalytic reaction, the reaction times is 65 hours; After reaction terminates, leach cell, carclazyte respectively, be extracted with ethyl acetate reaction solution, extraction carclazyte, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-phenyl-(2-pyridyl) methyl alcohol, reaction conversion ratio 96%, product yield 94%, enantiomeric excess rate (ee%) 99%.
embodiment 3
Wet Penicillium notatum ATCC 204049 cell is produced, as biocatalytic reaction catalyzer by ordinary method.
The carclazyte making method of having adsorbed substrate is, is absorbed by substrate 2-benzoyl pyridine carclazyte, namely obtains the carclazyte having adsorbed substrate, regulates substrate and carclazyte consumption, makes the quality ratio of substrate and carclazyte be 0.28.Carclazyte used is common carclazyte, and commercially, sterilizing, Preservation in sterile condition is stand-by, and other embodiment clay treatment is identical.
Phosphate buffered saline buffer is added in 500L bottom ventilation stirred tank, pH is 6.6, add the carclazyte having adsorbed substrate 2-benzoyl pyridine, substrate 2-benzoyl pyridine addition is made to be 80-90 g/L, corn steep liquor (with dry basis) content is 13g/L, and glucose content is 17g/L, wood sugar 14g/L, tween 80 content is 14g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 26.5 DEG C, add wet Penicillium cell and make concentration be 42g/L, ventilation ratio is 0.135V/(V minute) namely per minute air flow be 0.135 times of reaction solution volume, carry out biocatalytic reaction, the reaction times is 64 hours; After reaction terminates, leach cell, carclazyte respectively, be extracted with ethyl acetate reaction solution, extraction carclazyte, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-phenyl-(2-pyridyl) methyl alcohol, reaction conversion ratio 96-98%, product yield 94-96%, enantiomeric excess rate (ee%) 98-99%.
embodiment 4
Wet Penicillium notatum ATCC 204049 cell is produced, as biocatalytic reaction catalyzer by ordinary method.
The carclazyte making method of having adsorbed substrate is, is absorbed by substrate 2-benzoyl pyridine carclazyte, namely obtains the carclazyte having adsorbed substrate, regulates substrate and carclazyte consumption, makes the quality ratio of substrate and carclazyte be 0.3.Carclazyte used is common carclazyte, and commercially, sterilizing, Preservation in sterile condition is stand-by, and other embodiment clay treatment is identical.
Phosphate buffered saline buffer is added in 15L bottom ventilation stirred tank, pH is 6.6, add the carclazyte having adsorbed substrate 2-benzoyl pyridine, substrate 2-benzoyl pyridine addition is made to be 89 g/L, corn steep liquor (with dry basis) content is 15g/L, and glucose content is 17g/L, wood sugar 14g/L, tween 80 content is 14g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 27 DEG C, add wet Penicillium cell and make concentration be 44g/L, ventilation ratio is 0.14V/(V minute) namely per minute air flow be 0.14 times of reaction solution volume, carry out biocatalytic reaction, the reaction times is 65 hours; After reaction terminates, leach cell, carclazyte respectively, be extracted with ethyl acetate reaction solution, extraction carclazyte, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-phenyl-(2-pyridyl) methyl alcohol, reaction conversion ratio 97.5%, product yield 95.6%, enantiomeric excess rate (ee%) 98.7%.

Claims (2)

1. the method for Penicillium cell biocatalysis preparation (S)-phenyl-(2-pyridyl) methyl alcohol, it is characterized in that adding phosphate buffered saline buffer in retort, pH is 6.6, add the carclazyte having adsorbed substrate 2-benzoyl pyridine, substrate 2-benzoyl pyridine addition is made to be 80-90 g/L, corn steep liquor (with dry basis) content is 12-15g/L, glucose content is 15-18g/L, wood sugar 13-15g/L, tween 80 content is 12-15g/L, 121 DEG C of autoclavings 30 minutes; When being cooled to 26-27 DEG C, add wet Penicillium cell and make concentration be 40-44g/L, ventilation ratio is 0.13-0.14V/(V minute), carry out biocatalytic reaction, the reaction times is 60-65 hour; After reaction terminates, leach cell, carclazyte respectively, be extracted with ethyl acetate reaction solution, extraction carclazyte, combined ethyl acetate extraction liquid, steam ethyl acetate, obtain product (S)-phenyl-(2-pyridyl) methyl alcohol, 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 carclazyte making method of having adsorbed substrate is, absorbs substrate 2-benzoyl pyridine carclazyte, namely obtains the carclazyte having adsorbed substrate; regulate substrate and carclazyte consumption, make the quality ratio of substrate and carclazyte be 0.27-0.3.
CN201410594403.3A 2014-10-30 2014-10-30 Method for producing (S)-phenyl methanol from blue mold Pending CN104328154A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1028164A1 (en) * 1999-02-12 2000-08-16 Pfizer Products Inc. Microbial conversion of 2-methylquinoxaline
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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1028164A1 (en) * 1999-02-12 2000-08-16 Pfizer Products Inc. Microbial conversion of 2-methylquinoxaline
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)

* Cited by examiner, † Cited by third party
Title
MOHAN PAL ET AL.: "Bioreduction of methyl heteroaryl and aryl heteroaryl ketones in high enantiomeric excess with newly isolated fungal strains", 《BIORESOURCE TECHNOLOGY》 *

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Application publication date: 20150204