CN104560745B - A kind of Pichia yeast Pichia sp.SIT2014 and its cultural method and application - Google Patents
A kind of Pichia yeast Pichia sp.SIT2014 and its cultural method and application Download PDFInfo
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- CN104560745B CN104560745B CN201410734438.2A CN201410734438A CN104560745B CN 104560745 B CN104560745 B CN 104560745B CN 201410734438 A CN201410734438 A CN 201410734438A CN 104560745 B CN104560745 B CN 104560745B
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
Abstract
The present invention discloses a kind of Pichia yeastPichia Sp.SIT2014 and cultural method and application.The Pichia yeastPichia Sp.SIT2014, deposit number are CGMCC NO.9300.The method that the hydroxyaryl ethanone of biocatalyst catalyzed precursor 2 is chiral aryl vicinal diamines is utilized it as, i.e., by Pichia yeastPichia Sp.SIT2014 cells are suspended in phosphate buffer and 2 hydroxyaryl ethyl ketones are added to it, and 30 50 DEG C of progress 48h of asymmetric reduction reaction 3 of control temperature, gained reaction solution centrifuges, and supernatant is through extracting, being drying to obtain chiral aryl vicinal diamines.Utilize this kind of Pichia yeastPichia The method that sp.SIT2014 prepares chiral aryl vicinal diamines as biocatalyst, a variety of chiral aryl vicinal diamines can be synthesized, conversion ratio and enantiomeric excess value are more than 99%.
Description
Technical field
The present invention relates to Yeasts and application thereof, more particularly to a kind of Pichia yeast Pichia
Sp.SIT2014 and its cultural method and utilize it as the method that biocatalyst prepares chiral aryl vicinal diamines.
Background technology
Chipal compounds play the role of in human lives it is important, because two kinds of enantiomers of same compound are in medicine
Huge difference all be present above reason, toxicity and function, thus prepares optically pure handedness module compound it is medical,
The fields such as agricultural chemicals, environmental protection, material all have great importance.
And optically pure chiral aryl vicinal diamines, such as (S)-benzoglycols are not only indispensable chirality in liquid crystal material
Additive, and synthesize many optics drugs and the important intermediate of some functional materials.Chiral aryl vicinal diamines one
As have two kinds synthesis methods.
A kind of Kinetic Resolution synthetic method for racemic modification glycol.A deficiency is its reason existing for this method
There was only 50% by yield.
Second method is the asymmetric reduction synthetic method of precursor ketone.Compared to first method, the theory of this method
Yield can reach the compound of 100% optically pure chiral diol.At present, many methods may apply to chiral diol
Preparation on, focus primarily upon (Hatakeyama, the Taito etal.Process such as chemical method and bioanalysis, Hatakeyama
for producing optically active alcohols.U.S.Pat.Appl.Publ.,20110282077,17Nov
2011) precursors hydroxyl acetophenone is reduced using chemical catalyst ruthenium compound to obtain (S)-benzoglycols, optical purity
Value up to 94.6%, and chiral catalyst is used in course of reaction and organic solvent environmental pollution causes necessarily to influence.
(Wei Dongzhi, Lin Jinping, Gao Qiong, a kind of asymmetric split of microorganism prepare S- benzoglycols for 2008 Nian Wei Toshiba etc.
Method, Chinese patent, patent No. ZL200810038899.0, authorized announcement date, on January 2nd, 2013) a kind of microorganism of research
As catalyst preparation S- benzoglycols, although chipal compounds optical voidness angle value that this method obtains is higher, and reachable 99.9%,
Preparation process is environmentally friendly, but using the method for Kinetic Resolution, and the theoretical yield of Kinetic Resolution is up to 50%.It is real
Border conversion ratio only has 38%, and the reaction time is longer, it is necessary to 33-96h.
(Zhang Rongzhen, Xu Yan, Geng Yawei, a kind of carbonyl reductase recombinant bacterium efficiently prepare (S)-benzene to Zhang Rongzhen in 2009 etc.
The method of ethyl glycol, Chinese patent, patent No. ZL200910263147.9, authorized announcement date, on May 23rd, 2012) utilize
Carbonyl reductase recombinant bacterium, (S)-benzoglycols can be reduced to the catalysis hydroxy acetophenone of high yield high selectivity.But base
Its mitotic stability is poor caused by engineering bacteria has potential plasmid loss risk in succeeding generations, is needed in incubation
Inducer isopropylthio-β-D- the thiogalactosides and antibiotic of costliness are added, causes the environmental improvement cost of fermented waste fluid
Lifting, and have no that it is applied to the chiral aryl ethylene glycol of synthesis other structures.
In summary, the preparation method of chiral aryl vicinal diamines either exist synthesis step complex environment it is unfriendly or
Optical purity and low yield, or strain stability is poor, substrate composes the problems such as narrow and is difficult to industrialize.Therefore having must
A kind of carbonyl reduction enzyme biocatalyst with wider substrate application, with greater activity and selectivity is developed to come
Overcome above-mentioned technical barrier.
The content of the invention
It is an object of the present invention to provide a kind of Pichia pastoris Pichia sp.SIT2014CGMCC NO.9300.
The second object of the present invention is to provide a kind of above-mentioned Pichia pastoris Pichia sp.SIT2014CGMCC
NO.9300 cultural method.
The three of the object of the invention are that providing one kind makees Pichia pastoris Pichia sp.SIT2014CGMCC NO.9300
The method applied for biocatalyst, i.e., using Pichia pastoris Pichia sp.SIT2014CGMCC NO.9300 as living things catalysis
Agent, it is corresponding chiral aryl vicinal diamines to be catalyzed a variety of precursor 2- hydroxyaryl ethyl ketones asymmetric reductions, can obtain 99% with
On conversion ratio and optical purity.In the microbe preparation method of the chiral aryl vicinal diamines, when chiral aryl vicinal diamines react
Between it is short, it is only necessary to 3-24h, high conversion rate, reach as high as 99.9%.
Technical scheme
A kind of Pichia pastoris Pichia sp.SIT2014, are a kind of bacterial strains for belonging to fungi Parasaccharomyces, and the bacterial strain is
It is isolated from Chinese Soils of Shanghai Region, on 06 16th, 2014 in China Committee for Culture Collection of Microorganisms
Common micro-organisms center preservation, deposit number are CGMCC NO.9300, preservation organization address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number Institute of Microorganism, Academia Sinica of institute, postcode：100101.
Above-mentioned Pichia pastoris Pichia sp.SIT2014CGMCC NO.9300 bacterial strains have following microbial characteristic：
(1), morphological feature
Pichi strain cell is spherical in shape, and ellipse is tapered once in a while, but does not form pinnacle.Bacterium colony surface is smooth, wet
Moisten, be sticky, easily provoking, bacterium colony is homogeneous, and positive and negative and edge, the color of central part are all very homogeneous, and bacterium colony is milky white
Color；
(2), feature is learned in culture
Bacterium colony is opaque on dusty yeast-tryptone-agar glucose, glossy, and milky is presented；
(3), physiological and biochemical property
Facultative anaerobic bacterium；
(4), motility：Do not move
(5), carbon assimilation
It is positive：D- galactolipins, the pears of L- tri- sugar, D- xyloses, sucrose, maltose, methyl α-D- glucopyranosides, fiber two
Sugar, salicin, glycerine, ethanol；
It is negative：D- grapes amine, melibiose, lactose, gossypose, starch, galactitol, inositol, maltonic acid, D- glucose
Aldehydic acid, methanol；
(6) optimal temperature, is grown：25℃-32℃.
The strain still keeps character stable, therefore have good mitotic stability after passage 20 times.
Chiral aryl vicinal diamines are prepared as biocatalyst by the use of above-mentioned Pichia pastoris Pichia sp.SIT2014, i.e.,
Asymmetric reduction reaction is carried out using Pichia pastoris Pichia sp.SIT2014 as biocatalyst catalysis 2- hydroxyaryls ethyl ketone
Obtain chiral aryl vicinal diamines；
Described 2- hydroxyaryls ethyl ketone be 2- hydroxy acetophenones, 2- hydroxyls m chloroacetophenone, 2- hydroxyls o-chloroacetophenone,
2- hydroxyls parabromoacetophenone or 2- hydroxyl parachloroacetophenones；
The chiral aryl vicinal diamines of corresponding gained are (S)-benzoglycols, (S)-m-chloro benzoglycols, (S)-adjacent chlorobenzene second
Glycol, (S)-to bromobenzene ethylene glycol or (S)-to chlorobenzene ethylene glycol.
Above-mentioned Pichia pastoris Pichia sp.SIT2014 can be with high selectivity, high yield and low as biocatalyst
Cost carrys out catalytically synthesizing chiral aryl vicinal diamines.
Above-mentioned prepares chiral aryl neighbour two by the use of above-mentioned Pichia pastoris Pichia sp.SIT2014 as biocatalyst
The method of alcohol, specifically comprises the following steps
(1), Pichia pastoris Pichia sp.SIT2014 culture
The Pichia pastoris Pichia sp.SIT2014 slant strains for taking 4 DEG C of refrigerators to preserve, the ring strain of picking one are linked into dress
Have in the 100ml shaking flasks of 15ml seed culture mediums, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out seed culture
12-18h, obtain seed liquor；
Then seed liquor is pressed：The volume ratio of fermentation medium is 5% inoculum concentration, and seed liquor is inoculated into fermentation medium
In, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out fermented and cultured 15-24h, obtained zymotic fluid centrifugation, is received
It is Pichia pastoris Pichiasp.SIT2014 cells to obtain cell, is then placed in buffer solution and preserves；
Above-described seed culture medium, fermentation medium are identical, by every liter of calculating, 15g containing glucose, and tryptone
5g, dusty yeast 5g, K2HPO40.5g, KH2PO40.5g, NaCl 1g, MgSO4·7H2O0.5g, surplus are water；
Described buffer solution is the sodium phosphate aqueous solution of pH5.5-8.0 sodium phosphate aqueous solution, preferably pH5.5~7.0,
More preferably pH 6.5 sodium phosphate aqueous solution, its dosage press Pichia pastoris Pichia sp.SIT2014 dry weights：Buffer solution
For 5-100g：1L ratio calculates；
(2), hydroxyl is added into the buffer solution containing Pichia pastoris Pichia sp.SIT2014 cells obtained by step (1)
Base aryl methyl ketone, then control temperature to carry out reduction reaction 3-48h for 30-50 DEG C and obtain reaction solution；
The amount of hydroxyaryl ethyl ketone used in above-mentioned reaction, by Pichia pastoris Pichiasp.SIT2014 dry weights：Hydroxyl
Aryl methyl ketone is 5-100g：5-100mmol ratio calculates；
(3), rotating speed is controlled to be centrifuged for 10000r/min the reaction solution of step (2), obtained supernatant is used
After isometric ethyl acetate extraction three times, extract anhydrous sodium sulfate drying, then it is evaporated, that is, is gone smoothly with Rotary Evaporators
Property aryl vicinal diamines.
Beneficial effects of the present invention
A kind of Pichia pastoris Pichia sp.SIT2014CGMCC NO.9300 of the present invention, it is easy to train because it has
Support, mitotic stability is high, and carbonyl reduction enzymatic activity is higher in its thalline, to the reproducibilities of 2- hydroxyaryl ethyl ketones and selectivity compared with
By force, its be used for biocatalyst catalysis 2- hydroxyaryl ethyl ketones prepare chiral aryl vicinal diamines, there is high conversion rate, reaction time
The features such as short, final conversion ratio reach as high as 99.9%, and optical activity value is more than 99%, from input Pichia pastoris Pichia
Sp.SIT2014 cells are until obtain chiral aryl vicinal diamines, it is only necessary to 3-48h.And the Pichia pastoris of gained after passing on 20 times
Pichia sp.SIT2014 colonial morphologies keep constant, and fermentation gained cell is catalyzed 2- hydroxyaryl second as biocatalyst
Ketone prepares chiral aryl vicinal diamines, and its conversion ratio and optical purity are still all up more than 99%, therefore its mitotic stability is high.
Embodiment
Below by some embodiments, the present invention will be further described, but is not intended to limit the present invention.
Reagent used in the present invention is the pure or pure HPLC reagent of analysis respectively.
The detection of chiral aryl vicinal diamines optical voidness angle value, using Shimadzu high performance liquid chromatograph LC-20A, from island
Tianjin instrument company；
(R, S)-benzoglycols chromatographiccondition of target product is as follows in various embodiments of the present invention：
Mobile phase is n-hexane：Isopropanol=95:5, flow velocity 0.8ml/min, Detection wavelength 210nm, column temperature are 27 DEG C,
Using chiral OD-H posts (4.6mm × 250mm, being purchased from Daicel medicine company chiral technology (Shanghai) Co., Ltd.).
Optical purity ee value calculating methods are as follows:
Ee (%)=[S (configuration)-R (configuration)]/[S (configuration)+R (configuration)] × 100%
S (configuration)：Represent the concentration of S (configuration) product enantiomer；
R (configuration)：Represent the concentration of R (configuration) product enantiomer.
The definition of signified cellular carbonyl reductase vitality unit is in the present invention：At 30 DEG C, under conditions of pH6.5, often
Enzyme amount needed for minute 1.0 μm of ol of catalysis 2- hydroxy acetophenones reduction generation benzoglycols is 1 unit of activity, i.e. 1U.
Embodiment 1
The culture of Pichia yeast Pichia sp.SIT2014 thalline；
Slant medium used, calculated by every liter, 15.0g containing glucose, tryptone 5.0g, dusty yeast 5.0g,
KH2PO40.5g,K2HPO40.5g, MgSO4·7H2O 0.5g, agar 20.0g, surplus are water.
Seed culture medium used, calculated by every liter, 15.0g containing glucose, tryptone 5.0g, dusty yeast 5.0g,
KH2PO40.5g,K2HPO40.5g, MgSO4·7H2O 0.5g, surplus are water.
Fermentation medium used, calculated by every liter, 15.0g containing glucose, tryptone 5.0g, dusty yeast 5.0g,
KH2PO40.5g,K2HPO40.5g, MgSO4·7H2O 0.5g, surplus are water.
The Pichia pastoris Pichia sp.SIT2014 slant strains for taking 4 DEG C of refrigerators to preserve, the ring strain of picking one are linked into dress
Have in the 100ml shaking flasks of 15ml seed culture mediums, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out seed culture
18h, obtain seed liquor；
Then seed liquor is pressed：The volume ratio of fermentation medium is 5% inoculum concentration, and seed liquor is inoculated into fermentation medium
In, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out fermented and cultured 24h, obtained zymotic fluid centrifugation, harvest
Cell is Pichia pastoris Pichia sp.SIT2014 cells.
After testing, it is about 20~30U/L that it produces carbonyl reduction enzyme activity to the zymotic fluid of final gained, and cell is dense in zymotic fluid
Degree calculates about 4~5g/L by dry weight, i.e., unit cell production carbonyl reduction enzyme activity is about 4-5U/g stem cells.On it is indicated above that
The Pichia pastoris Pichia sp.SIT2014 cells for stating gained have stronger reproducibility and selectivity to aryl hydroxy-ketone.
Embodiment 2
The Secondary Culture of Pichia yeast Pichia sp.SIT2014 thalline；
Formula as described in embodiment 1 prepares slant medium, seed culture medium and fermentation medium.
The Pichia pastoris Pichia sp.SIT2014 slant strains of 4 DEG C of refrigerators preservations are taken, aseptically picking single bacterium
Fall on a fresh inclined-plane, after cultivating 48h at 30 DEG C in biochemical cultivation case, the bacterium colony grown was 1 generation.In 1 generation bacterium colony
Picking single bacterium is fallen on a fresh inclined-plane, and after cultivating 48h at 30 DEG C in biochemical cultivation case, the bacterium colony grown was 2 generations.By upper
State method pass on 20 times after, the ring in picking 20 generation bacterium colony is linked into the 100ml shaking flasks equipped with 15ml seed culture mediums,
Temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out seed culture 18h, obtains seed liquor；
Then seed liquor is pressed：The volume ratio of fermentation medium is 5% inoculum concentration, and seed liquor is inoculated into fermentation medium
In, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out fermented and cultured 24h, obtained zymotic fluid centrifugation, harvest
Cell is Pichia pastoris Pichia sp.SIT2014 cells.
After testing, it is about 20~30U/L that it produces carbonyl reduction enzyme activity to the zymotic fluid of final gained, and cell is dense in zymotic fluid
Degree calculates about 4~5g/L by dry weight, i.e., unit cell production carbonyl reduction enzyme activity is about 4-5U/g stem cells.On it is indicated above that
The Pichia pastoris Pichia sp.SIT2014 cells for stating gained still keep higher activity after passing on 20 times.
Embodiment 3
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxy acetophenones, its catalytic reaction equation
It is as follows：
The Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1 are catalyzed 2- hydroxy benzenes as biocatalyst
Ethyl ketone carries out asymmetric reduction reaction and prepares the chiral aryl vicinal diamines i.e. method of (S)-benzoglycols, specifically includes following step
Suddenly：
The Pichia pastoris Pichia sp.SIT2014 cells of the gained of embodiment 1 are placed in buffer solution；
Described buffer solution is the sodium phosphate aqueous solution that pH is 6.5, and its dosage presses Pichia pastoris Pichia sp.SIT2014
Dry cell weight：Buffer solution is 10g：1L ratio calculates；
2- hydroxy benzenes second is added into the buffer solution containing Pichia pastoris Pichia sp.SIT2014 cells of above-mentioned gained
Ketone, then to control temperature be 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 20h and obtains reaction solution；
The amount of 2- hydroxy acetophenones used in above-mentioned reaction, by Pichia pastoris Pichia sp.SIT2014 dry cell weights：2- hydroxyls
Benzoylformaldoxime is 0.5g：0.15mmol ratio calculates；
Rotating speed is controlled to be centrifuged for 10000r/min the reaction solution of above-mentioned gained, the bodies such as obtained supernatant is used
After long-pending ethyl acetate extraction three times, extract anhydrous sodium sulfate drying, then it is evaporated with Rotary Evaporators, produces target production
Thing (S)-benzoglycols, conversion ratio 99.9%, ee values are 99.5%.
Embodiment 4
Pichia pastoris Pichia sp.SIT2014 by the use of the passage 20 times of the gained of embodiment 2 urge as biocatalyst
Change 2- hydroxyaryl ethyl ketones and prepare chiral aryl vicinal diamines, described 2- hydroxyaryls ethyl ketone is 2- hydroxy acetophenones, and it is catalyzed
Reaction equation is as follows：
The Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 2 are catalyzed 2- hydroxy benzenes as biocatalyst
Ethyl ketone carries out asymmetric reduction reaction and prepares the chiral aryl vicinal diamines i.e. method of (S)-benzoglycols, specifically includes following step
Suddenly：
The Pichia pastoris Pichia sp.SIT2014 cells of the gained of embodiment 2 are placed in buffer solution；
Described buffer solution is the sodium phosphate aqueous solution that pH is 6.5, and its dosage presses Pichia pastoris Pichia sp.SIT2014
Dry cell weight：Buffer solution is 10g：1L ratio calculates；
2- hydroxy benzenes second is added into the buffer solution containing Pichia pastoris Pichia sp.SIT2014 cells of above-mentioned gained
Ketone, then to control temperature be 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 20h and obtains reaction solution；
The amount of 2- hydroxy acetophenones used in above-mentioned reaction, by Pichia pastoris Pichia sp.SIT2014 dry cell weights：2- hydroxyls
Benzoylformaldoxime is 0.5g：0.15mmol ratio calculates；
Rotating speed is controlled to be centrifuged for 10000r/min the reaction solution of above-mentioned gained, the bodies such as obtained supernatant is used
After long-pending ethyl acetate extraction three times, extract anhydrous sodium sulfate drying, then it is evaporated with Rotary Evaporators, produces target production
Thing (S)-benzoglycols, conversion ratio 99.9%, ee values are 99.6%.
Embodiment 5
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxy acetophenones, i.e., by the gained of embodiment 1
Pichia pastoris Pichia sp.SIT2014 carry out asymmetric reduction reaction preparation as biocatalyst catalysis 2- hydroxy acetophenones
Chiral aryl vicinal diamines are the method for (S)-benzoglycols, are specifically comprised the following steps：
The Pichia pastoris Pichia sp.SIT2014 cells of the gained of embodiment 1 are placed in buffer solution；
Described buffer solution is the sodium phosphate aqueous solution that pH is 5.5, and its dosage presses Pichia pastoris Pichia sp.SIT2014
Dry cell weight：Buffer solution is 10g：1L ratio calculates；
2- hydroxy benzenes second is added into the buffer solution containing Pichia pastoris Pichia sp.SIT2014 cells of above-mentioned gained
Ketone (R therein1For-C6H5), then to control temperature be 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 2h and obtains reaction solution；
The amount of 2- hydroxy acetophenones used in above-mentioned reaction, by Pichia pastoris Pichia sp.SIT2014 dry cell weights：2- hydroxyls
Benzoylformaldoxime is 0.5g：0.15mmol ratio calculates；
Rotating speed is controlled to be centrifuged for 10000r/min the reaction solution of above-mentioned gained, the bodies such as obtained supernatant is used
After long-pending ethyl acetate extraction three times, extract anhydrous sodium sulfate drying, then it is evaporated with Rotary Evaporators, produces target production
Thing chirality benzoglycols, conversion ratio 42.8%, ee values are 96.3%.
Embodiment 6
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxy acetophenones, i.e., by the gained of embodiment 1
Pichia pastoris Pichia sp.SIT2014 carry out asymmetric reduction reaction preparation as biocatalyst catalysis 2- hydroxy acetophenones
Chiral aryl vicinal diamines are the method for (S)-benzoglycols, are specifically comprised the following steps：
The Pichia pastoris Pichia sp.SIT2014 cells of the gained of embodiment 1 are placed in buffer solution；
Described buffer solution is the sodium phosphate aqueous solution that pH is 7.0, and its dosage presses Pichia pastoris Pichia sp.SIT2014
Dry cell weight：Buffer solution is 10g：1L ratio calculates；
2- hydroxy benzenes second is added into the buffer solution containing Pichia pastoris Pichia sp.SIT2014 cells of above-mentioned gained
Ketone, then to control temperature be 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 2h and obtains reaction solution；
The amount of 2- hydroxy acetophenones used in above-mentioned reaction, by Pichia pastoris Pichia sp.SIT2014 dry cell weights：2- hydroxyls
Benzoylformaldoxime is 0.5g：0.15mmol ratio calculates；
Rotating speed is controlled to be centrifuged for 10000r/min the reaction solution of above-mentioned gained, the bodies such as obtained supernatant is used
After long-pending ethyl acetate extraction three times, extract anhydrous sodium sulfate drying, then it is evaporated with Rotary Evaporators, produces target production
Thing chirality benzoglycols, conversion ratio 72.3%, ee values are 98.3%.
Embodiment 7
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxyl parachloroacetophenones, i.e., by the institute of embodiment 1
The Pichia pastoris Pichia sp.SIT2014 obtained carry out asymmetric reduction as biocatalyst catalysis 2- hydroxyls parachloroacetophenone
Reaction prepares chiral aryl vicinal diamines i.e. (S)-to the method for chlorobenzene ethylene glycol, specifically comprises the following steps：
2- hydroxy acetophenones therein are simply replaced with 2- hydroxyl parachloroacetophenones, finally by specific steps with embodiment 3
Target product (S)-to chlorobenzene ethylene glycol is obtained, it is 99.1% that its conversion ratio, which is more than 99.9%, ee values,.
Embodiment 8
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxyl parabromoacetophenones, i.e., by the institute of embodiment 1
The Pichia pastoris Pichia sp.SIT2014 obtained carry out asymmetric reduction as biocatalyst catalysis 2- hydroxyls parabromoacetophenone
Reaction prepares chiral aryl vicinal diamines i.e. (S)-to the method for bromobenzene ethylene glycol, specifically comprises the following steps：
2- hydroxy acetophenones therein are simply replaced with 2- hydroxyl parabromoacetophenones, finally by specific steps with embodiment 3
Target product (S)-to bromobenzene ethylene glycol is obtained, it is 99.3% that its conversion ratio, which is more than 99.9%, ee values,.
Embodiment 9
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxyl o-chloroacetophenones, i.e., by the institute of embodiment 1
The Pichia pastoris Pichia sp.SIT2014 obtained carry out asymmetric reduction as biocatalyst catalysis 2- hydroxyls o-chloroacetophenone
The method that reaction prepares chiral aryl vicinal diamines i.e. (S)-adjacent chlorobenzene ethylene glycol, specifically comprises the following steps：
2- hydroxy acetophenones therein are simply replaced with 2- hydroxyl o-chloroacetophenones, finally by specific steps with embodiment 3
Target product (S)-adjacent chlorobenzene ethylene glycol is obtained, it is 99.2% that its conversion ratio, which is more than 99.9%, ee values,.
Embodiment 10
Biocatalyst catalysis 2- hydroxyl virtues are used as by the use of the Pichia pastoris Pichia sp.SIT2014 of the gained of embodiment 1
Base ethyl ketone prepares chiral aryl vicinal diamines, and described 2- hydroxyaryls ethyl ketone is 2- hydroxyl m chloroacetophenones, i.e., by the institute of embodiment 1
The Pichia pastoris Pichia sp.SIT2014 obtained carry out asymmetric reduction as biocatalyst catalysis 2- hydroxyls m chloroacetophenone
Reaction prepares the chiral aryl vicinal diamines i.e. method of (S)-m-chloro benzoglycols, specifically comprises the following steps：
2- hydroxy acetophenones therein are simply replaced with 2- m-chloro hydroxy acetophenones, finally by specific steps with embodiment 3
Target product (S)-m-chloro benzoglycols is obtained, it is 99.4% that its conversion ratio, which is more than 99.9%, ee values,.
Above said content is only the basic explanation under present inventive concept, and is appointed according to what technical scheme was made
What equivalent transformation, all should belong to protection scope of the present invention.
Claims (7)
- A kind of 1. Pichia yeastPichia Sp.SIT2014, deposit number are CGMCC NO.9300.
- 2. Pichia yeast as claimed in claim 1Pichia Sp.SIT2014 cultural method, it is characterised in that step is such as Under：The Pichia pastoris for taking 4 DEG C of refrigerators to preservePichia Sp.SIT2014 slant strains, the ring strain of picking one, which is linked into, to be equipped with In the 100ml shaking flasks of 15ml seed culture mediums, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm cultures 12-18h is obtained Seed liquor；Then seed liquor is pressed：The volume ratio of fermentation medium is 5% inoculum concentration, and seed liquor is inoculated into fermentation medium, Temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out fermented and cultured 15-24h, and obtained zymotic fluid centrifugation, harvest obtains Pichia pastorisPichiaSp.SIT2014 cells；Above-described seed culture medium, fermentation medium are identical, by every liter of calculating, 15g containing glucose, tryptone 5g, ferment Female powder 5g, K2HPO40.5g, KH2PO40.5g, NaCl 1g, MgSO4·7H2O 0.5g, surplus are water.
- 3. utilize Pichia yeast as claimed in claim 1Pichia Sp.SIT2014 is as biocatalyst catalyzed precursor 2- hydroxyaryl ethyl ketones asymmetric reduction is chiral aryl vicinal diamines；Described 2- hydroxyaryls ethyl ketone is 2- hydroxy acetophenones, 2- hydroxyl m chloroacetophenones, 2- hydroxyls o-chloroacetophenone, 2- hydroxyls Base parabromoacetophenone or 2- hydroxyl parachloroacetophenones；The chiral aryl vicinal diamines of corresponding gained are the benzoglycols of S configurations, the m-chloro benzoglycols of S configurations, the adjacent chlorine of S configurations Benzoglycols, S configurations to bromobenzene ethylene glycol or S configurations to chlorobenzene ethylene glycol.
- 4. utilize Pichia yeast as claimed in claim 3Pichia Sp.SIT2014 is as biocatalyst catalyzed precursor 2- hydroxyaryl ethyl ketones asymmetric reduction is the method for chiral aryl vicinal diamines, it is characterised in that is specifically comprised the following steps：（1）, culture Pichia pastorisPichia sp.SIT2014The Pichia pastoris for taking 4 DEG C of refrigerators to preservePichia Sp.SIT2014 slant strains, the ring strain of picking one, which is linked into, to be equipped with In the 100ml shaking flasks of 15ml seed culture mediums, temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out seed culture 12-18h, obtain seed liquor；Then seed liquor is pressed：The volume ratio of fermentation medium is 5% inoculum concentration, and seed liquor is inoculated into fermentation medium, Temperature is controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out fermented and cultured 15-24h, obtained zymotic fluid centrifugation, harvest it is thin Born of the same parents are Pichia pastorisPichiaSp.SIT2014 cells, then it is placed in buffer solution and preserves；Above-described seed culture medium, fermentation medium are identical, by every liter of calculating, 15g containing glucose, tryptone 5g, ferment Female powder 5g, K2HPO40.5g, KH2PO40.5g, NaCl 1g, MgSO4·7H2O 0.5g, surplus are water；Described buffer solution is the sodium phosphate aqueous solution that pH is 5.5-8.0, and its dosage presses Pichia pastorisPichia Sp.SIT2014 dry weights：Buffer solution is 5-100g：1L ratio calculates；（2）, to step（1）Gained contains Pichia pastorisPichia2- hydroxyls are added in the buffer solution of sp.SIT2014 cells Aryl methyl ketone, then control temperature to carry out reduction reaction 3-48h for 30-50 DEG C and obtain reaction solution；The amount of 2- hydroxyaryls ethyl ketone used in above-mentioned reaction, by Pichia pastorisPichiaSp.SIT2014 dry cell weights：2- hydroxyls Aryl methyl ketone is 5-100g：5-100mmol ratio calculates；（3）, by step（2）Reaction solution control rotating speed to be centrifuged for 10000r/min, obtained supernatant with etc. body After long-pending ethyl acetate extraction three times, extract is evaporated with after anhydrous sodium sulfate drying with Rotary Evaporators, produces chiral aryl Vicinal diamines.
- 5. utilize Pichia yeast as claimed in claim 4Pichia Sp.SIT2014 is as biocatalyst catalyzed precursor 2- hydroxyaryl ethyl ketones asymmetric reduction is the method for chiral aryl vicinal diamines, it is characterised in that step（1）Described in buffering Liquid is the sodium phosphate aqueous solution that pH is 5.5~7.0.
- 6. utilize Pichia yeast as claimed in claim 4Pichia Sp.SIT2014 is as biocatalyst catalyzed precursor 2- hydroxyaryl ethyl ketones asymmetric reduction is the method for chiral aryl vicinal diamines, it is characterised in that step（1）Described in buffering Liquid pH is 6.5 sodium phosphate aqueous solution.
- 7. utilize Pichia yeast as claimed in claim 4Pichia Sp.SIT2014 is as biocatalyst catalyzed precursor 2- hydroxyaryl ethyl ketones asymmetric reduction is the method for chiral aryl vicinal diamines, it is characterised in that step（1）Described in fermentation Culture, i.e., by seed liquor：The volume ratio of fermentation medium is 5% inoculum concentration, and seed liquor is inoculated into equipped with 50ml fermented and cultureds In the 250ml shaking flasks of base, pH=6.5, temperature are controlled on shaking table is 30 DEG C, rotating speed is that 180rpm carries out fermented and cultured 24h.
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| CN109536423B (en) * | 2019-01-11 | 2022-06-07 | 宁夏医科大学 | Method for culturing bacillus cereus and special culture medium thereof |
| CN110283733B (en) * | 2019-06-20 | 2020-10-27 | 浙江工业大学 | Tupistra yeast ZJPH1807 and application thereof |
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| WO2007014544A2 (en) * | 2005-08-02 | 2007-02-08 | Julich Chiral Solutions Gmbh | Stereoselective synthesis of chiral diols |
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