CN104560745A - Pichia sp.SIT2014 and culture method and application thereof - Google Patents
Pichia sp.SIT2014 and culture method and application thereof Download PDFInfo
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Abstract
The invention discloses Pichia sp.SIT2014 and a culture method and an application thereof. The preservation number of the Pichia sp.SIT2014 is CGMCC NO.9300. The method for reducing precursor 2-hydroxyaryl ethanone into chiral aryl-o-diol by using Pichia sp.SIT2014 as a biocatalyst comprises the steps of suspending Pichia sp.SIT2014 cells in phosphate buffer, adding 2-hydroxyaryl ethanone, controlling the temperature to 30-50 DEG C, carrying out an asymmetric reduction reaction for 3-48 hours, carrying out centrifugal separation on the obtained reaction liquid, and extracting and drying supernatant to obtain the chiral aryl-o-diol. The method for preparing the chiral aryl-o-diol by using the Pichia sp.SIT2014 as the biocatalyst can be used for synthesizing a variety of chiral aryl-o-diols, and both of the conversion rate and the enantiomer excess value are 99% or higher.
Description
Technical field
The present invention relates to Yeasts and uses thereof, relate to a kind of Pichia yeast in particular
pichia spand cultural method and utilize it to prepare the method for chiral aryl vicinal diamines as biological catalyst .SIT2014.
Background technology
Chipal compounds has important effect in human lives, because two kinds of enantiomorphs of same compound all exist huge difference on pharmacology, toxicity and function, therefore prepare optically pure handedness module compound and all have great importance in fields such as medicine, agricultural chemicals, environmental protection, materials.
And optically pure chiral aryl vicinal diamines, as (S)-benzoglycols is not only indispensable chiral additives in liquid crystal material, be also the important intermediate of the many optics drugs of synthesis and some functional materialss.Chiral aryl vicinal diamines generally has two kinds of methods of synthesizing.
A kind of is the kinetic resolution synthetic method of racemic modification glycol.The deficiency that the method exists is that its theoretical yield only has 50%.
Second method is the asymmetric reduction synthetic method of precursor ketone.Compared to first method, the theoretical yield of the method can reach the compound of the optically pure chiral diol of 100%.At present, there is a lot of method can be applied in the preparation of chiral diol, mainly concentrate on chemical method and biological process, (the Hatakeyama such as Hatakeyama, Taito etal. Process for producing optically active alcohols. U.S. Pat. Appl. Publ., 20110282077, 17 Nov 2011) utilize chemical catalyst ruthenium mixture to carry out reduction to precursors hydroxyl methyl phenyl ketone to obtain (S)-benzoglycols, optical purity angle value is up to 94.6%, and use chiral catalyst in reaction process and organic solvent environmental pollution causes certain influence.
(Wei Dongzhi, Lin Jinping, the Gao Qiong such as 2008 Nian Wei Toshiba, a kind of microorganism is asymmetric splits the method preparing S-benzoglycols, Chinese patent, patent No. ZL200810038899.0, authorized announcement date, on January 2nd, 2013) study a kind of microorganism as catalyst preparing S-benzoglycols, although the chipal compounds optical purity angle value that the method obtains is higher, can 99.9% be reached, preparation process is environmentally friendly, but adopt the method for kinetic resolution, and the theoretical yield of kinetic resolution is up to 50%.Actual conversion only has 38%, and the reaction times is longer, needs 33-96h.
(Zhang Rongzhen, Xu Yan, the Geng Yawei such as Zhang Rongzhen in 2009, a kind of carbonyl reductase recombinant bacterium efficiently prepares the method for (S)-phenylglycol, Chinese patent, patent No. ZL200910263147.9, authorized announcement date, on May 23rd, 2012) utilize carbonyl reductase recombinant bacterium, the catalysis hydroxy acetophenone of high yield highly selective can be reduced to (S)-benzoglycols.But genetic engineering bacterium has potential plasmid loss risk and causes its mitotic stability poor in succeeding generations, need to add expensive inducer isopropylthio-β-D-thiogalactoside and microbiotic in culturing process, cause the environmental improvement cost of fermented waste fluid, and have no the chiral aryl ethylene glycol that it is applied to other structure of synthesis.
In sum, the preparation method of chiral aryl vicinal diamines or to there is synthesis step complex environment unfriendly, or optical purity and productive rate low, or the problem such as bacterial classification poor stability, substrate spectrum be narrow and be difficult to realize industrialization.Therefore be necessary develop a kind of have wider substrate range of application, have greater activity and optionally carbonyl reduction enzyme biocatalyst to overcome above-mentioned technical barrier.
Summary of the invention
One of the object of the invention is to provide a kind of pichia spp
pichiasp.SIT2014 CGMCC NO.9300.
Two of object of the present invention is to provide a kind of above-mentioned pichia spp
pichiathe cultural method of sp.SIT2014 CGMCC NO.9300.
Three of the object of the invention is to provide a kind of by pichia spp
pichiathe method that sp.SIT2014 CGMCC NO.9300 applies as biological catalyst, by pichia spp
pichiasp.SIT2014 CGMCC NO.9300 is as biological catalyst, and catalysis multiple precursor 2-hydroxyaryl ethyl ketone asymmetric reduction is corresponding chiral aryl vicinal diamines, all can obtain transformation efficiency and the optical purity of more than 99%.In the microbe preparation method of this chiral aryl vicinal diamines, the chiral aryl vicinal diamines reaction times is short, only needs 3-24h, and transformation efficiency is high, reaches as high as 99.9%.
Technical scheme of the present invention
A kind of pichia spp Pichia sp.SIT2014, it is a kind of bacterial strain belonging to Mycophyta Saccharomycodes, this bacterial strain is separated and obtains from Chinese Soils of Shanghai Region, in on 06 16th, 2014 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, deposit number is CGMCC NO.9300, preservation organization address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100101.
Above-mentioned pichia spp
pichiasp.SIT2014 CGMCC NO.9300 bacterial strain has following microbial characteristic:
(1), morphological specificity
Pichi strain cell is spherical in shape, ellipse, tapered once in a while, but does not form pinnacle.Bacterium colony smooth surface, moistening, thickness, easily provoke, bacterium colony is homogeneous, and the color of pros and cons and edge, central part is all very homogeneous, and bacterium colony is oyster white;
(2) feature, is cultivated
On yeast powder-Tryptones-agar glucose, bacterium colony is opaque, glossy, presents oyster white;
(3), physiological and biochemical property
Facultative anaerobic bacterium;
(4), mobility: do not move
(5), carbon assimilation
Positive: D-semi-lactosi, L-tri-pears sugar, D-wood sugar, sucrose, maltose, methyl α-D-glycopyranoside, cellobiose, saligenin, glycerine, ethanol;
Negative: D-grape amine, melibiose, lactose, raffinose, starch, melampyrum, inositol, maltonic acid, D-glucuronic acid, methyl alcohol;
(6), optimal temperature is grown: 25 DEG C-32 DEG C.
This bacterial classification, through going down to posterity after 20 times, still keeps proterties to stablize, therefore has good mitotic stability.
Utilize above-mentioned pichia spp
pichiasp.SIT2014 prepares chiral aryl vicinal diamines, by pichia spp as biological catalyst
pichiasp.SIT2014 carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxyaryl ethyl ketone and obtains chiral aryl vicinal diamines;
Described 2-hydroxyaryl ethyl ketone is 2-hydroxy acetophenone, 2-hydroxyl m chloroacetophenone, 2-hydroxyl o-chloroacetophenone, 2-hydroxyl parabromoacetophenone or 2-hydroxyl parachloroacetophenone;
The chiral aryl vicinal diamines of corresponding gained is (S)-benzoglycols, (S)-m-chloro benzoglycols, (S)-adjacent chlorobenzene ethylene glycol, (S)-to bromobenzene ethylene glycol or (S)-to chlorobenzene ethylene glycol.
Above-mentioned pichia spp
pichiasp.SIT2014 as biological catalyst can highly selective, high yield and low cost carry out catalytically synthesizing chiral aryl vicinal diamines.
Above-mentioned utilizes above-mentioned pichia spp
pichiasp.SIT2014 prepares the method for chiral aryl vicinal diamines as biological catalyst, specifically comprises the steps
(1), pichia spp
pichiathe cultivation of sp.SIT2014
Get the pichia spp of 4 DEG C of Refrigerator stores
pichiasp.SIT2014 slant strains, picking one ring bacterial classification is linked into and is equipped with in the 100ml shaking flask of 15ml seed culture medium, and on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out seed culture 12-18h, obtains seed liquor;
Then by seed liquor: the volume ratio of fermention medium is the inoculum size of 5%, seed liquor is inoculated in fermention medium, on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out fermentation culture 15-24h, and the fermented liquid obtained is centrifugal, and harvested cell is pichia spp
pichiasp.SIT2014 cell, is then placed in damping fluid and preserves;
Above-described seed culture medium, fermention medium are identical, by often liter of calculating, containing glucose 15g, and Tryptones 5g, yeast powder 5g, K
2hPO
40.5g, KH
2pO
40.5g, NaCl 1g, MgSO
47H
2o 0.5g, surplus is water;
Described damping fluid is the sodium phosphate aqueous solution of pH5.5-8.0, and be preferably the sodium phosphate aqueous solution of pH5.5 ~ 7.0, be more preferably the sodium phosphate aqueous solution of pH 6.5, its consumption presses Pichia pastoris
pichiasp.SIT2014 dry weight: damping fluid is that the ratio of 5-100g:1L calculates;
(2), to step (1) gained containing pichia spp
pichiaadd hydroxyaryl ethyl ketone in the damping fluid of sp.SIT2014 cell, then control temperature is 30-50 DEG C and carries out reduction reaction 3-48h and obtain reaction solution;
The amount of above-mentioned reaction hydroxyaryl ethyl ketone used, by Pichia pastoris
pichiasp.SIT2014 dry weight: hydroxyaryl ethyl ketone is that the ratio of 5-100g:5-100mmol calculates;
(3), the reaction solution of step (2) being controlled rotating speed is that 10000r/min carries out centrifugation, the supernatant liquor obtained is with after isopyknic extraction into ethyl acetate three times, extraction liquid anhydrous sodium sulfate drying, then uses Rotary Evaporators evaporate to dryness, obtains chiral aryl vicinal diamines.
Beneficial effect of the present invention
A kind of pichia spp of the present invention
pichiasp.SIT2014 CGMCC NO.9300, be easy to cultivate because it has, mitotic stability is high, and in its thalline, carbonyl reduction enzymic activity is higher, to the reductibility of 2-hydroxyaryl ethyl ketone and selectivity stronger, it prepares chiral aryl vicinal diamines for biological catalyst catalysis 2-hydroxyaryl ethyl ketone, have transformation efficiency high, the features such as the reaction times is short, final transformation efficiency reaches as high as 99.9%, optical activity value more than 99%, from input pichia spp
pichiasp.SIT2014 cell, until obtain chiral aryl vicinal diamines, only needs 3-48h.And the pichia spp of the gained after 20 times of going down to posterity
pichiasp.SIT2014 colonial morphology remains unchanged, and fermentation gained cell prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and its transformation efficiency and optical purity still all can reach more than 99%, and therefore its mitotic stability is high.
Embodiment
Below by some embodiments, the present invention will be further described, but do not limit the present invention.
The present invention's reagent used is the reagent that analytical pure or HPLC are pure respectively.
The detection of chiral aryl vicinal diamines optical purity angle value, adopts Shimadzu high performance liquid chromatograph LC-20A, comes from Shimadzu instrument company;
In various embodiments of the present invention, (R, S)-benzoglycols chromatographiccondition of target product is as follows:
Moving phase is normal hexane: Virahol=95:5, flow velocity 0.8ml/min, and determined wavelength is 210nm, and column temperature is 27 DEG C, adopts chirality OD-H post (4.6mm × 250mm is purchased from Daicel medicine company chiral technology (Shanghai) Co., Ltd.).
Optical purity ee value calculating method is as follows:
Ee (%)=[S (configuration)-R(configuration)]/[S (configuration)+R(configuration)] × 100%
S (configuration): the concentration representing S (configuration) product enantiomorph;
R (configuration): the concentration representing R (configuration) product enantiomorph.
In the present invention, the cellular carbonyl reductase vitality unit of indication is defined as: at 30 DEG C, under the condition of pH6.5, and the enzyme amount needed for benzoglycols of per minute catalysis 2-hydroxy acetophenone reduction generation 1.0 μm of ol is 1 unit of activity, i.e. 1U.
embodiment 1
Pichia yeast
pichiathe cultivation of sp.SIT2014 thalline;
Slant medium used, by often liter of calculating, containing glucose 15.0g, Tryptones 5.0g, yeast powder 5.0g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o 0.5g, agar 20.0g, surplus is water.
Seed culture medium used, by often liter of calculating, containing glucose 15.0g, Tryptones 5.0g, yeast powder 5.0g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o 0.5g, surplus is water.
Fermention medium used, by often liter of calculating, containing glucose 15.0g, Tryptones 5.0g, yeast powder 5.0g, KH
2pO
40.5g, K
2hPO
40.5g, MgSO
47H
2o 0.5g, surplus is water.
Get the pichia spp of 4 DEG C of Refrigerator stores
pichiasp.SIT2014 slant strains, picking one ring bacterial classification is linked into and is equipped with in the 100ml shaking flask of 15ml seed culture medium, and on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out seed culture 18h, obtains seed liquor;
Then by seed liquor: the volume ratio of fermention medium is the inoculum size of 5%, seed liquor is inoculated in fermention medium, on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out fermentation culture 24h, and the fermented liquid obtained is centrifugal, and the cell of results is pichia spp
pichiasp.SIT2014 cell.
The fermentation liquor of final gained detects, and it produces carbonyl reduction enzyme activity and is about 20 ~ 30U/L, and in fermented liquid, cell concn calculates about 4 ~ 5g/L by dry weight, and namely unit cell product carbonyl reduction enzyme activity is about 4-5U/g stem cell.Show thus, the pichia spp of above-mentioned gained
pichiasp.SIT2014 cell has stronger reductibility and selectivity to aryl hydroxyketone.
embodiment 2
Pichia yeast
pichiathe Secondary Culture of sp.SIT2014 thalline;
By formulated slant medium, seed culture medium and fermention medium described in embodiment 1.
Get the pichia spp of 4 DEG C of Refrigerator stores
pichiasp.SIT2014 slant strains, aseptically picking list bacterium colony is on a fresh inclined-plane, and cultivate 48h at 30 DEG C in biochemical cultivation case after, the bacterium colony grown was 1 generation.In 1 generation bacterium colony, picking list bacterium colony is on a fresh inclined-plane, and cultivate 48h at 30 DEG C in biochemical cultivation case after, the bacterium colony grown was 2 generations.Go down to posterity after 20 times as stated above, the ring in picking 20 generation bacterium colony is linked into and is equipped with in the 100ml shaking flask of 15ml seed culture medium, and on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out seed culture 18h, obtains seed liquor;
Then by seed liquor: the volume ratio of fermention medium is the inoculum size of 5%, seed liquor is inoculated in fermention medium, on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out fermentation culture 24h, and the fermented liquid obtained is centrifugal, and the cell of results is pichia spp
pichiasp.SIT2014 cell.
The fermentation liquor of final gained detects, and it produces carbonyl reduction enzyme activity and is about 20 ~ 30U/L, and in fermented liquid, cell concn calculates about 4 ~ 5g/L by dry weight, and namely unit cell product carbonyl reduction enzyme activity is about 4-5U/g stem cell.Show thus, the pichia spp of above-mentioned gained
pichiasp.SIT2014 cell still keeps higher activity through going down to posterity after 20 times.
embodiment 3
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and described 2-hydroxyaryl ethyl ketone is 2-hydroxy acetophenone, and its catalyzed reaction equation is as follows:
By the pichia spp of embodiment 1 gained
pichiasp.SIT2014 carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxy acetophenone and prepares the chiral aryl vicinal diamines i.e. method of (S)-benzoglycols, specifically comprises the steps:
By the pichia spp of embodiment 1 gained
pichiasp.SIT2014 cell is placed in damping fluid;
Described damping fluid to be pH be 6.5 sodium phosphate aqueous solution, its consumption presses pichia spp
pichiasp.SIT2014 dry cell weight: damping fluid is that the ratio of 10g:1L calculates;
To above-mentioned gained containing pichia spp
pichiaadd 2-hydroxy acetophenone in the damping fluid of sp.SIT2014 cell, then control temperature is 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 20h and obtains reaction solution;
The amount of above-mentioned reaction 2-hydroxy acetophenone used, by pichia spp
pichiasp.SIT2014 dry cell weight: 2-hydroxy acetophenone is that the ratio of 0.5g:0.15mmol calculates;
It is that 10000r/min carries out centrifugation that the reaction solution of above-mentioned gained is controlled rotating speed, the supernatant liquor obtained is with after isopyknic extraction into ethyl acetate three times, extraction liquid anhydrous sodium sulfate drying, then Rotary Evaporators evaporate to dryness is used, obtain target product (S)-benzoglycols, transformation efficiency is 99.9%, ee value is 99.5%.
embodiment 4
Utilize the pichia spp of 20 times of going down to posterity of embodiment 2 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and described 2-hydroxyaryl ethyl ketone is 2-hydroxy acetophenone, and its catalyzed reaction equation is as follows:
By the pichia spp of embodiment 2 gained
pichiasp.SIT2014 carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxy acetophenone and prepares the chiral aryl vicinal diamines i.e. method of (S)-benzoglycols, specifically comprises the steps:
By the pichia spp of embodiment 2 gained
pichiasp.SIT2014 cell is placed in damping fluid;
Described damping fluid to be pH be 6.5 sodium phosphate aqueous solution, its consumption presses pichia spp
pichiasp.SIT2014 dry cell weight: damping fluid is that the ratio of 10g:1L calculates;
To above-mentioned gained containing pichia spp
pichiaadd 2-hydroxy acetophenone in the damping fluid of sp.SIT2014 cell, then control temperature is 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 20h and obtains reaction solution;
The amount of above-mentioned reaction 2-hydroxy acetophenone used, by pichia spp
pichiasp.SIT2014 dry cell weight: 2-hydroxy acetophenone is that the ratio of 0.5g:0.15mmol calculates;
It is that 10000r/min carries out centrifugation that the reaction solution of above-mentioned gained is controlled rotating speed, the supernatant liquor obtained is with after isopyknic extraction into ethyl acetate three times, extraction liquid anhydrous sodium sulfate drying, then Rotary Evaporators evaporate to dryness is used, obtain target product (S)-benzoglycols, transformation efficiency is 99.9%, ee value is 99.6%.
embodiment 5
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and described 2-hydroxyaryl ethyl ketone is 2-hydroxy acetophenone, by the pichia spp of embodiment 1 gained
pichiasp.SIT2014 carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxy acetophenone and prepares the chiral aryl vicinal diamines i.e. method of (S)-benzoglycols, specifically comprises the steps:
By the pichia spp of embodiment 1 gained
pichiasp.SIT2014 cell is placed in damping fluid;
Described damping fluid to be pH be 5.5 sodium phosphate aqueous solution, its consumption presses pichia spp
pichiasp.SIT2014 dry cell weight: damping fluid is that the ratio of 10g:1L calculates;
To above-mentioned gained containing pichia spp
pichia2-hydroxy acetophenone (R is wherein added in the damping fluid of sp.SIT2014 cell
1for-C
6h
5), then control temperature is 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 2h and obtains reaction solution;
The amount of above-mentioned reaction 2-hydroxy acetophenone used, by pichia spp
pichiasp.SIT2014 dry cell weight: 2-hydroxy acetophenone is that the ratio of 0.5g:0.15mmol calculates;
It is that 10000r/min carries out centrifugation that the reaction solution of above-mentioned gained is controlled rotating speed, the supernatant liquor obtained is with after isopyknic extraction into ethyl acetate three times, extraction liquid anhydrous sodium sulfate drying, then Rotary Evaporators evaporate to dryness is used, obtain target product chirality benzoglycols, transformation efficiency is 42.8%, ee value is 96.3%.
embodiment 6
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and described 2-hydroxyaryl ethyl ketone is 2-hydroxy acetophenone, by the pichia spp of embodiment 1 gained
pichiasp.SIT2014 carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxy acetophenone and prepares the chiral aryl vicinal diamines i.e. method of (S)-benzoglycols, specifically comprises the steps:
By the pichia spp of embodiment 1 gained
pichiasp.SIT2014 cell is placed in damping fluid;
Described damping fluid to be pH be 7.0 sodium phosphate aqueous solution, its consumption presses pichia spp
pichiasp.SIT2014 dry cell weight: damping fluid is that the ratio of 10g:1L calculates;
To above-mentioned gained containing pichia spp
pichiaadd 2-hydroxy acetophenone in the damping fluid of sp.SIT2014 cell, then control temperature is 30 DEG C, rotating speed is that 180r/min carries out reduction reaction 2h and obtains reaction solution;
The amount of above-mentioned reaction 2-hydroxy acetophenone used, by pichia spp
pichiasp.SIT2014 dry cell weight: 2-hydroxy acetophenone is that the ratio of 0.5g:0.15mmol calculates;
It is that 10000r/min carries out centrifugation that the reaction solution of above-mentioned gained is controlled rotating speed, the supernatant liquor obtained is with after isopyknic extraction into ethyl acetate three times, extraction liquid anhydrous sodium sulfate drying, then Rotary Evaporators evaporate to dryness is used, obtain target product chirality benzoglycols, transformation efficiency is 72.3%, ee value is 98.3%.
embodiment 7
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and described 2-hydroxyaryl ethyl ketone is 2-hydroxyl parachloroacetophenone, by the pichia spp of embodiment 1 gained
pichiasp.SIT2014 carries out as biological catalyst catalysis 2-hydroxyl parachloroacetophenone the method that asymmetric reduction reaction prepares chiral aryl vicinal diamines i.e. (S)-to chlorobenzene ethylene glycol, specifically comprises the steps:
2-hydroxy acetophenone wherein, with embodiment 3, is just replaced with 2-hydroxyl parachloroacetophenone by concrete steps, and finally obtain target product (S)-to chlorobenzene ethylene glycol, it is 99.1% that its transformation efficiency is greater than 99.9%, ee value.
embodiment 8
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, described 2-hydroxyaryl ethyl ketone is 2-hydroxyl parabromoacetophenone, pichia spp Pichia sp.SIT2014 by embodiment 1 gained carries out as biological catalyst catalysis 2-hydroxyl parabromoacetophenone the method that asymmetric reduction reaction prepares chiral aryl vicinal diamines i.e. (S)-to bromobenzene ethylene glycol, specifically comprises the steps:
2-hydroxy acetophenone wherein, with embodiment 3, is just replaced with 2-hydroxyl parabromoacetophenone by concrete steps, and finally obtain target product (S)-to bromobenzene ethylene glycol, it is 99.3% that its transformation efficiency is greater than 99.9%, ee value.
embodiment 9
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, described 2-hydroxyaryl ethyl ketone is 2-hydroxyl o-chloroacetophenone, pichia spp Pichia sp.SIT2014 by embodiment 1 gained carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxyl o-chloroacetophenone and prepares the chiral aryl vicinal diamines i.e. method of (S)-adjacent chlorobenzene ethylene glycol, specifically comprises the steps:
Concrete steps are with embodiment 3, and just 2-hydroxy acetophenone is wherein replaced with 2-hydroxyl o-chloroacetophenone, finally obtain target product (S)-adjacent chlorobenzene ethylene glycol, it is 99.2% that its transformation efficiency is greater than 99.9%, ee value.
embodiment 10
Utilize the pichia spp of embodiment 1 gained
pichiasp.SIT2014 prepares chiral aryl vicinal diamines as biological catalyst catalysis 2-hydroxyaryl ethyl ketone, and described 2-hydroxyaryl ethyl ketone is 2-hydroxyl m chloroacetophenone, by the pichia spp of embodiment 1 gained
pichiasp.SIT2014 carries out asymmetric reduction reaction as biological catalyst catalysis 2-hydroxyl m chloroacetophenone and prepares the chiral aryl vicinal diamines i.e. method of (S)-m-chloro benzoglycols, specifically comprises the steps:
Concrete steps are with embodiment 3, and just 2-hydroxy acetophenone is wherein replaced with 2-m-chloro hydroxy acetophenone, finally obtain target product (S)-m-chloro benzoglycols, it is 99.4% that its transformation efficiency is greater than 99.9%, ee value.
Above said content be only the present invention conceive under basic explanation, and according to any equivalent transformation that technical scheme of the present invention is done, all should protection scope of the present invention be belonged to.
Claims (7)
1. a Pichia yeast
pichiasp.SIT2014, deposit number is CGMCC NO.9300.
2. Pichia yeast as claimed in claim 1
pichiathe cultural method of sp.SIT2014, is characterized in that step is as follows:
Get the pichia spp of 4 DEG C of Refrigerator stores
pichiasp.SIT2014 slant strains, picking one ring bacterial classification is linked into and is equipped with in the 100ml shaking flask of 15ml seed culture medium, on shaking table control temperature be 30 DEG C, rotating speed be 180rpm cultivate 12-18h obtain seed liquor;
Then by seed liquor: the volume ratio of fermention medium is the inoculum size of 5%, seed liquor is inoculated in fermention medium, on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out fermentation culture 15-24h, and the fermented liquid obtained is centrifugal, and results obtain pichia spp
pichiasp.SIT2014 cell;
Above-described seed culture medium, fermention medium are identical, by often liter of calculating, containing glucose 15g, and Tryptones 5g, yeast powder 5g, K
2hPO
40.5g, KH
2pO
40.5g, NaCl 1g, MgSO
47H
2o 0.5g, surplus is water.
3. utilize Pichia yeast as claimed in claim 1
pichiasp.SIT2014 is chiral aryl vicinal diamines as biological catalyst catalyged precursor 2-hydroxyaryl ethyl ketone asymmetric reduction;
Described 2-hydroxyaryl ethyl ketone is 2-hydroxy acetophenone, 2-hydroxyl m chloroacetophenone, 2-hydroxyl o-chloroacetophenone, 2-hydroxyl parabromoacetophenone or 2-hydroxyl parachloroacetophenone;
The chiral aryl vicinal diamines of corresponding gained be the benzoglycols of S configuration, the m-chloro benzoglycols of S configuration, the adjacent chlorobenzene ethylene glycol of S configuration, S configuration to bromobenzene ethylene glycol or S configuration to chlorobenzene ethylene glycol.
4. utilize Pichia yeast as claimed in claim 3
pichiasp.SIT2014 is the method for chiral aryl vicinal diamines as biological catalyst catalyged precursor 2-hydroxyaryl ethyl ketone asymmetric reduction, it is characterized in that specifically comprising the steps:
(1), Pichia pastoris is cultivated
pichiasp.SIT2014
Get the pichia spp of 4 DEG C of Refrigerator stores
pichiasp.SIT2014 slant strains, picking one ring bacterial classification is linked into and is equipped with in the 100ml shaking flask of 15ml seed culture medium, and on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out seed culture 12-18h, obtains seed liquor;
Then by seed liquor: the volume ratio of fermention medium is the inoculum size of 5%, seed liquor is inoculated in fermention medium, on shaking table, control temperature is 30 DEG C, rotating speed is that 180rpm carries out fermentation culture 15-24h, and the fermented liquid obtained is centrifugal, and the cell of results is Pichia pastoris
pichiasp.SIT2014 cell, is then placed in damping fluid and preserves;
Above-described seed culture medium, fermention medium are identical, by often liter of calculating, containing glucose 15g, and Tryptones 5g, yeast powder 5g, K
2hPO
40.5g, KH
2pO
40.5g, NaCl 1g, MgSO
47H
2o 0.5g, surplus is water;
The sodium phosphate aqueous solution of described damping fluid to be pH be 5.5-8.0, its consumption presses Pichia pastoris
pichiasp.SIT2014 dry weight: damping fluid is that the ratio of 5-100g:1L calculates;
(2), to step (1) gained containing pichia spp
pichiaadd 2-hydroxyaryl ethyl ketone in the damping fluid of sp.SIT2014 cell, then control temperature is 30-50 DEG C and carries out reduction reaction 3-48h and obtain reaction solution;
The amount of above-mentioned reaction 2-hydroxyaryl used ethyl ketone, by pichia spp
pichiasp.SIT2014 dry cell weight: 2-hydroxyaryl ethyl ketone is that the ratio of 5-100g:5-100mmol calculates;
(3), the reaction solution of step (2) being controlled rotating speed is that 10000r/min carries out centrifugation, the supernatant liquor obtained is with after isopyknic extraction into ethyl acetate three times, use Rotary Evaporators evaporate to dryness after extraction liquid anhydrous sodium sulfate drying, obtain chiral aryl vicinal diamines.
5. utilize Pichia yeast as claimed in claim 4
pichiasp.SIT2014 is the method for chiral aryl vicinal diamines as biological catalyst catalyged precursor 2-hydroxyaryl ethyl ketone asymmetric reduction, it is characterized in that the damping fluid described in step (1) to be pH be the sodium phosphate aqueous solution of 5.5 ~ 7.0.
6. utilize Pichia yeast as claimed in claim 4
pichiasp.SIT2014 is the method for chiral aryl vicinal diamines as biological catalyst catalyged precursor 2-hydroxyaryl ethyl ketone asymmetric reduction, it is characterized in that the pH of buffer described in step (1) is the sodium phosphate aqueous solution of 6.5.
7. utilize Pichia yeast as claimed in claim 4
pichiasp.SIT2014 is the method for chiral aryl vicinal diamines as biological catalyst catalyged precursor 2-hydroxyaryl ethyl ketone asymmetric reduction, it is characterized in that the fermentation culture described in step (1), namely by seed liquor: the volume ratio of fermention medium is the inoculum size of 5%, seed liquor be inoculated into and be equipped with in the 250ml shaking flask of 50ml fermention medium, on shaking table, control pH=6.5, temperature are 30 DEG C, rotating speed is that 180rpm carries out fermentation culture 24h.
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| CN115710158A (en) * | 2021-08-23 | 2023-02-24 | 凯特立斯（深圳）科技有限公司 | Method for preparing ticagrelor intermediate through asymmetric catalysis |
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