CN103773724B - Rhodococcus XS1012 and preparing the application in chiral alcohol - Google Patents
Rhodococcus XS1012 and preparing the application in chiral alcohol Download PDFInfo
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- CN103773724B CN103773724B CN201410024110.1A CN201410024110A CN103773724B CN 103773724 B CN103773724 B CN 103773724B CN 201410024110 A CN201410024110 A CN 201410024110A CN 103773724 B CN103773724 B CN 103773724B
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- Prior art keywords
- trifluoromethyl
- phenyl
- bis
- ethanol
- rhodococcus
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- 230000003474 anti-emetic effect Effects 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
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- 239000003054 catalyst Substances 0.000 description 1
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- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
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- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- DLRVVLDZNNYCBX-CQUJWQHSSA-N gentiobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-CQUJWQHSSA-N 0.000 description 1
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- 235000011090 malic acid Nutrition 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Do you the invention discloses strain microorganism novel bacterial--a Rhodococcus (Rhodococcus? erythropolis) XS1012 and the application in preparation (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol thereof; Adopting this novel bacterial catalysis to prepare, that optical purity (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol has stereoselectivity is good, product optical purity advantages of higher; The present invention is the chirality biocatalysis of catalyzer through the wet thallus that fermentation obtains by employing Rhodococcus XS1012, when concentration of substrate be 20mmol/L, cosubstrate is glucose (50g/L) and Virahol (v/v, 10%) time, object product (S)-[3,5-bis-(trifluoromethyl) phenyl] ee of ethanol reaches 99.9%, and productive rate reaches 92.0%.
Description
(1) technical field
The present invention relates to a strain microorganism novel bacterial---Rhodococcus (Rhodococcuserythropolis) XS1012, and at asymmetric reduction [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone prepares application in optical purity (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol.
(2) background technology
(S)-[3,5-bis-(trifluoromethyl) phenyl] chemical structural formula of ethanol is:
(S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is important chiral intermediate for the synthesis of new chemotherapeutic antiemetic nk 1 receptor antagonist class medicine, the antidepressant drug prepared by it has good potential curative effect in a series of maincenter for the treatment of and peripheral nervous system suppress.Research shows, active [3,5-bis-(trifluoromethyl) phenyl] ethanol apparently higher than R-configuration of [3,5-bis-(trifluoromethyl) phenyl] ethanol of S-configuration, and it is very strong to the avidity of nk 1 receptor, has good perviousness to brain barrier.
The method utilizing 3,5-bis trifluoromethyl methyl phenyl ketone to prepare optical purity (S)-3,5-bis trifluoromethyl phenylethyl alcohol at present mainly contains chemical method and biological process.When adopting chemical method synthesis, need to use the chemical catalysts such as expensive transition metal Ru, and complex steps, severe reaction conditions, energy consumption is high, and pollute large, productive rate is on the low side.Biological rule utilizes resolvase or full cell to carry out catalysis preparation.Be applied to oxydo-reductase mainly alcoholdehydrogenase or the aldehyde ketone reductase enzyme of catalytic asymmetric reduction carbonyl compound, they all need to add coenzyme (NAD (P) H) in catalytic process, but coenzyme is expensive, and the purification procedures of aldehyde ketone reductase enzyme or alcoholdehydrogenase is complicated, limits its industrial applications to a certain extent.Adopt microbe whole-cell biocatalytic reduction can overcome above-mentioned shortcoming, because not only containing for redox enzyme in microorganism cells, also can realize regenerating coenzyme simultaneously, the separation and purification process of complicated enzyme can be saved.
At present, biocatalysis synthesis (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol that document has been reported relates generally to following several microorganism or enzyme.(the PollardD such as Pollard, TruppoM, PollardJ, etal., TetrahedronAsymmetry, 2006, 17 (4): 554-559) from rhodococcus (Rhodococcuserythropolis), separation obtains alcoholdehydrogenase, and use it for the important pharmaceutical intermediate (S)-3 of synthesis, 5-bis trifluoromethyl phenylethyl alcohol, ee value is greater than 99.9%, transformation efficiency is greater than 98%, but also need to add formate dehydrogenase (formatedehydrogenase in the alcoholdehydrogenase enzyme catalysis reduction process needing cofactor NADH, FDH) to realize the in-situ regeneration of cofactor, (the ZhuD such as Zhu, YangY, JohnD, etal., OrgBiomolChem, 2006,4:2690-2695) adopt reddish brown shadow yeast (Sporobolomycessalmonicolor) carbonyl reductase to prepare (S)-3,5-bis trifluoromethyl phenylethyl alcohol, optical activity reaches 99%, (the Zhang Fang such as Zhang Fang, Jiang Shu, Liu Chunmei, etc. Pharmaceutical Biotechnology, 2007,16 (1): 411-414) catalysis 3 in the two-phase system utilizing rhodotorula (Saccharomycesrhodotorula) resting cell to form at water/octane (volume fraction 20%), 5-bis trifluoromethyl acetophenone reduction obtains (S)-3,5-bis trifluoromethyl phenylethyl alcohol, after reaction 36h, transformation efficiency is 81.7%, but the e.e. value of product is only up to 89.5%, the applicant (WangP, SuHZ, SunLM, eta1., ChineseJChemEng, 2011,19 (6): 1028-1032) strain novel bacterial-candida tropicalis (Candidatropicalis) 104 catalytic asymmetric reduction preparation (S)-3 is also reported, the method of 5-bis trifluoromethyl phenylethyl alcohol, the method is carried out in aqueous phase system, and ee value is greater than 99.9%.
(3) summary of the invention
The object of the invention is to provide strain microorganism novel bacterial-Rhodococcus (Rhodococcuserythropolis) XS1012, and its catalytic asymmetric reduction [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone prepares the application of optically pure (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol.
The technical solution used in the present invention is:
The invention provides a strain new strains--Rhodococcus (Rhodococcuserythropolis) XS1012, this bacterial strain is preserved in China typical culture collection center, preservation date is on December 11st, 2013, preservation address: China, Wuhan, Wuhan University, deposit number: CCTCCNO:M2013650.
Rhodococcus XS1012 of the present invention screens as follows and obtains:
1) bacterial screening:
Bacterial classification is originated: Rhodococcus XS1012 system obtains from separation screening the soil sample picking up from area, Xiaoshan, Zhejiang Province.Concrete screening method is as follows: will gather soil sample (Xiaoshan, Zhejiang) and join and be equipped with in the 250mL shaking flask of 50mL enrichment medium, 25 ~ 32 DEG C, 150 ~ 250rpm, cultivate 4 ~ 5d, become after muddiness until nutrient solution, getting 1mL nutrient solution is forwarded in fresh enrichment medium, continue cultivation 4 ~ 5d, so repeat enrichment culture 3 ~ 4 times.In enrichment medium with [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone for sole carbon source.Enrichment medium final concentration consists of: [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone 10 ~ 100mmol/L, KH
2pO
41.0 ~ 3.0g/L, (NH
4)
2sO
42.0 ~ 6.0g/L, NaCl1.0 ~ 4.0g/L, MgSO
47H
2o1.0 ~ 3.0g/L, solvent is water, pH6.5 ~ 7.0.
By spread plate substratum after last enrichment culture liquid dilution, after separating for several times is cultivated, obtain single bacterium colony bacterial strain.Plate culture medium consists of in enrichment medium the agar adding 15 ~ 20g/L.
Picking list colony inoculation is to seed culture medium, and 25 ~ 32 DEG C, 150 ~ 250rpm cultivates 24 ~ 48h, getting seed liquor is forwarded in fermention medium with the inoculum size of volumetric concentration 6 ~ 10%, 25 ~ 32 DEG C, and 150 ~ 250rpm cultivates 24 ~ 48h, fermented liquid is centrifugal, obtain wet thallus.
With [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone is substrate, screening the microorganism cells (wet thallus namely after fermentation culture) obtained is catalyzer, in phosphoric acid buffer (pH value is 7.0) after 20 ~ 35 DEG C of bio-transformation 12 ~ 60h, chiral gas chromatography is adopted to detect object product (S)-[3 in conversion fluid, 5-bis-(trifluoromethyl) phenyl] enantiomeric excess value (ee value) of ethanol, therefrom screening obtains having the microorganism strains of high antimer excessive value (ee value > 99%)-be designated as strain X S1012.
2) physiological and biochemical property
Colonial morphology: under 25 ~ 32 DEG C of conditions, slant medium cultivates 48h, colony diameter is about 2 ~ 5mm, neat in edge, circular, and protuberance, bacterium colony is safran.
Physiological and biochemical property: Gram-positive, in shaft-like, size (0.6 ~ 1.2) μm × (4 ~ 7) μm atrichia.Utilizable carbon source has: dextrin, D-raffinose, alpha-D-glucose, D-glucitol, pectin, polysorbate40, D-MANNOSE, PEARLITOL 25C, D-galactosonic acid, γ-aminobutyric acid, D-melibiose, D-Fructose, D-R alcohol, ALANINE, D-ALPHA-Hydroxypropionic acid methyl esters, alpha-hydroxybutyric acid, Beta-methyl-D-glucosides, L-arginine, maltonic acid, Pfansteihl, beta-hydroxy-D, L-butyric acid, D-fibres sugars, glycerine, D-Glucose aldehydic acid, glucose amide, citric acid, alpha-ketoacid, gentiobiose, trehalose, D-Glucose-6-phosphoric acid salt, glucose amide, α-ketoglutaric acid, etheric acid, sucrose, L-fucose, D-Fructose-6-phosphoric acid, L-Histidine, propionic acid, D-turanose, N-acetyl-D-semi-lactosi Portugal amine, quinic acid, L MALIC ACID, acetic acid, creatinine, D-Ser, succsinic acid, L MALIC ACID, acetic acid.Unavailable carbon source has: the acid of gelatin, beta-hydroxyphenyl acetic acid, α-D-lactose, L-PROLINE, Pyruvic Acid Methyl ester, maltose, L-GaA lactone, D-trehalose, D-semi-lactosi, D-Whitfield's ointment, 3-methyl glucoside, L-Aspartic acid, N-ACETYL-D-GLUCOSAMINE, D-malic acid, L-rhamnosyl, D-Asp, L-Glutimic acid, stachyose, Serine, D-granulated sugar, formic acid.
3) 16SrDNA sequence characteristic
Be template with the cell STb gene that Ezup pillar bacterial genomes DNA extraction agent box extracts, universal primer 7F (5'-CAGAGTTTGATCCTGGCT-3') and 1540R (5'-AGGAGGTGATCCAGCCGCA-3') is utilized to increase the 16SrDNA gene of bacterial strain, (PCR reaction system is 25 μ l, comprising genomic dna 10 ~ 25ng, 5 × Buffer2.5 μ l, archaeal dna polymerase 0.2 μ l, each 5mM of forward and reverse primer; PCR reaction conditions: 98 DEG C of denaturation 3min, then 25s is kept at 98 DEG C, 55 DEG C keep 25s, 30 circulations are carried out under the condition of 72 DEG C of maintenance 1min, finally extend 10min at 72 DEG C), PCR primer is carried out the agarose gel electrophoresis of 1%, found that at the single band of 1500bp place appearance, by to after the recovery of band and purifying, order-checking (the raw work in Shanghai) confirms that the 16SrDNA gene order of described bacterial strain is for shown in SEQIDNO:1:
CATGCAAGTCGAGCGGTAAGGCCTTTCGGGGTACACGAGCGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTCCTATCGCATGGTGGGTGGTGGAAAGATTTATCGGTGCAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCAAGCGTTGTCCGGAATTACTGGGCGTAAAGAGTTCGTAGGCGGTTTGTCGCGTCGTTTGTGAAAACCAGCAGCTCAACTGCTGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTCCTTCCACGGAATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGAAAGCTGCAGAGATGTGGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCACGTTATGGTGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAGACCGTGAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCTTAACCCCTTGTGGGAGGGAGCCGTCGAAGGTGGGATCGGCGATGGGACGAAG
This sequence has submitted to GenBank(GenBank accession number to be No.KJ000875), the 16SrDNA sequence of strain X S1012 is carried out sequence analysis (BLAST) on NCBI website (http://www.ncbi.nlm.nih.gov), and result shows: the part strain sequence homology of strain X S1012 and Rhod (Rhodococcussp.) is higher.The sequence homology of strain X S1012 and Rhodococcuserythropolis bacterial strain (GenBank accession number is No.HQ179128.1) reaches 100%.
According to physio-biochemical characteristics and binding molecule biological assay, this strain X S1012 is accredited as Rhodococcus (Rhodococcuserythropolis), called after Rhodococcus (Rhodococcuserythropolis) XS1012.
By response surface experimental design, the Optimal Medium obtaining Rhodococcus XS1012 bacterial strain consists of: glucose 10 ~ 25g/L, yeast extract paste 3 ~ 15g/L, KH
2pO
41.0 ~ 4.5g/L, (NH
4)
2sO
41.5 ~ 6.0g/L, peptone 5 ~ 20g/L, NaCl0.5 ~ 2.5g/L, MgSO
47H
2o0.5 ~ 1.5g/L, solvent is water, and pH is 6.0 ~ 7.5.
The culture condition of Rhodococcus XS1012 is: initial pH6.0 ~ 7.5, shaking flask liquid amount 80 ~ 120mL/250mL Erlenmeyer flask, culture temperature 25 ~ 35 DEG C, shaking speed 150 ~ 250rpm, inoculum size 4 ~ 10%, incubation time 36 ~ 48h.
The present invention also provides a kind of described Rhodococcus XS1012 in preparation (S)-[3, 5-bis-(trifluoromethyl) phenyl] application in ethanol, described is applied as: with [3, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is substrate, the wet thallus obtained through fermentation culture with Rhodococcus XS1012 is for enzyme source, in pH be 6.0 ~ 8.5 damping fluid or distilled water form reaction system in, react under 20 DEG C ~ 35 DEG C conditions, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, extraction liquid is the mixed solution containing target product, mixed solution obtains (S)-[3 through separation and purification, 5-bis-(trifluoromethyl) phenyl] ethanol.In described reaction system, the initial final concentration of substrate is 10 ~ 200mmol/L(preferably 10 ~ 30mmol/L), the consumption of wet thallus counts 15 ~ 70g/L(preferably 20 ~ 60g/L with dry cell weight).
Further, preferred described reaction is conversion reaction 12 ~ 60h, preferably 24 ~ 48h under 25 DEG C ~ 35 DEG C conditions.
The method of mixed solution separation and purification of the present invention is: mixed solution is through rotatory evaporator distillation and concentration 5 times of volumes, then after adding the silica gel mixing of column volume 1 ~ 5%, be transferred in the chromatography column containing silica gel, add the silica gel of column volume 1 ~ 5% again, then be that the sherwood oil of 8:1 is separated for eluent carries out silica gel column chromatography with ethyl acetate mixtures with volume ratio, collect the elutriant merged containing target product, by the elutriant containing target product through rotatory evaporator evaporate to dryness, obtain (S)-[two (trifluoromethyl) phenyl of 3,5-] ethanol.(S)-[two (trifluoromethyl) phenyl of 3,5-] ethanol
1hNMR (600MHz, CDCl
3): 7.85 (s, 2H), 7.79 (s, 1H), 5.06-5.03 (q, 1H, J=6.48Hz), 1.54 (d, 3H, J=6.48Hz).
Further, for promoting regenerating coenzyme, improve reaction efficiency, also be added with cosubstrate in described reaction system and form transformation system, described cosubstrate is the mixing of following one or both and above arbitrary proportion: 1. methyl alcohol, 2. ethanol, 3. Virahol, 4. glycerine, 5. glucose, 6. fructose, 7. sucrose or 8. maltose, and preferred described cosubstrate is one of following: 1. glucose, 2. Virahol or 3. the mixing of glucose and Virahol.
Further, when preferred described cosubstrate is glucose, fructose, sucrose or maltose, adding final concentration is 20 ~ 200g/L transformation system (preferred 50g/L), and when cosubstrate is methyl alcohol, ethanol, Virahol or glycerine, adding volume final concentration is 10 ~ 30%(preferably 10%).
Further, preferred described cosubstrate be glucose and Virahol for two cosubstrate time, optical purity and the productive rate of products therefrom are the highest, and adding glucose final concentration in transformation system is the preferred 50g/L of 20 ~ 100g/L(), the volume final concentration 10 ~ 20%(adding Virahol preferably 10%).
Further, the application of preferred described Rhodococcus XS1012 in preparation (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is carried out as follows:
With [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone is substrate, the wet thallus obtained through fermentation culture with Rhodococcus XS1012 is for enzyme source, add cosubstrate, in pH be 6.0 ~ 8.5 phosphoric acid buffer or distilled water form transformation system in, 24 ~ 48h is reacted under 25 DEG C ~ 35 DEG C conditions, after reaction terminates, obtain the reaction solution of the target product contained, reaction solution obtains (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol through separation and purification; In described transformation system, the initial final concentration of substrate is 10 ~ 200mmol/L, and the consumption of wet thallus counts 20 ~ 60g/L with dry cell weight; Described preferred cosubstrate is glucose and Virahol, and in transformation system, the interpolation final concentration of glucose is the preferred 50g/L of 20 ~ 100g/L(), the volume final concentration of Virahol is 10 ~ 20%(preferably 10%).
Enzyme source of the present invention obtains as follows:
1) slant culture: the mono-colony inoculation of picking Rhodococcus XS1012, to slant medium, cultivates 2 ~ 3d, 4 DEG C of Refrigerator stores for 25 ~ 32 DEG C; Slant medium final concentration consists of: glucose 10 ~ 25g/L, peptone 5 ~ 20g/L, yeast extract paste 3 ~ 15g/L, (NH
4)
2sO
41.5 ~ 6.0g/L, KH
2pO
41.0 ~ 4.5g/L, NaCl0.5 ~ 2.5g/L, MgSO
47H
2o0.5 ~ 6.0g/L, agar 15 ~ 20g/L, solvent is water, pH6.0 ~ 7.5;
2) seed culture: choose a ring thalline access seed culture medium from cultivating ripe inclined-plane, 25 ~ 32 DEG C, 150 ~ 250rpm cultivates 24 ~ 48h, obtains seed liquor; Described seed culture medium final concentration consists of: glucose 10 ~ 25g/L, yeast extract paste 3 ~ 15g/L, KH
2pO
41.0 ~ 4.5g/L, (NH
4)
2sO
41.5 ~ 6.0g/L, peptone 5 ~ 20g/L, NaCl0.5 ~ 2.5g/L, MgSO
47H
2o0.5 ~ 6.0g/L, solvent is water, pH6.0 ~ 7.5;
3) fermentation culture: seed liquor is transferred in fermention medium with the inoculum size of volumetric concentration 6 ~ 10%, 25 ~ 32 DEG C, 150 ~ 250rpm cultivates 24 ~ 48h, obtain fermented liquid, fermented liquid is centrifugal, phosphoric acid buffer (pH7.0) washing of gained precipitation, obtain wet thallus, be enzyme source; Fermentative medium formula is with seed culture medium prescription.
Damping fluid preferably phosphoric acid salt buffer of the present invention, described phosphoric acid salt is Na
2hPO
4-citric acid, Na
2hPO
4-NaH
2pO
4, K
2hPO
4-KH
2pO
4, Na
2hPO
4-KH
2pO
4, K
2hPO
4-NaOH, more preferably pH is the Na of 7.0
2hPO
4-KH
2pO
4damping fluid.
The concentration of the product in extraction liquid and unreacted substrate adopts gas chromatographic analysis, uses inner mark method ration.Internal standard substance is dodecane.Get 1ml extraction liquid to add 2 μ l dodecanes and analyze.GC conditions: Japanese Shimadzu GC-2014 gas chromatograph; U.S. Varian CP-Chirasil-Dex chiral capillary gas chromatography post (25m × 0.25mm × 0.25 μm).Carrier gas is high pure nitrogen, and flow is 2mL/min; Sample size 1 μ L, splitting ratio is 15:1; Detector and injector temperature are 250 DEG C; Column temperature 80 DEG C retains 2min, is warming up to 180 DEG C with 4 DEG C/min, maintains 10min; Detector is FID.According to gas chromatographic detection spectrogram, calculate concentration and the ee value of product in reaction solution by relative correction factor method.
Calculation of yield method is:
Marker method: be internal standard substance with dodecane, records production concentration typical curve.Adding a certain amount of dodecane during mensuration is in the sample to which internal standard substance, calculates production concentration according to internal standard substance concentration.
Typical curve preparation method: the substrate or the product standard substance that accurately take a series of different concns, be dissolved in normal hexane, be mixed with a series of mixing solutions, use gas chromatographic detection respectively.Gained color atlas integration is obtained peak area, with the peak area ratio (S of substrate or product and n-dodecane
substrate/ S
dodecaneor S
product/ S
dodecane) be X-coordinate, concentration ratio (C
substrate/ C
dodecaneor C
product/ C
dodecane) be ordinate zou, make typical curve, thus the relative correction factor that the relative correction factor obtaining 3,5-bis trifluoromethyl methyl phenyl ketone and internal standard substance is 1.31,3,5-bis trifluoromethyl phenylethyl alcohol and internal standard substance is 1.69.
Calculation of yield formula is as follows:
C in formula (1)
pfor (S)-3,5-dual-trifluoromethyl benzene alcohol concn, C
0it is 3,5-bis trifluoromethyl methyl phenyl ketone starting point concentration.
The optical purity of product is characterized by enantiomeric excess value (enantiomericexcess, ee).Calculation formula is:
C in formula (2)
sand C
rbe respectively the volumetric molar concentration of S type and R type 3,5-bis trifluoromethyl phenylethyl alcohol.
Beneficial effect of the present invention is mainly reflected in: the invention provides a strain microorganism novel bacterial--Rhodococcus (Rhodococcuserythropolis) XS1012, it can prepare chiral aryl secondary alcohol by asymmetric reduction, thus provide useful reference in microorganisms legal system for crucial chiral intermediate (S)-[3,5-bis-(trifluoromethyl) phenyl] the ethanol aspect of nk 1 receptor antagonist class medicine; Adopting this novel bacterial catalysis to prepare, that optical purity (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol has stereoselectivity is good, product optical purity advantages of higher; The present invention is the chirality biocatalysis of catalyzer through the wet thallus that fermentation obtains by employing Rhodococcus XS1012, when concentration of substrate be 20mmol/L, cosubstrate is glucose (50g/L) and Virahol (v/v, 10%) time, object product (S)-[3,5-bis-(trifluoromethyl) phenyl] ee of ethanol reaches 99.9%, and productive rate reaches 92.0%.
(4) accompanying drawing explanation
Fig. 1 is the standard substance vapor detection collection of illustrative plates of 3,5-bis trifluoromethyl methyl phenyl ketone substrate and [two (trifluoromethyl) phenyl of 3,5-] ethanol product.
Fig. 2 is Rhodococcus XS1012 bioreduction extraction liquid gas chromatogram.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the acquisition of wet thallus
Fermented liquid culture medium prescription: glucose 15g/L, peptone 7.5g/L, yeast extract paste 6g/L, (NH
4)
2sO
43g/L, KH
2pO
41.5g/L, NaCl0.75g/L, MgSO
47H
2o0.75g/L, solvent is water, pH6.5.
The same fermentative medium formula of seed culture based formulas.
1) slant culture: the mono-colony inoculation of picking Rhodococcus XS1012, to slant medium, cultivates 2 ~ 3d, 4 DEG C of Refrigerator stores for 30 DEG C; Slant medium final concentration consists of: glucose 15g/L, peptone 7.5g/L, yeast extract paste 6g/L, (NH
4)
2sO
43g/L, KH
2pO
41.5g/L, NaCl0.75g/L, MgSO
47H
2o0.75g/L, agar 20g/L, solvent is water, pH6.5.
2) seed culture: choose a ring thalline access seed culture medium from cultivating ripe inclined-plane, 30 DEG C, 200rpm cultivates 24h, acquisition seed liquor;
3) fermentation culture: seed liquor is transferred in fermention medium with the inoculum size of volumetric concentration 6%, 30 DEG C, 200rpm cultivates 48h, obtains fermented liquid, and fermented liquid is centrifugal, the phosphoric acid buffer (Na of gained precipitation pH7.0
2hPO
4-KH
2pO
4damping fluid) washing, obtain wet thallus, be enzyme source.
Embodiment 2 ~ 4:
The wet thallus of embodiment 1 gained is suspended in the phosphate buffered saline buffer (Na of the different pH of 10mL
2hPO
4-KH
2pO
4damping fluid) in, wet thallus in dry cell weight concentration for 35g/L; Add [3 of final concentration 20mmol/L, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is as substrate, add the glucose of final concentration 50g/L again as cosubstrate, be placed in 30 DEG C, the shaking table of 200rpm reacts 24h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, get extraction liquid and carry out gas chromatographic analysis, use inner mark method ration.Internal standard substance is dodecane.Get 1ml extraction liquid to add 2 μ l dodecanes and analyze.GC conditions: Japanese Shimadzu GC-2014 gas chromatograph, Zhejiang University N2000 chromatographic working station; U.S. Varian CP-Chirasil-Dex chiral capillary gas chromatography post (25m × 0.25mm × 0.25 μm).Carrier gas is high pure nitrogen, and flow is 2mL/min; Sample size 1 μ L, splitting ratio is 15:1; Detector and injector temperature are 250 DEG C; Column temperature 80 DEG C retains 2min, is warming up to 180 DEG C with 4 DEG C/min, maintains 10min; Detector is FID.According to gas chromatographic detection spectrogram, calculate concentration and the ee value of product in reaction solution by relative correction factor method.Concentration and the ee value of product (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol are shown in Table 1.As shown in Table 1, when the pH of damping fluid is 7.0, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is the highest, be 8.4mmol/L, ee value is 99.9%, and productive rate is 42%.
The concentration of table 1 product and ee value
Embodiment 5 ~ 10:
The wet thallus of embodiment 1 gained is suspended in 10mL different phosphate phthalate buffer system (pH7.0), wet thallus in dry cell weight concentration for 35g/L; Add [3 of final concentration 20mmol/L, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is as substrate, add the glucose of final concentration 50g/L again as cosubstrate, be placed in 30 DEG C, in the shaking table of 200rpm, react 24h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, get extraction liquid and carry out gas chromatographic analysis, use inner mark method ration.Concentration and the ee value of product (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol are shown in Table 2.As shown in Table 2, when buffering liquid is Na
2hPO
4-KH
2pO
4time, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is the highest, be 14.6mmol/L, ee value is 99.9%, and productive rate is 73.0%.
The concentration of table 2 product and ee value
Embodiment 11 ~ 13:
The wet thallus of embodiment 1 gained is suspended in 10mLNa
2hPO
4-KH
2pO
4in phosphate buffered saline buffer (pH7.0), wet thallus in dry cell weight concentration for 35g/L; Add [3 of final concentration 20mmol/L, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is as substrate, add the glucose of final concentration 50g/L again as cosubstrate, under differing temps, in the shaking table of 200rpm, react 24h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, get extraction liquid and carry out gas chromatographic analysis, use inner mark method ration.Concentration and the ee value of product (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol are shown in Table 3.As shown in Table 3, when temperature is 30 DEG C, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is the highest, be 14.3mmol/L, ee value is 99.9%, and productive rate is 71.5%.
The concentration of table 3 product and ee value
Embodiment 14 ~ 16:
The wet thallus of embodiment 1 gained is suspended in 10mLNa
2hPO
4-KH
2pO
4in phosphate buffered saline buffer (pH7.0), the wet thallus (with dry weight basis) of different concns; Add [3 of final concentration 20mmol/L, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is as substrate, add the glucose of final concentration 50g/L again as cosubstrate, be placed in 30 DEG C, in the shaking table of 200rpm, react 24h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, get extraction liquid and carry out gas chromatographic analysis, use inner mark method ration.The concentration of product (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol and ee value are in table 4.As shown in Table 4, when biomass is 45g/L, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is the highest, be 15.8mmol/L, ee value is 99.9%, and productive rate is 79.0%.
The concentration of table 4 product and ee value
Embodiment 17 ~ 26:
The wet thallus of embodiment 1 gained is suspended in 10mLNa
2hPO
4-KH
2pO
4in phosphate buffered saline buffer (pH7.0), wet thallus in dry cell weight concentration for 45g/L; Add [3 of final concentration 20mmol/L, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is as substrate, add the alcohols of the different carbohydrate of quality final concentration or different volumes final concentration again as cosubstrate, be placed in 30 DEG C, in the shaking table of 200rpm, react 24h, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, get extraction liquid and carry out gas chromatographic analysis, use inner mark method ration.The concentration of product (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol and ee value are in table 5.As shown in Table 5, when not adding cosubstrate, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is only 1.0mmol/L, and ee value is 82.8%, and productive rate is 5.0%; When cosubstrate be glucose (50g/L) and Virahol (v/v, 10%) time, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is the highest, be 18.4mmol/L, ee value is 99.9%, and productive rate is 92.0%.
The concentration of table 5 product and ee value
Embodiment 27 ~ 29:
The wet thallus of embodiment 1 gained is suspended in 10mLNa
2hPO
4-KH
2pO
4in phosphate buffered saline buffer (pH7.0), wet thallus in dry cell weight concentration for 45g/L; Add [3 of different Final substrate concentrations, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is as substrate, add glucose (final concentration 50g/L) and the two cosubstrate of Virahol (final concentration 10%, v/v) conduct again, be placed in 30 DEG C, 24h is reacted in the shaking table of 200rpm, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, get extraction liquid and carry out gas chromatographic analysis, use inner mark method ration.The concentration of product (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol and ee value are in table 6.As shown in Table 6, when concentration of substrate is 20mmol/L, the concentration of (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol is the highest, be 18.4mmol/L, ee value is 99.9%, and productive rate is 92.0%.
The concentration of table 6 product and ee value
Claims (10)
1. Rhodococcus (Rhodococcuserythropolis) XS1012, is preserved in China typical culture collection center, and preservation date is on December 11st, 2013, preservation address: China, Wuhan, Wuhan University, deposit number: CCTCCNO:M2013650.
2. Rhodococcus XS1012 is preparing (S)-[3 as claimed in claim 1, 5-bis-(trifluoromethyl) phenyl] application in ethanol, it is characterized in that described being applied as: with [3, 5-bis-(trifluoromethyl) phenyl] ethyl ketone is substrate, the wet thallus obtained through fermentation culture with Rhodococcus XS1012 is for enzyme source, in pH be 6.0 ~ 8.5 damping fluid or distilled water form reaction system in, react under 20 DEG C ~ 35 DEG C conditions, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, extraction liquid is the mixed solution containing target product, mixed solution obtains (S)-[3 through separation and purification, 5-bis-(trifluoromethyl) phenyl] ethanol.
3. Rhodococcus XS1012 is preparing (S)-[3 as claimed in claim 2,5-bis-(trifluoromethyl) phenyl] application in ethanol, it is characterized in that in described reaction system, the initial final concentration of substrate is 10 ~ 200mmol/L, and the consumption of wet thallus counts 15 ~ 70g/L with dry cell weight.
4. Rhodococcus XS1012 is preparing (S)-[3 as claimed in claim 2,5-bis-(trifluoromethyl) phenyl] application in ethanol, it is characterized in that also being added with in described reaction system cosubstrate and form transformation system, described cosubstrate is the mixing of following one or both and above arbitrary proportion: 1. methyl alcohol, 2. ethanol, 3. Virahol, 4. glycerine, 5. glucose, 6. fructose, 7. sucrose or 8. maltose.
5. Rhodococcus XS1012 is preparing (S)-[3 as claimed in claim 4,5-bis-(trifluoromethyl) phenyl] application in ethanol, it is characterized in that described cosubstrate is one of following: 1. glucose, 2. Virahol or 3. the mixing of glucose and Virahol.
6. Rhodococcus XS1012 is preparing (S)-[3 as claimed in claim 4,5-bis-(trifluoromethyl) phenyl] application in ethanol, when it is characterized in that described cosubstrate is glucose, fructose, sucrose or maltose, adding final concentration is 20 ~ 200g/L transformation system, when cosubstrate is methyl alcohol, ethanol, Virahol or glycerine, adding volume final concentration is 10 ~ 30%.
7. Rhodococcus XS1012 is preparing (S)-[3 as claimed in claim 6,5-bis-(trifluoromethyl) phenyl] application in ethanol, it is characterized in that described cosubstrate is glucose and Virahol, in transformation system, glucose adds final concentration is 20 ~ 100g/L, and the volume final concentration of Virahol is 10 ~ 20%.
8. the application of Rhodococcus XS1012 in preparation (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol as claimed in claim 2, is characterized in that described application is carried out as follows:
With [3,5-bis-(trifluoromethyl) phenyl] ethyl ketone is substrate, the wet thallus obtained through fermentation culture with Rhodococcus XS1012 is for enzyme source, add cosubstrate, in pH be 6.0 ~ 8.5 phosphoric acid buffer or distilled water form transformation system in, 24 ~ 48h is reacted under 25 DEG C ~ 35 DEG C conditions, after reaction terminates, reaction solution is centrifugal, get supernatant liquor and add isopyknic n-hexane extraction, extraction liquid is the mixed solution containing target product, and mixed solution obtains (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol through separation and purification; In described transformation system, the initial final concentration of substrate is 10 ~ 200mmol/L, and the consumption of wet thallus counts 20 ~ 60g/L with dry cell weight; Described cosubstrate is glucose and Virahol, and in transformation system, the interpolation final concentration of glucose is 20 ~ 100g/L, and the interpolation volume final concentration of Virahol is 10 ~ 20%.
9. as described in claim 2 or 8, Rhodococcus XS1012 is preparing (S)-[3, 5-bis-(trifluoromethyl) phenyl] application in ethanol, it is characterized in that the method for described mixed solution separation and purification is: mixed solution is through rotatory evaporator distillation and concentration 5 times of volumes, then after adding the silica gel mixing of column volume 1 ~ 5%, be transferred in the chromatography column containing silica gel, add the silica gel of column volume 1 ~ 5% again, then be that the sherwood oil of 8:1 is separated for eluent carries out silica gel column chromatography with ethyl acetate mixtures with volume ratio, collect the elutriant merged containing target product, by the elutriant containing target product through rotatory evaporator evaporate to dryness, obtain (S)-[3, two (trifluoromethyl) phenyl of 5-] ethanol.
10. the application of Rhodococcus XS1012 in preparation (S)-[3,5-bis-(trifluoromethyl) phenyl] ethanol as described in claim 2 or 4, is characterized in that described enzyme source obtains as follows:
1) slant culture: the mono-colony inoculation of picking Rhodococcus XS1012, to slant medium, cultivates 2 ~ 3d, 4 DEG C of Refrigerator stores for 25 ~ 32 DEG C; Slant medium final concentration consists of: glucose 10 ~ 25g/L, peptone 5 ~ 20g/L, yeast extract paste 3 ~ 15g/L, (NH
4)
2sO
41.5 ~ 6.0g/L, KH
2pO
41.0 ~ 4.5g/L, NaCl0.5 ~ 2.5g/L, MgSO
47H
2o0.5 ~ 6.0g/L, agar 15 ~ 20g/L, solvent is water, pH6.0 ~ 7.5;
2) seed culture: choose a ring thalline access seed culture medium from cultivating ripe inclined-plane, 25 ~ 32 DEG C, 150 ~ 250r/min cultivates 24 ~ 48h, obtains seed liquor; Described seed culture medium final concentration consists of: glucose 10 ~ 25g/L, yeast extract paste 3 ~ 15g/L, KH
2pO
41.0 ~ 4.5g/L, (NH
4)
2sO
41.5 ~ 6.0g/L, peptone 5 ~ 20g/L, NaCl0.5 ~ 2.5g/L, MgSO
47H
2o0.5 ~ 6.0g/L, solvent is water, pH6.0 ~ 7.5;
3) fermentation culture: seed liquor is transferred in fermention medium with the inoculum size of volumetric concentration 6 ~ 10%, 25 ~ 32 DEG C, 150 ~ 250rpm cultivates 24 ~ 48h, obtain fermented liquid, fermented liquid is centrifugal, and precipitation phosphoric acid buffer washs, obtain wet thallus, be enzyme source; The same seed culture medium of fermentative medium formula.
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