CN1212403C

CN1212403C - Method for preparing optical pure phenylethanediol by utilizing microbial stereoselectivity transformation and its special-purpose microbe

Info

Publication number: CN1212403C
Application number: CN 03132140
Authority: CN
Inventors: 徐岩; 王栋; 穆晓清; 聂尧
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2003-06-27
Filing date: 2003-06-27
Publication date: 2005-07-27
Anticipated expiration: 2023-06-27
Also published as: CN1477203A

Abstract

The utility model relates to a method for preparing optical pure phenylethanediol by utilizing microbial stereo-selectivity transformation and special microbe thereof, which belongs to the technical field for splitting recemic compounds by a biological method. The method sieves to obtain two strains of candida parapsilosis (CCTCC M203011) and bacillus polymyxa (CCTCC M203010). Racemic phenyl ethanediol is asymmetrically converted by the two strains to obtain (S)-phenyl ethanediol as a product of which the optical purity is high, and the optical purity is within 90 to 100%e. e on average. The component and the culture condition of a majorized nutrient medium are determined, so the optical purity of the product is also improved. A reaction system and a reaction condition of the racemic phenyl ethanediol which is asymmetrically converted by whole cells of the two stains are determined. The method is helpful to know the split biodiversity of the recemic compounds, which has important meanings for developing how to split special enzyme sources in the future and researching a split method.

Description

A kind of using microbe stereoselectivity transforms method and the special microorganism thereof for preparing the optical purity phenylglycol

Technical field

A kind of using microbe stereoselectivity transforms method and the special microorganism thereof for preparing the optical purity phenylglycol, belongs to biological process resolution of racemic compound technical.

Background technology

The optical purity phenylglycol, i.e. (S)-phenylglycol, can remember work (S)-PED again, be not only indispensable important chiral additives in the liquid crystal material, and become the preparation have the important intermediate of optically active medicine, agricultural chemicals and functional materials, the research of carrying out the phenylglycol method for splitting is extremely meaningful.

Chipal compounds has vital role in people's life, because two enantiomorphs are all different in each side such as pharmacology, toxicity and functions, therefore, prepare optically pure chirality module compound and all have value widely in fields such as medicine, agricultural, material and environmental protection.

The chemical structure of phenylglycol is:

Owing to contain two hydroxyls in the phenylglycol structure; utilizing chemical method to prepare the optical purity phenylglycol needs hydroxyl selective protection; easily generate other derivatives; it is bigger to split difficulty; chiral catalyst that is added in the reaction and protective material have toxicity; can work the mischief to environment, therefore utilize biological process to transform the focus that the optical purity phenylglycol just becomes research.

At present, resolution of racemic compound common methods mainly comprises in the world: chemical resolution method, chromatogram Split Method, the film Split Method that liquid membrane Split Method and chirality solid film split, and biological process.

The comparison of the several frequently seen method for splitting of table 1

Chromatography	The film Split Method	Chemical method	Biological process
Chromatography	The film Split Method	Chemical method	Biological process	Narrow, the many additive of separated sample variable range costs an arm and a leg, is subjected to the restriction of context of detection	Equipment requirements height, process be numerous and diverse, cost an arm and a leg, be difficult to be accepted by suitability for industrialized production	Yield is low, optical purity is low, production process is numerous and diverse, energy consumption is big, environmental pollution is serious, toxicity is big, cost is high	The catalytic efficiency height, the optical purity height, technology is easy, equipment is simple, save energy, environmental friendliness, with low cost

Utilize biological process to transform the optically pure chipal compounds of preparation and have the reaction conditions gentleness, product is single, and stereoselectivity, regioselectivity and chemo-selective are higher, and can finish some chemosynthesis and be difficult to the advantages such as reaction of carrying out.The bioconversion reaction of synthesis of optically active material is broadly divided into two classes: a class is racemic modification to be split as two have optically active enantiomorph; Another kind of is from racemize or prochiral precursor, obtains asymmetric optical activity product by catalyzed reaction.

Rise the nineties in the world microorganism and enzyme resolving chiral compound are carried out a large amount of research.Enzyme is made of L-amino acid, and its active centre has constituted an asymmetric environment, helps the identification to raceme, is a kind of catalyzer of height chirality.Its catalytic efficiency height has stronger specificity.The enzymatic resolving racemic is more satisfactory selection.Utilize intact cell that racemic compound is transformed, can obtain the optical purity enantiomorph, in nonaqueous phase and organic-water biphasic reaction system, but also Enzymatic transformation prepares optical pure compound.

It is reported that the method that present biological process prepares the optical purity phenylglycol mainly comprises:

Aldo etc. utilize lipase-catalyzed asymmetric transesterification resolution of racemic phenylglycol, and the esterification after product is formed more complicated, after basic hydrolysis and enzymic hydrolysis, obtains product (S)-PED, can only reach 50% but productive rate is the highest;

Andreas etc. utilize the reduction of glycerol dehydrogenase (GDH) catalysis asymmetric oxidation, but reaction needs to add coenzyme (NAD in system ⁺) and the reprocessing cycle system.

Kyoung etc. utilize two oxydase (NDO) the selective oxidation vinylbenzene of naphthalene, but the products obtained therefrom optical purity is lower, only is 78.6%e.e..

Summary of the invention

(1) technical problem that will solve

The purpose of this invention is to provide a kind of microorganism stereoselectivity and transform method and the special microorganism thereof for preparing the optical purity phenylglycol.The object of the invention not only is to prepare the phenylglycol of high-optical-purity, but seeks the novel bacterial of research different from the past by screening, thereby investigates the diversity of microbial strains aspect chiral separation.And then the difference of comparison different strain on path for transformation and reaction mechanism.

(2) technical scheme

The present invention is at first from microorganism, and screening can efficiently transform the bacterial classification of racemize phenylglycol.On this basis, the reaction conditions of this culture of strains condition and asymmetric conversion is optimized, and investigates the zymologic property of the conversion in this bacterial classification source with thick enzyme, the reaction path of asymmetric conversion and mechanism.

The approach that designs this method for transformation is as follows:

Phenylglycol abbreviates PED as

One, racemize PED transforms the screening with bacterial strain

Substratum

Microbial culture based component (g/100ml): glucose 0.5～2.5, meat extract 0.5～2.0, peptone 0.5～2.0, yeast extract paste 0.15～0.5, inorganic salt solution 3.5～7.0% (V/V), pH6.0～7.5.

Yeast culture based component (g/100ml): glucose 3.5～5.0, yeast extract paste 0.15～0.5, inorganic salt solution 7.5～10% (V/V), pH6.0～7.5.

Mould medium composition (g/100ml): glucose 0.5～2.5, meat extract 0.5～2.0, peptone 0.5～2.0, yeast extract paste 0.15～0.5, inorganic salt solution 3.5～7.0% (V/V), pH6.0～7.5.

Inorganic salt solution composition (g/100ml): (NH ₄) ₂HPO ₄10～15, KH ₂PO ₄5～10, MgSO ₄7H ₂O0.5～1.8, NaCl 0.025～0.1, ZnSO ₄7H ₂O 0.025～0.1, FeSO ₄7H ₂O 0.025～0.1, CuSO ₄5H ₂O 0.0025～0.01, MnSO ₄4H ₂O 0.025～0.1.

Main agents

The racemize phenylglycol is purchased in Beijing chemical reagents corporation

(R)-PED, (S)-PED, (R, S)-PED purchases the Sigma-Aldrich company in the U.S.

Culture of strains

Bacterial classification is that 10% 50ml shakes in the bottle in 30 ℃, 150rpm shaking culture 48 hours at liquid amount.After cultivate finishing, thalline is centrifugal and use the physiological saline washed twice, and collecting cell is used for resolution reaction.

The catalytic conversion reaction of microorganism cells

In the buffer solution of potassium phosphate of 2ml, 0.1mol/L (pH6.5), add 0.2g somatic cells and 10mg racemize phenylglycol, oscillatory reaction is 48 hours on 30 ℃ constant temperature shaking table.After the reaction, mixture is centrifugal, gets supernatant liquor 5ml ethyl acetate extraction, and the ethyl acetate layer that obtains is used for analyzing.

Above-mentioned reaction is carried out mixture after 12 hours through centrifugal and ethyl acetate extraction, and ethyl acetate layer is used for the analytical reaction intermediate.

(R)-the determining of PED and (S)-PED analytical procedure

(R)-PED, (S)-PED, (R, S)-the PED standard specimen analyzes by chiral stationary phase high performance liquid chromatography (CSPHPLC) on Chiralcel OB-H post, (R)-retention time of PED is 13.9min, (S)-retention time of PED is 16.9min.Used high performance liquid chromatograph is HPl 100, UV-detector, and (250 * 4.6mm) purchase in Japanese Daicel Chemical Ind., Ltd. chiral column Chiralcel OB-H

Determine that concrete chromatographic condition is: chiral column Chiralcel OB-H (250 * 4.6mm); Moving phase normal hexane/Virahol=9/1; Flow velocity 0.5ml/min; 30 ℃ of column temperatures; Post is pressed normal pressure; Ultraviolet detection wavelength 215nm; Sample size 5 μ l.

The optical purity of product is estimated by mapping excessive value (%e.e.).

(S)-PED％e.e.＝[(S _S-S _R)/(S _S+S _R)]×100％

Remaining phenylglycol Residual (%)=[(S _S+ S _R)/S ₀] * 100%

Productive rate yield (%)=Residual * [1-(100-%e.e.)/200].

S _S: the peak area of reaction back (S)-enantiomorph; S _R: the peak area of reaction back (R)-enantiomorph;

S ₀: the peak area sum of (S)-and (R)-enantiomorph before the reaction.

The analysis of reaction intermediate

Utilize gas chromatography mass spectrometer analytical reaction intermediate.12 hours sample is answered in negate, carries out GC-MS and analyzes, and determines the chemical structure of reaction intermediate.Used gas chromatography mass spectrometer Trace MS U.S. Finigon mass spectrum company produces.

The GC-MS chromatographic condition is: chromatographic column ov17 (30m * 0.25mm * 0.25 μ m); 60 ℃ of starting temperatures; 240 ℃ of final temperatures; Temperature programming speed 10deg/min; Residence time 15min; Carrier gas He; Threshold speed 0.80ml/min.

Screen through a large amount of microorganisms such as bacterium, yeast and mould that comprise, consider that from optical purity and two aspects of productive rate of gained converted product selected two strain bacterial classifications are the catalytic conversion reaction starting strain the laboratory preservation.

Candida parapsilosis CCTCC M203011, product (S)-PED, optical purity is 90～100%e.e., productive rate is 85～100%.Bacillus polymyxa CCTCC M203010, product (S)-PED, optical purity is 90～100%e.e., productive rate is 45～50%.

Two, the cultivation of bacterial strain and condition of enzyme production optimization

Medium component is optimized

Investigated carbon source, nitrogenous source, phosphorus source respectively to transforming and the influence of biomass, and finally determined two strain starting strains through orthogonal experiment:

1, Candida parapsilosis C.parapsilosis CCTCC M203011 substratum consists of (g/100ml): glucose 1～10, yeast extract paste 0.1～3, (NH ₄) ₂HPO ₄1.0～3.0, KH ₂PO ₄0.1～1.5, MgSO ₄7H ₂O0.01～0.25, NaCl 0.001～0.025, ZnSO ₄7H ₂O0.001～0.025, FeSO ₄7H ₂O 0.001～0.025, CuSO ₄5H ₂O 0.0001～0.025, MnSO ₄4H ₂O 0.0001～0.025;

Culture condition is optimized

Initial pH, liquid amount, temperature, incubation time have been investigated respectively to transforming and the influence of biomass.

Determined that finally starting strain C.parapsilosis CCTCC M203011 culture condition is: initial pH5.0～8.0, liquid amount 5～30%, 20～35 ℃ of culture temperature, incubation time 24～72 hours.

2 or select for use starting strain bacillus polymyxa B.polymyxa CCTCC M203010 substratum to consist of (g/100ml): glucose 0.25～1, meat extract 0.5～5, peptone 0.5～5, yeast extract paste 0.1～1, (NH ₄) ₂HPO ₄0.5～5, KH ₂PO ₄0.15～0.5, MgSO ₄7H ₂O 0.01～0.25, and NaCl 0.005～0.05, ZnSO ₄7H ₂O0.001～0.05, FeSO ₄7H ₂O 0.001～0.05, CuSO ₄5H ₂O 0.0001～0.005, MnSO ₄4H ₂O0.0005～0.005;

Culture condition is: initial pH6.5～8.0, liquid amount 10～30%, 25～35 ℃ of culture temperature, incubation time 24～72 hours;

After the optimization, the optical purity of C.parapsilosis CCTCC M203011 converted product (S)-PED is 94～100%e.e.; The optical purity of B.polymyxa CCTCC M203010 converted product (S)-PED is 94～100%e.e..

Three, the optimization of whole-cell catalytic conversion reaction system and reaction conditions

The preparation of culture of strains and full cell

It is that 5～30% 250ml shakes in the bottle in 20～35 ℃, 100～300rpm shaking culture 24～72 hours that C.parapsilosis CCTCC M203011 is seeded in liquid amount.It is that 10～30% 250ml shakes in the bottle in 25～35 ℃, 100～300rpm shaking culture 24～72 hours that B.polymyxa CCTCC M203010 is seeded in liquid amount.After cultivate finishing, that the thalline of two bacterial strains is centrifugal respectively and use physiological saline washed twice, the cell of collection to obtain full cell through lyophilize to be used for resolution reaction.

The catalytic conversion reaction of microorganism cells

C.parapsilosis CCTCC M203011 whole-cell catalytic conversion reaction conditions is: cell concn 1～15% (W/V), concentration of substrate 0.05～10% (W/V), pH value in reaction 5.0～8.0,20～40 ℃ of temperature of reaction, 24～72 hours reaction times.

B.polymyxa CCTCC M203010 whole-cell catalytic conversion reaction conditions is: cell concn 1～10% (W/V), concentration of substrate 0.05～10% (W/V), pH value in reaction 5.0～8.0,25～40 ℃ of temperature of reaction, 24～72 hours reaction times.

Analyze by 12 hours reaction mixture of microorganism cells catalyzed conversion being carried out GC-MS, find except that phenylglycol (retention time 9.85min), also have another material to exist and accumulation at 9.00min.By above-mentioned substance is carried out mass spectroscopy, find that its molion is m/z 136.1, can determine that according to the structure of its fragment this material is the beta-hydroxyphenyl ethyl ketone.Therefore, the reaction intermediate of determining asymmetric conversion racemize phenylglycol is the beta-hydroxyphenyl ethyl ketone.

Investigate the conversion reaction process, find that C.parapsilosis CCTCC M203011 is converted into intermediate beta-hydroxyphenyl ethyl ketone with (R)-phenylglycol earlier, and then be (S)-phenylglycol beta-hydroxyphenyl ethyl ketone asymmetric reduction.Therefore, the content of reaction back (S)-phenylglycol is higher than the content of (S)-enantiomorph in the preceding racemic mixture of reaction, and the productive rate of product is higher than 50%.And B.polymyxa CCTCC M203010 is no longer to be reduced to (S)-phenylglycol behind the intermediate beta-hydroxyphenyl ethyl ketone with (R)-phenylglycol oxidation conversion.Therefore, the content of reaction back (S)-phenylglycol keeps constant substantially, and productive rate is lower than 50%.

By the thick enzyme zymologic property of three-dimensional selective oxidation reductase enzyme is studied, think:

The reaction mechanism that the oxydo-reductase that derives from C.parapsilosis CCTCC M203011 will (R)-phenylglycol is asymmetric be converted into (S)-phenylglycol is: at NADP ⁺Existence under, is the beta-hydroxyphenyl ethyl ketone earlier with (R)-phenylglycol selective oxidation, while NADP ⁺Be reduced to NADPH; In the presence of NADH, be (S)-phenylglycol with intermediate beta-hydroxyphenyl ethyl ketone asymmetric reduction then, NADH is oxidized to NAD simultaneously ⁺

The reaction mechanism that derives from the asymmetric conversion of oxydo-reductase (R)-phenylglycol of B.polymyxa CCTCC M203010 is: at NAD ⁺Existence under, be the beta-hydroxyphenyl ethyl ketone only with (R)-phenylglycol selective oxidation, the while coenzyme NAD ⁺Be reduced to NADH.

The thick enzyme of C.parapsilosis CCTCC M203011 oxydo-reductase is stronger to the primary alconol specificity of 5C.In the chiral alcohol that comprises secondary alcohol and dibasic alcohol, stronger for the specificity of dibasic alcohol.This enzymatic oxidation reaction pH value is pH8.0, and temperature is 50 ℃.Metal chelating and heavy metal ion can produce restraining effect to the vigor of this enzyme.

During the thick enzyme catalysis reduction reaction of C.parapsilosis CCTCC M203011 oxydo-reductase, stronger to the 2-butanone specificity.The optimum pH of this enzyme catalysis reduction reaction is pH6.0, and optimum temperuture is 40 ℃.

Candida parapsilosis (Candida parapsilosis CCTCC M203011): colonial morphology is an oyster white, smooth surface, and glossy, bacterium colony is opaque.Thalli morphology is an oval, the cell vegetative propagation, and budding has pseudohypha and septate hypha sometimes.Physio-biochemical characteristics are glucose fermentation, can utilize the D-semi-lactosi, D-wood sugar, L-arabinose, sucrose, maltose, trehalose, methyl-a-D-glycopyranoside, melizitose, glycerine, D-sorbyl alcohol, D-N.F,USP MANNITOL, D-glyconic acid-1,5-lactone, 2-ketone-D-glyconic acid, succsinic acid, citric acid, ethanol is as sole carbon source, can utilize ethamine, L-Methionin, cadaverine is as only nitrogen source.Starch forms test: feminine gender, acetic acid produces test: feminine gender, hydrolysis of urea test: feminine gender, the blue B reaction of diazo: feminine gender.30 ℃ of optimum growth temperatures, the suitableeest initiation pH6.5.33 ℃ of the optimum temperutures of the full cell transformation almond of this bacterium alcohol, the suitableeest action pH 6.5.

At NADP ⁺Existence under, is the beta-hydroxyphenyl ethyl ketone earlier with (R)-phenylglycol selective oxidation, while NADP ⁺Be reduced to NADPH; In the presence of NADH, be (S)-phenylglycol with intermediate beta-hydroxyphenyl ethyl ketone asymmetric reduction then, NADH is oxidized to NAD simultaneously ⁺

Bacillus polymyxa (Bacillus polymyxa CCTCC M203010):

Colonial morphology is irregular form, and the edge is wavy, and smooth surface is glossy, and bacterium colony is opaque, the no wrinkle in surface, grey.Thalli morphology is a rod-short, and it is random to distribute, and terminal spore is arranged, the gemma ellipse, and sporangium has the phenomenon of expanding, Gram-variable.Physio-biochemical characteristics are the catalase positive, and glucose fermentation produces sour aerogenesis.33 ℃ of optimum growth temperatures, the suitableeest initiation pH7.0.33 ℃ of the optimum temperutures of the full cell transformation almond of this bacterium alcohol, the suitableeest action pH 7.0.In the presence of NAD+, be the beta-hydroxyphenyl ethyl ketone with (R)-phenylglycol selective oxidation, coenzyme NAD+be reduced to NADH simultaneously.

(3) beneficial effect: the present invention obtains two strain changing effects bacterial strain preferably by screening, and they are Candidaparapsilosis CCTCC M203011 and Bacillus polymyxa CCTCC M203010.

Carry out asymmetric conversion racemize phenylglycol with this two strains bacterial strain and obtain product (S)-phenylglycol, optical purity is 90-100%e.e..

After substratum composition and culture condition optimization, the asymmetric conversion racemize of bacterial strain uses therefor phenylglycol, the optical purity of product (S)-phenylglycol also improves to some extent, has determined substratum composition and the culture condition optimized.

Determine the reaction system and the reaction conditions of the asymmetric conversion racemize of this two strains strain whole-cell phenylglycol.

Enzyme to the thick enzyme of oxydo-reductase of this two strains bacterial strain is that character is studied; Study the reaction path and the coenzyme dependency of its asymmetric conversion, inquired into conversion reaction mechanism.These work help to understand the species diversity that racemic compound splits, and have the meaning of outbalance for the research of exploitation that will split specific enzyme source from now on and method for splitting.

The biological material specimens preservation

Candida parapsilosis: preservation date on March 1st, 2003, depositary institution's title and abbreviation: Chinese typical culture collection center C CTCC, deposit number: NO.M203011.

Bacillus polymyxa: preservation date on March 1st, 2003, depositary institution's title and abbreviation: Chinese typical culture collection center C CTCC, deposit number: NO.M203010.

Embodiment

Embodiment 1

Adopt Candida parapsilosis C.parapsilosis CCTCC M203011 whole-cell catalytic to transform:

In the buffer solution of potassium phosphate of 2ml, 0.1mol/L (pH6.5), add 0.1g somatic cells and 16mg racemize phenylglycol, oscillatory reaction is 48 hours on 33 ℃ constant temperature shaking table, and after the reaction, mixture is centrifugal.Product (S)-PED optical purity 98.80%e.e., productive rate 92.86%.

Embodiment 2

Adopt bacillus polymyxa B.polymyxa CCTCC M203010 whole-cell catalytic to transform:

In the buffer solution of potassium phosphate of 2ml, 0.1mol/L (pH7.0), add 0.16g somatic cells and 10mg racemize phenylglycol, oscillatory reaction is 60 hours on 33 ℃ constant temperature shaking table, and after the reaction, mixture is centrifugal.Product (S)-PED optical purity is increased to 95.61%e.e., and productive rate is increased to 47.98%.

Claims

1. A method for preparing optically pure phenylethylene glycol by a biological method, characterized in that the starting bacterial strain adopts Bacillus polymyxa CCTCC NO.M203010, after the cultivation of the bacterial strain and the optimization of the enzyme production conditions, the cultivation of the bacterial classification And the preparation of whole cells, the catalytic conversion reaction of microbial cells is carried out with racemic phenylethylene glycol as a substrate: the mass volume concentration of cells is 1-10%, the mass volume concentration of substrates is 0.05-10%, and the reaction pH value is 5.0-8.0 , the reaction temperature is 25-40°C, and the reaction time is 24-72 hours.

2. The method according to claim 1, characterized in that the cultivation of bacterial strains and the optimization of enzyme production conditions

The medium composition is calculated in g/100ml: glucose 0.25~1, meat extract 0.5~5, peptone 0.5~5, yeast extract 0.1~1, (NH ₄ ) ₂ HPO ₄ 0.5~5, KH ₂ PO ₄ 0.15~0.5 , MgSO ₄ 7H ₂ O 0.01～0.25, NaCl 0.005～0.05, ZnSO ₄ 7H ₂ O 0.001～0.05, FeSO ₄ 7H ₂ O 0.001～0.05, CuSO ₄ 5H ₂ O 0.0001～0.005, MnSO ₄ 4H ₂ O 0.0005～0.005;

The culture conditions are as follows: the initial pH is 6.5-8.0, the filling volume is 10-30%, the culture temperature is 25-35 DEG C, and the culture time is 24-72 hours.

3. The method according to claim 1, characterized in that the culture of strains and the preparation of whole cells

B.polymyxa CCTCC M203010 was inoculated in a 250ml shaker flask with a liquid volume of 10-30% at 25-35°C and 100-300rpm shaking culture for 24-72 hours; after the culture, the bacteria of the strain were centrifuged and washed with normal saline Once, the collected cells were freeze-dried to obtain whole cells for splitting reactions.

4. The method according to claim 1, characterized in that the optical purity of (S)-phenylethylene glycol obtained by asymmetric transformation of whole cells of B. polymyxa CCTCC M203010 strain is increased to 90-100% e.e., the yield increased to 45-50%.

5. A strain used for preparing optically pure phenylethylene glycol according to claim 1, which is Bacillus polymyxa SYB-2, and its preservation number is CCTCC, NO.M203010.