CN101134943A - Bacillus alcaligenes and method for preparing single enantiomer amygdalic acid - Google Patents

Abstract

The present invention discloses one kind of Bacillus alcaligenes and the process of preparing single antimer mandelic acid therewith. The resting cell of the Bacillus alcaligenes, ECU0401 CGMCC No.2009, is used as the biological catalyst in selectively converting racemized mandelic acid and its derivative to prepare optically active mandelic acid and its derivative. The Bacillus alcaligenes strain of the present invention has high catalyzing effect, and the product (R)-(-)-mandelic acid has optical purity over 99.9 %, obviously higher than that obtained with catalyzing yellow Bacillus brevis AS1.818 and with catalyzing Saccharomyces cervisiae AS2.150.

Description

A kind of Alcaligenes and prepare the method for single enantiomer amygdalic acid

Technical field

The present invention relates to strain Alcaligenes and uses thereof, especially prepare the method for single enantiomer amygdalic acid.

Technical background

Amygdalic acid (Alpha-hydroxy toluylic acid) is a kind of important chiral intermediate.Than the raceme of amygdalic acid, the use value of its single enantiomer (dextrorotation or left-handed amygdalic acid) is higher.Therefore, the single enantiomer that how can simple and effective ground obtains amygdalic acid becomes the focus of research.

(R)-(-)-amygdalic acid and derivative thereof are the chiral synthons of the important synthetic various medicines of a class, be widely used in the synthetic of multiple medicine, for example, the important intermediate of semisynthetic antibiotics, cephalo, antitumor and antiaging agent, also can be used as the chirality chiral reagent, be used to split other chipal compounds.

(R)-(-)-chemical structure of amygdalic acid is:

Traditional chemical industry Split Method mainly is to utilize the Chiral Amine compounds, as Alpha-Methyl benzylamine, (-)-ephedrine and cinchonine etc., by forming the method for diastereomeric salt, obtains single enantiomer.Once reported that the enantiomorph that has many physico-chemical processes to can be used for amygdalic acid split, as chromatography, crystallization process, embrane method and supercritical extraction also can utilize certain to go back original reagent asymmetric synthesis (R)-(-)-amygdalic acid.

Biological process also is used to synthesize (R)-(-)-amygdalic acid.For example, utilize Alcaligenes faecalis ATCC8750 to transform racemic mandelonitrile; Utilize Streptococcus faecalis asymmetric reduction benzoyl formic acid; Utilize the derivative of Candida antarctica B resolution of racemic amygdalic acid; Utilize Pseudomonas putida ATCC12336; Utilize the glycerol dehydrogenase oxydasis racemic 1, the 2-benzoglycols; Utilize GLO-I and the asymmetric conversion alpha-carbonyl of GLO-I I phenylacetic aldehyde etc.

Yet aforesaid method is not suitable for commercially producing (R)-(-)-amygdalic acid, and this is because reducing catalyst or resolution reagent cost an arm and a leg, and physico-chemical process demands strict technology, and needs the synthetic of derivative in the biochemical method or need the NADH circulation.

On the other hand, racemic amygdalic acid can be produced by lower cost on a large scale.Therefore, if (R)-(-)-amygdalic acid can simply and effectively be isolated, then be expected to realize the scale operation of (R)-(-)-amygdalic acid from racemic amygdalic acid.

Can utilize in the asymmetric degraded racemize of the amygdalic acid desaturase amygdalic acid method of (S)-enantiomorph to prepare and obtain (R)-(-)-amygdalic acid.The theoretical yield of producing amygdalic acid in this way is 50%.This desaturase energy specificity ground degraded (S)-(+)-amygdalic acid, thus (R)-(-)-amygdalic acid is retained in the reaction system.More existing bibliographical informations utilize desaturase selectivity degraded (S)-(+)-amygdalic acid and obtain (R)-(-)-amygdalic acid.For example, and people such as Miyamoto (Biotechnol.Lett., 1992,14:1137-1142) utilize Alcaligenes bronchisepticus resolution of racemic amygdalic acid, productive rate is 42%, e.e. is 97%.People such as Jamaluddin (J.Bacterial., 1970,101:786-793) utilize the Aspergillus niger desaturase racemic amygdalic acid of degrading to obtain optically pure (R)-(-)-amygdalic acid, desaturase relies on NAD and NADP.(J.Ferment.Bioeng. 19953:247-250) utilizes Pseudomonas polycolor to transform 300mM racemize amygdalic acid to people such as Takahashi, obtains (R)-(-)-amygdalic acid, and productive rate is 35%, and e.e. is 99.9%.Patent report (CN1840671,2006) strain screening obtains the asymmetric conversion racemize of brevibacterium flavum (Brevibacterium flavum) AS1.818 amygdalic acid and obtains product (R)-(-)-amygdalic acid, and e.e. is 90～96%.

Summary of the invention

The technical issues that need to address of the present invention are the methods that disclose a kind of Alcaligenes and prepare the single enantiomer amygdalic acid, to overcome the above-mentioned defective that prior art exists.

The said Alcaligenes of the present invention (Alcaligenes sp.) ECU0401 is a kind of bacterial classification that belongs to Alkaligenes, be the soil of contriver in the East China University of Science campus, obtain through primary dcreening operation, multiple sieve and separation and purification, this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on 04 13rd, 2007, address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica, preserving number is CGMCC No.2009;

Screening method comprises the steps:

Gathered the following 200 parts of pedotheques of varying environment condition, utilized amygdalic acid to carry out two-wheeled liquid enrichment culture for sole carbon source, screening (S)-(+)-amygdalic acid desaturase produces bacterium.By repeated screening, (S)-(+)-amygdalic acid desaturase that separates the plant height vigor that obtains produces bacterium.

Bacterial classification of the present invention has following microbial characteristic:

1. morphological specificity:

(1) form of cell and size: sphere, 0.5～1.2 * 0.5～2.6 μ m;

(2) throw the formation of sporozoite: do not have;

2. the growth conditions on various substratum:

(1) the beef extract agar plate is cultivated (30 ℃, 36 hours):

Colony shape: circle;

Bacterium colony surface elevation: level and smooth, glossy, bacterium colony thickness;

Size: 2～4mm;

Tone: white;

(2) beef extract gelatin stab culture (20 ℃, 6 days):

Liquefy gelatin not;

3. physiological and biochemical property:

(1) nitrate utilization (28 ℃, 5 days): can utilize;

(2) the outer kind of starch compound of born of the same parents produces: do not produce;

(3) hydrolysis of urea: the positive;

(4) methyl red test: feminine gender;

(5) to the demand of oxygen: good gas;

(6) height oozes the substratum tolerance: can tolerate the height that contains 20%NaCl and ooze substratum;

(7) carbon assimilation:

Positive: glucose, sucrose, maltose, glycerine, starch and inositol;

Negative: lactose;

(8) carbon source through fermentation: nonfermented carbon source;

(9) growth pH scope: 5.0～8.0;

(10) growth optimal temperature: 20～37 ℃.

Show that according to " the outstanding Bacteria Identification handbook of uncle " authentication method that provides and above-mentioned test-results this bacterial classification belongs to Alcaligenes (Alcaligenes sp.).But having significantly differently with the Alcaligenes of former report, its main difference part is: catalysis racemize amygdalic acid and derivative thereof (o-Chloromelic acid, a chloro mandelic acid, parachloromandelic acid and parahydroxymandelic acid) have higher productive rate and good selectivity (e.e.〉99.9%).

Identify through 16S rDNA, confirm that this bacterial strain is Alcaligenes (Alcaligenes sp.), is numbered Alcaligenes sp.ECU0401

Alcaligenes of the present invention (Alcaligenes sp.) ECU0401 can be used to produce single enantiomer amygdalic acid or derivatives thereof, and production method comprises the steps:

(1) cultivation of bacterial strain:

With the method for said Alcaligenes Alcaligenes sp.ECU0401 employing this area routine, in fermention medium, carry out amplification cultivation 24～36h, 20～50 ℃ of temperature;

Again with this nutrient solution as seed, be 1～10% (v/v) based on the inoculum size of fermention medium volume, be seeded in the 200mL fermention medium, cultivate 24～48h, the centrifugal resting cell that obtains on the shaking table of 100～160rpm down at 20～50 ℃;

Said fermention medium can adopt conventional substratum, and its component and content are as follows:

Glycerine 10～50g, peptone 1～20g, yeast extract paste 1～20g, KH ₂PO ₄1～10g, NaCl0.1～2g, MgSO ₄0.1～2g, FeSO ₄7H ₂O0.01～0.05g, water 1000mL, pH3～8,

(2) with the resting cell of gathering in the crops, be suspended in pH and be in 6.0～8.0 the buffer solution of potassium phosphate, add racemic amygdalic acid or derivatives thereof, concentration is 25～100mM, the constant temperature shaking table oscillatory reaction of 25～40 ℃ and 100～160rpm 2～14 hours, from reaction product, collect the pure product of (R)-(-)-amygdalic acid then: white powder, productive rate 41.5%, e.e.〉99.9%, specific rotatory power [α] _D ²⁵=-150.0 (c1.0, H ₂O);

The content of resting cell in buffer solution of potassium phosphate is 0.01～0.1g resting cell/mL;

The preferred o-Chloromelic acid of said racemize mandelic acid derivatives, a chloro mandelic acid, parachloromandelic acid or parahydroxymandelic acid.

Analytical procedure

Amygdalic acid, benzoyl formic acid and benzoic content C ₁₈Carry out HPLC and analyze, moving phase is 10mM KH ₂PO ₄The aqueous solution: (13: 87, v/v), flow velocity was 0.8ml/min to methyl alcohol, detects wavelength 215nm.Peak sequence is followed successively by: amygdalic acid (8.2min), benzoyl formic acid (13.5min) and phenylformic acid (43.1min).

(e.e. is %) with chirality liquid-phase chromatographic column Chiralcel OD-H (250 *  4.6mm, Daicel for enantiomeric excess value, Japan) analyze, moving phase is normal hexane/Virahol/trifluoroacetic acid (volume ratio 90: 10: 0.2), and flow velocity is 0.8mL/min, detects wavelength 228nm.Peak sequence is followed successively by (S)-(+)-amygdalic acid (11.2min) and (R)-(-)-amygdalic acid (12.2min).

Enantiomeric excess value $e.e.=\frac{\[R-\mathrm{MA}\]-\[S-\mathrm{MA}\]}{\[R-\mathrm{MA}\]+\[S-\mathrm{MA}\]}\×100\%$

(R)-(-)-amygdaline yield $Y=\frac{\[R-\mathrm{MA}\]}{{\[\mathrm{RS}-\mathrm{MA}\]}_0}\×100\%$

[R-MA] is the concentration of (R)-(-)-amygdalic acid in the formula, and [S-MA] is the concentration of (S)-(+)-amygdalic acid, [RS-MA] ₀Starting point concentration for the racemize amygdalic acid.

By above-mentioned disclosed technical scheme as seen, bacterial strain Alcaligenes of the present invention (Alcaligenes sp.) ECU0401, has catalytic effect preferably, transform the racemize amygdalic acid with this bacterial strain and obtain product (R)-(-)-amygdalic acid, its optical purity reaches e.e.〉99.9%, optical purity obviously is better than forefathers (CN1840671,2006) report utilizes the brevibacterium flavum AS1.818 catalysis resulting product of racemize amygdalic acid (R)-(-)-amygdalic acid (90～96%e.e.) and (CN1840670,2006) report utilize the yeast saccharomyces cerevisiae AS2.150 catalysis resulting product of racemize amygdalic acid (R)-(-)-amygdalic acid (〉 90%e.e.).

Embodiment

Embodiment 1 microorganism culturing

To from soil, separate the also Alcaligenes Alcaligenes sp.ECU0401 (preserving number: CGMCC No.2009) be seeded to 50mL fermention medium (glycerine 10g, peptone 15g, yeast extract paste 8g, KH of preservation ₂PO ₄4g, NaCl2g, MgSO ₄0.2g, FeSO ₄7H ₂O0.05g, tap water 1000mL, pH7.0) in the pre-15h that cultivates, as seed, the inoculum size with 10% is seeded in the 200mL fermention medium with this nutrient solution, cultivates 48h on 30 ℃ of following 160rpm shaking tables.

Embodiment 2 utilizes resting cell preparation (R)-(-)-amygdalic acid

With results Alcaligenes Alcaligenes sp.ECU0401 cell with 12, the centrifugal 15min of 000 * g obtains about weight in wet base 100g resting cell, with cell suspension in the 1000mL buffer solution of potassium phosphate, add the racemic amygdalic acid of 3.8g, final concentration is 25mM, in the constant temperature shaking table oscillatory reaction of 30 ℃ and 160rpm after 14 hours, with reaction solution with 12, the centrifugal 15min of 000 * g removes cell.In supernatant liquor, add 2M H ₂SO ₄Being acidified to pH is 1.0～2.0, adds salt to saturated, uses the equal-volume ethyl acetate extraction, triplicate, combining extraction liquid adds anhydrous sodium sulfate drying and spends the night, and rotary evaporation is removed organic solvent, obtain crystal crude product, by obtaining the pure product of (R)-(-)-amygdalic acid behind silica gel column chromatography (benzene: ethyl acetate: formic acid, volume ratio 10: 2: the 1) purifying: white powder, productive rate 41.5%, e.e.〉99.9%, specific rotatory power [α] $\stackrel{25}{D}=-150.0(c1.0,H_{2}O).$

Embodiment 3 utilizes grown cell catalysis to synthesize (R)-(-)-amygdalic acid

At 200mL substratum (peptone 15g, yeast extract paste 8g, KH ₂PO ₄₄G, NaCl2g, MgSO ₄0.2g, FeSO ₄7H ₂O0.05g, tap water 1000mL adds 2g racemize amygdalic acid in pH7.0), the constant temperature shaking table shaking culture Alcaligenes Alcaligenes of 30 ℃ and 160rpm sp.ECU040148 hour, fermented liquid with the centrifugal 15min of 12,000 * g, is removed cell.In supernatant liquor, add 2M H ₂SO ₄Being acidified to pH is 1.0～2.0, add salt to saturated, use the equal-volume ethyl acetate extraction, triplicate, combining extraction liquid, add anhydrous sodium sulfate drying and spend the night, rotary evaporation is removed organic solvent, obtains crystal crude product, by silica gel column chromatography (benzene: ethyl acetate: formic acid, volume ratio 10: 2: 1) obtain the pure product of (R)-(-)-amygdalic acid behind the purifying: white powder, productive rate 40.5%, e.e.〉99.9%.

Synthesizing of embodiment 4～8 optical pure mandel derivatives

Take by weighing the Alcaligenes sp.ECU0401 resting cell of the weight in wet base 10g of embodiment 1, below a series of racemize amygdalic acids and the derivative thereof in the tabulation 1 is substrate, concentration of substrate is 25mM, reaction volume 100mL, the constant temperature shaking table oscillatory reaction of 30 ℃ and 160rpm 24～48 hours, sampling analysis, these 5 kinds of racemic amygdalic acids and derivative thereof have generated optically active (R)-(-)-amygdalic acid and derivative (R)-(-)-o-Chloromelic acid thereof after transforming as a result, (S)-(+)-chloro mandelic acid, (S)-(+)-parachloromandelic acid and (R)-(-)-parahydroxymandelic acid, optical purity all is better than 99.9%e.e., does not promptly detect enantiomeric impurity under the analysis condition that is adopted.

Table 1 utilizes asymmetric degraded amygdalic acid of Alcaligenessp.ECU0401 resting cell and derivative thereof

Claims (6)

1. an Alcaligenes (Alcaligenes sp.) ECU0401 CGMCC No.2009.

2. adopt the described Alcaligenes of claim 1 (Alcaligenes sp.) ECU0401 CGMCCNo.2009 to produce the method for single enantiomer amygdalic acid or derivatives thereof, comprise the steps:

(1) said Alcaligenes Alcaligenes sp.ECU0401 is carried out amplification cultivation in fermention medium, the centrifugal resting cell that obtains;

(2) with the resting cell of results, be suspended in pH and be in 6.0～8.0 the buffer solution of potassium phosphate, add racemic amygdalic acid or derivatives thereof, 25～40 ℃ of reactions 2～14 hours, from reaction product, collect the pure product of (R)-(-)-amygdalic acid then.

3. method according to claim 2 is characterized in that, the concentration of racemic amygdalic acid or derivatives thereof is 25～100mM.

4. method according to claim 2 is characterized in that, the content of resting cell in buffer solution of potassium phosphate is 0.01～0.1g resting cell/mL.

5. method according to claim 2 is characterized in that, said fermention medium component and content are as follows: glycerine 10～50g, peptone 1～20g, yeast extract paste 1～20g, KH ₂PO ₄1～10g, NaCl0.1～2g, MgSO ₄0.1～2g, FeSO ₄7H ₂O 0.01～0.05g, water 1000mL, pH 3～8.

6. method according to claim 2 is characterized in that, said racemize mandelic acid derivatives is selected from o-Chloromelic acid, a chloro mandelic acid, parachloromandelic acid or parahydroxymandelic acid.