CN101613666B - Bacillus and application thereof - Google Patents

Bacillus and application thereof Download PDF

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CN101613666B
CN101613666B CN2009100543581A CN200910054358A CN101613666B CN 101613666 B CN101613666 B CN 101613666B CN 2009100543581 A CN2009100543581 A CN 2009100543581A CN 200910054358 A CN200910054358 A CN 200910054358A CN 101613666 B CN101613666 B CN 101613666B
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benzoglycols
mandelic acid
bacillus
cyclic carbonate
carbonate ester
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CN101613666A (en
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许建和
常磊
潘江
徐毅
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East China University of Science and Technology
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Abstract

The invention discloses a Bacillus sp.ECU0015 with the conservation number as CGMCC No.2874. Resting cells and growing cells of the Bacillus strain are taken as bio-catalyst to catalyzes benzoglycols cyclic carbonate, acetyl mandelic acid and derivatives thereof to carry out unsymmetrical hydrolysis to prepare chiral compounds such as chiral benzoglycols, mandelic acid and the like which obtain optical activity. The Bacillus strain of the invention has high three-dimension selectivity, simple and safe bio-transformation technique, low cost and environment protection, and the product is easy to purify and has prospect in industrial application.

Description

One bacillus and purposes
Technical field
The invention belongs to biological chemical field, relate to a bacillus and be used to transform the method that compounds such as benzoglycols cyclic carbonate ester, acetyl mandelic acid obtain the optical homochiral compound.
Technical background
Chipal compounds is the staple product of present biocatalysis, is mainly used in fields such as pharmacy.Optical homochiral glycol and cyclic carbonate ester thereof have a wide range of applications as important chiral intermediate, and its synthetic preparation more and more comes into one's own.For example, (S)-benzoglycols can be used as the chiral additives of liquid crystal material, is used to shorten the time of response of liquid crystal panel; (R)-and p-nitrophenyl ethylene glycol and (S)-3-chloro-1, the 2-propylene glycol is the chiral intermediate of cardiovascular agent beta-Blocking agent; (R)-propylene carbonate and (1S, 2R)-the indane glycol is respectively reverse transcriptase inhibitors and proteinase inhibitor synthetic chiral intermediate.Many in recent years enterprises and research group all drop into the new synthesis technology that great enthusiasm is studied these chipal compounds.
Known chiral diol synthetic method has chemical method and biological process.Chemical method comprises: utilize the asymmetric bishydroxy of organo-metallic catalyst catalyzed alkene, the selective hydrolysis of epoxide or the selective reduction of chiral hydroxy acid etc.; Biological rule comprises the selective hydrolysis of the asymmetric bishydroxy of the catalytic alkene of dioxygenase, the catalytic epoxide enantioselective hydrolysis of epoxide hydrolase, the catalytic diol carboxylic acid ester of ester hydrolase and the selective reduction of enzymatic glycol selective oxidation of redox or corresponding ketone etc.What the chemosynthesis report of chirality cyclic carbonate ester was more is to adopt round-about way: first synthesis of chiral glycol or epoxide, further then synthesis of chiral cyclic carbonate ester; The report that the direct synthesis of chiral cyclic carbonate ester of asymmetric carbonylation reaction that utilizes epoxide is also arranged in recent years, but the optical purity of product lower (being up to 70%).
From material characteristic and source, the chemical method synthesizing cyclic carbonate ester can be made by carbonic acid gas and corresponding epoxide, and present domestic propylene carbonate etc. have realized successfully that large-tonnage is synthetic, and raw material sources are very abundant; Compare with other carboxylicesters, carbonic acid gas that the cyclic carbonate ester hydrolysis produces has avoided organic acid to the corrosion of equipment with to the pollution of environment.In conjunction with biocatalytic reaction gentleness, characteristics that selectivity is high, the catalytic cyclic carbonate ester stereo selective hydrolysis of cyclic carbonate ester lytic enzyme will be the green novel method of chirality cyclic carbonate ester and chiral diol synthetic.
At present, the stereo selective hydrolysis of cyclic carbonate ester lytic enzyme catalysis cyclic carbonate ester belongs to a newer research field in the world, and the work aspect a lot of all is in the infancy.Source from enzyme, kind is less, mostly be animal-origin, research contents relates to the purifying of enzyme, characterize and secondary structure, utilize research that its catalysis cyclic carbonate ester enantioselectivity splits seldom, the result neither very desirable (Yang Y.L., Ramaswamy S., and Jakoby W.B.Enzymatic hydrolysis of organic cyclic carbonates.J.Biol.Chem.1998,273:7814-7817), microbe-derived cyclic carbonate ester lytic enzyme only limits to indivedual reports of Japanese scholar, its research object limitation at one end is disubstituted cyclic carbonate ester (the Matsumoto K of methyl, Sato Y, Shimojo M and Hatanaka M.Highly enantioselective preparation of C 2-symmetricaldiols:microbial hydrolysis of cyclic carbonates.Tetrahedron:Asymmetry.2000 11:1965-1973), but also has very big development space aspect Screening of Bioflocculant-producing Bacteria.Aspect the research of substrate spectrum, focusing mostly at a long-chain fat family cyclic carbonate ester and an end is two substituted ring carbonic ethers of methyl, research to short-chain fat family and fragrant same clan cyclic carbonate ester is less, so screen new cyclic carbonate ester lytic enzyme, preparation optical purity benzoglycols has great importance.
Relate to another kind of chiral molecules chiral mandelic acid and derivative thereof among the present invention and possess the wide development prospect equally.Optical pure mandel is mainly used in medicine industry, can be used for the chiral intermediate of medicine such as synthetic Cyclelate, hexamine mandelate, hydrochloric acid Ao Xibuning.
The main method of preparation optical pure mandel and derivative thereof comprises that high pressure crystal method, chromatography, enzyme process split.Enzyme process splits the stereo selective hydrolysis that the racemize mandelonitrile is carried out in the combination that comprises use nitrilase or Nitrile hydratase and Ntn hydrolase; Or use the hydrolysis of lipase-catalyzed mandelate to split.Up to the present, the report that uses the hydrolysis of enzyme process catalysis acetyl mandelic acid to split is considerably less.The bacterial strain that the present invention screening obtains can enantioselective hydrolysis acetyl mandelic acid and derivative thereof, obtains optical pure mandel and derivative thereof, thereby has expanded the substrate spectrum, enlarged the application of biological catalyst.
Summary of the invention
The invention discloses the new isolating genus bacillus of a strain and be used to prepare the method for optical activity glycol, amygdalic acid and o-Chloromelic acid, to overcome the above-mentioned defective that prior art exists.
Genus bacillus of the present invention (Bacillus sp.ECU0015) is to belong to bacillus, be that the contriver is from pedotheque, the highly selective cyclic carbonate ester lytic enzyme that obtains through primary dcreening operation, multiple sieve and separation and purification produces bacterium, this bacterial strain was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on 02 06th, 2009, and preserving number is CGMCC No.2874.
Above-mentioned bacterial strain screens part soil surplus Shanghai, Hebei, Jiangsu, Shandong, Shanxi, Hubei etc. geographic 300 and obtains, and the concrete steps of screening are as follows:
Gather pedotheque from different environment, utilize the benzoglycols cyclic carbonate ester to be sole carbon source, carry out the two-wheeled enrichment culture, screening cyclic carbonate ester lytic enzyme produces bacterium.By repeated screening, separate the cyclic carbonate ester lytic enzyme that obtains a plant height vigor and highly selective and produce bacterium.
Bacterial classification of the present invention has following microbial characteristic:
1, form size
Shaft-like or oval, 0.7～1.0 μ m * 3～5 μ m;
2, suitable growth environment
Suitable growth temperature is 25～35 ℃, can survive in pH 5～9 environment;
3, the dull and stereotyped bacterium colony characteristic of cultivating
Cultivate 12h and can form little bacterium colony on 30 ℃ of flat boards, 36h forms drying, equatorial grey bacterium colony, and the edge is irregular, and is middle outstanding.
According to " uncle Jie Shi identification handbook " authentication method that provides and above-mentioned microbial characteristic, and through the 16SrDNA evaluation, confirm that this bacterial strain is bacillus (Bacillus sp.), label is Bacillus sp.ECU0015.
Genus bacillus strain Bacillus sp.ECU0015 of the present invention can be used to produce benzoglycols, amygdalic acid or the o-Chloromelic acid of single enantiomer, and the method for production comprises following step:
(1) preparation of bacterial classification
With genus bacillus strain Bacillus sp.ECU0015 (121 ℃ of the bacterium of going out, 20～40min) rich medium (for example: glycerine 1%, peptone 0.5%, extractum carnis 0.8%, agar 1.5%) line on the flat board, in 25～30 ℃ leave standstill cultivate about 1-2 days after picking list bacterium colony, carry out slant culture (culture condition is the same) as seed, be stored in 4 ℃ of refrigerators standby after about 2 days.
(2) cultivation of bacterial strain
With the method for said genus bacillus strain Bacillus sp.ECU0015 employing this area routine, in fermention medium, carry out amplification cultivation 24～48h, 20～50 ℃ of temperature;
Again with above-mentioned nutrient solution as seed, be 1～10% (v/v) based on the inoculum size of fermention medium volume, be seeded in the fermention medium of 50ml, cultivating 24～48h, the centrifugal resting cell that obtains on 100～180rpm shaking tables down at 20～50 ℃;
Said fermention medium can adopt conventional substratum, and wherein each components contents is as follows: glycerine 10～50g/L, extractum carnis 1～20g/L, peptone 1～20g/L, KH 2PO 41～10g/L, Na 2HPO 41～10g/L, NaCl 0.1～2g/L, MgSO 40.1～2g/L, pH 3～8.
(3) bio-transformation of substrate
With the resting cell of results, be suspended in pH and be in 6.0～8.0 the phosphate buffer solution, the content of resting cell is 10～100g (weight in wet base)/L; Add benzoglycols cyclic carbonate ester or other substrate, ultimate density is 5～100mM, oscillatory reaction 12～48h on the constant temperature shaking table of 25～40 ℃ and 100～160rpm, extract product (S)-(-)-benzoglycols, (R)-amygdalic acid or (R)-chloro mandelic acid then from reaction solution, optical purity of products is close to or higher than 98%.
By above-mentioned disclosed technical scheme as seen; adopt genus bacillus strain catalysis of the present invention to split benzoglycols cyclic carbonate ester and amygdalic acid acylate; not only has catalytic effect preferably; can synthesize the benzoglycols of high-optical-purity (being close to or higher than 98%) and amygdalic acid etc.; and catalyzer is easy to preparation, reaction conditions gentleness, substrate wide adaptability, has good industrial application DEVELOPMENT PROSPECT.
Embodiment
The following example will help further to understand the present invention, but can not limit content of the present invention.
The screening of embodiment 1 bacterial strain
Determine to screen microorganism from environment such as soil, at first prepare enrichment medium, substratum consists of: (NH 4) 2SO 41.0g/L, KH 2PO 43.0g/L, K 2HPO 46.0g/L, MgSO 40.5g/L, CaCl 20.05g/L; Other prepares rich medium, consists of: glucose (or glycerine) 15g/L, extractum carnis 5g/L, peptone 5g/L, KH 2PO 41.0g/L, Na 2HPO 41.0g/L, MgSO 40.5g/L pH 7.0.Take a morsel different sources soil sample be suspended in the enrichment medium of 2ml, (concentration is 0.2～1g/L) to be sole carbon source to add benzoglycols cyclic carbonate ester or diethyl carbonate, enrichment culture is 1～2 day under 25～40 ℃, 200rpm jolting condition, treat to be forwarded to fresh culture after microorganism growth gets up, continue to cultivate 1～2 day.The thin plate chromatography is carried out in sampling after the enrichment culture, and sxemiquantitative ground detects having or not or content of hydrolysate benzoglycols, to determine whether exist and can carry out microorganism transformed to carbonic ether in the soil sample.To the enrichment culture liquid of obvious substrate conversion is arranged, the dilution that takes a morsel is coated and was cultivated on the rich medium flat board 1～2 day, distinguish the picking form single bacterium colony different then with color, be inoculated in the 2ml rich medium, the methanol solution (final concentration of benzoglycols cyclic carbonate ester is 10mM) that adds the benzoglycols cyclic carbonate ester behind the cultivation 24h, after the bio-transformation 24h, the reaction solution ethyl acetate extraction, carry out the thin plate chromatography, detect the growing amount of benzoglycols.Preservation has the bacterial strain of obvious benzoglycols cyclic carbonate ester hydrolytic activity.
Obvious active inoculation is arranged in the 50ml rich medium with what preserve, behind the cultivation 36h, centrifugal collection thalline.The centrifugal thalline resuspending that gets off in the potassium phosphate buffer (50mM, pH 7.3) of 10ml, is added substrate benzoglycols cyclic carbonate ester (concentration is 10mM) and carries out bio-transformation.After transforming 24h, analyze with carrying out HPLC after the equal volume of ethyl acetate, use Chiracel OD-H chiral chromatographic column (the φ 0.46cm * 25cm) of Japanese Daicel company, moving phase is normal hexane: Virahol=95: 5 (v/v), the enantiomeric excess value (ee of flow velocity 1ml/min analytical calculation product p) and the transformation efficiency of substrate.In the candidate strain of strain more than 400, filtered out at last and can stereoselectivity transform the microorganism Bacillus sp.ECU0015 that the benzoglycols cyclic carbonate ester generates (S)-benzoglycols.
The cultivation of embodiment 2 microorganisms
Culture medium prescription: glycerine 15g/L, extractum carnis 8g/L, peptone 5g/L, KH 2PO 41.0g/L, Na 2HPO 41.0g/L, MgSO 40.5g/L pH 7.0.121 ℃ of high-temperature sterilization 20min.
Get the genus bacillus inclined-plane of 4 ℃ of preservations, picking one ring is seeded to shaking in the bottle of 250ml that the 50ml substratum is housed.Under 30 ℃, the 160rpm shaking table is cultivated 12h, is forwarded to shaking in the bottle of 500ml that the 100ml substratum is housed by the inoculum size of 5% (v/v), continues cultivation 36h, centrifugal cell harvesting in 30 ℃, 160rpm.The enzyme activity that records fermented liquid is about 5.08U/L, and the about 25g of cell concn (weight in wet base)/L, the enzyme of unit wet cell live and be the 0.20U/g wet cell.
Whole cell viability is defined as: at 30 ℃, pH 7.3, under the condition of concentration of substrate 10mM, per minute catalysis benzoglycols cyclic carbonate ester generates the needed enzyme amount of 1.0 μ mol benzoglycols and is defined as a unit of activity (U).
Embodiment 3 utilizes resting cell preparation (S)-(-)-benzoglycols
The centrifugal bacillus sp.ECU0015 resting cell 12g that obtains is suspended in 100ml phosphoric acid buffer (100mM, pH 7.3) in, add substrate benzoglycols cyclic carbonate ester, making its final concentration is 10mM, behind oscillatory reaction 24h on the constant temperature shaking table of 30 ℃ and 160rpm, reaction solution with the centrifugal 10min of 12,000 * g, is removed cell.In supernatant liquor, add NaCl to saturated, with the anhydrous diethyl ether extraction of 50ml, triplicate.The centrifugal cell that obtains soaks with the 20ml anhydrous diethyl ether, repeats twice, this two portions diethyl ether solution is merged, and with saturated NaCl solution washing twice, each 10ml.The ether extraction liquid that obtains spends the night with the anhydrous sodium sulfate drying of 10% (w/v), rotary evaporation is removed ether, obtain the crystal crude product of product, by silica gel column chromatography (sherwood oil: ethyl acetate, volume ratio is 4: 1) obtain (S)-benzoglycols behind the purifying, obtain white needle-like crystals after refining, productive rate 27.8%, ee are 99.8%.
Figure G2009100543581D00061
Embodiment 4～5 utilizes conversion of resting cells acetyl mandelic acid and m-chloro acetyl mandelic acid
Take by weighing 12g Bacillus sp.ECU0015 resting cell and be suspended in 100ml phosphoric acid buffer (100mM, pH 7.3) in, add acetyl mandelic acid or m-chloro acetyl mandelic acid, concentration of substrate is 10mM, at 30 ℃ of about 36h of constant temperature shaking table oscillatory reaction with 160rpm, intermittent sampling is measured the enantiomeric excess value of product, finishes reaction when the highest to the enantiomeric excess value of converted product.In reaction solution, add NaCl to saturated, divide three extractions with the equal-volume anhydrous diethyl ether, after the extraction liquid drying, rotary evaporation removes and desolvates, cross the silicagel column purifying, obtain (R)-amygdalic acid and (R)-pure product of a chloro mandelic acid, during productive rate and optical purity of products are listed in the table below.
Figure G2009100543581D00062
Embodiment 6 utilizes conversion of resting cells higher concentration benzoglycols cyclic carbonate ester
Take by weighing 3g Bacillus sp.ECU0015 resting cell and be suspended in 10ml phosphoric acid buffer (100mM, pH7.3) in, add the benzoglycols cyclic carbonate ester, concentration of substrate is 50mM, oscillatory reaction 36h on the constant temperature shaking table of 30 ℃ and 160rpm, intermittent sampling is measured the enantiomeric excess value of product.Transformation efficiency is 32.4% o'clock, ee pThe highest, be 97.4%.
Embodiment 7 utilizes Bacillus sp.ECU0015 grown cell gram level preparation (S)-benzoglycols
40 500ml that the 100ml rich medium is housed shake bottle, inoculate as embodiment 2 described seed culture fluids by the inoculum size of 5% (v/v), respectively add the methanol solution that 2ml is dissolved with the benzoglycols cyclic carbonate ester behind the inoculation 12h, the final concentration of substrate is 5mM in the substratum.Oscillatory reaction on the constant temperature shaking table of 30 ℃ and 160rpm adds stopped reaction behind the substrate 48h, centrifugal removal cell.Add NaCl in the supernatant liquor to saturated, the equal-volume anhydrous diethyl ether divides three extractions (anhydrous diethyl ether reclaims the back recycling), the anhydrous diethyl ether extraction liquid that obtains spends the night with 10% (w/v) anhydrous sodium sulfate drying, rotary evaporation is removed anhydrous diethyl ether, by silica gel column chromatography (sherwood oil: ethyl acetate, volume ratio 4: 1) obtain (S)-benzoglycols 0.98g behind the purifying, yield is 35.6%, ee p=98.3%.

Claims (6)

1. a bacillus Bacillus sp.ECU0015, preserving number is CGMCC No.2874.
2. the purposes of a genus bacillus as claimed in claim 1 is characterized in that being used for catalytic production optical homochiral benzoglycols, amygdalic acid or a chloro mandelic acid.
3. the purposes of bacillus sp.ECU0015 catalytic production optical homochiral compound according to claim 2 comprises the steps:
(1) the described bacillus sp.ECU0015 of claim 1 is carried out amplification cultivation in fermention medium;
(2) resting cell of cultivating in the centrifugal collection (1) is suspended in pH again and is in 6.0～8.0 the potassium phosphate buffer, adds benzoglycols cyclic carbonate ester, acetyl mandelic acid or m-chloro acetyl mandelic acid; Or directly in the nutrient solution of (1), add benzoglycols cyclic carbonate ester, acetyl mandelic acid or m-chloro acetyl mandelic acid; At 25～40 ℃ of reaction 10～40h, adopt conventional separation method from reaction mixture, to collect optically pure benzoglycols, amygdalic acid or a chloro mandelic acid then.
4. according to right 3 described purposes, it is characterized in that the weight in wet base content of described resting cell in phosphate buffered saline buffer is that 10～150g/L, grown cell weight in wet base content in fermented liquid is 10～50g/L.
5. according to right 3 described purposes, it is characterized in that the concentration of benzoglycols cyclic carbonate ester, acetyl mandelic acid or m-chloro acetyl mandelic acid is 5～100mmol/L.
6. according to right 3 described purposes, it is characterized in that described fermention medium is composed as follows: glycerine 10～50g/L, peptone 1～20g/L, extractum carnis 1～20g/L, KH 2PO 41～10g/L, Na 2HPO 41～10g/L, NaCl 0.1～2g/L, MgSO 40.1～2g/L, pH 4～8.
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