CN102161978B - Rhodococcus ZJPH1003 and application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid - Google Patents
Rhodococcus ZJPH1003 and application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid Download PDFInfo
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- CN102161978B CN102161978B CN2011100259864A CN201110025986A CN102161978B CN 102161978 B CN102161978 B CN 102161978B CN 2011100259864 A CN2011100259864 A CN 2011100259864A CN 201110025986 A CN201110025986 A CN 201110025986A CN 102161978 B CN102161978 B CN 102161978B
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- CN
- China
- Prior art keywords
- rhodococcus
- carboxylic acid
- cyclopropane carboxylic
- dinethyl cyclopropane
- wet thallus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 241000316848 Rhodococcus <scale insect> Species 0.000 title claims abstract description 33
- JWEFQVVDPVVSGR-UHFFFAOYSA-J carbanide;cyclopenta-1,3-diene;tantalum;tetrachloride Chemical compound [CH3-].[CH3-].[CH3-].[CH3-].[CH3-].[Cl-].[Cl-].[Cl-].[Cl-].[Ta].C1C=CC=[C-]1 JWEFQVVDPVVSGR-UHFFFAOYSA-J 0.000 title abstract 3
- 239000000758 substrate Substances 0.000 claims abstract description 35
- 238000000034 method Methods 0.000 claims abstract description 17
- 230000007062 hydrolysis Effects 0.000 claims abstract description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 12
- YMGUBTXCNDTFJI-UHFFFAOYSA-N cyclopropanecarboxylic acid Chemical compound OC(=O)C1CC1 YMGUBTXCNDTFJI-UHFFFAOYSA-N 0.000 claims description 40
- LDDOSDVZPSGLFZ-UHFFFAOYSA-N ethyl cyclopropanecarboxylate Chemical compound CCOC(=O)C1CC1 LDDOSDVZPSGLFZ-UHFFFAOYSA-N 0.000 claims description 28
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 22
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- 238000004166 bioassay Methods 0.000 description 1
- 230000002210 biocatalytic effect Effects 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
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- 239000004568 cement Substances 0.000 description 1
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- 238000012937 correction Methods 0.000 description 1
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- 238000003810 ethyl acetate extraction Methods 0.000 description 1
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- 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
- 229950006191 gluconic acid Drugs 0.000 description 1
- 229950004542 glucuronamide Drugs 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229960002989 glutamic acid Drugs 0.000 description 1
- KZNQNBZMBZJQJO-YFKPBYRVSA-N glyclproline Chemical compound NCC(=O)N1CCC[C@H]1C(O)=O KZNQNBZMBZJQJO-YFKPBYRVSA-N 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
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- 229960000367 inositol Drugs 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
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- 239000002728 pyrethroid Substances 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
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- 239000002994 raw material Substances 0.000 description 1
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a new strain-Rhodococcus sp. ZJPH1003 and an application thereof in preparing S-(+)-2,2-dimethylcyclopropane carboxylic acid through microbial catalytic asymmetric hydrolysis of 2,2-dimethylcyclopropane ethyl formate. The strain is conserved at China Center for Type Culture Collection, the conservation address is Wuhan University, Wuhan, China, postcode 430072, the conservation number is CCTCC M 2010371, and the conservation date is December 29, 2010. In the invention, the adopted method for preparing the S-(+)-2,2-dimethylcyclopropane carboxylic acid through microbial catalytic asymmetric hydrolysis of the 2,2-dimethylcyclopropane ethyl formate by utilizing the new microbial strain has the advantages of novel route, high yield, environmental friendliness and the like; and by utilizing a chiral biocatalysis method which takes a Rhodococcus ZJPH1003 cell as a catalyst, for the target product-S-(+)-2,2-dimethylcyclopropane carboxylic acid, the e.e can reach 82.5% and the yield can reach 41.3% when the substrate concentration is 30mmol/L.
Description
(1) technical field
The present invention relates to a strain and can be used for microorganism catalysis asymmetric hydrolysis 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester prepares S-(+)-2; The new bacterial strain of 2-dinethyl cyclopropane carboxylic acid---rhodococcus (Rhodococcus sp.) ZJPH1003 and at microorganism catalysis asymmetric hydrolysis 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester prepares S-(+)-2, the application in the 2-dinethyl cyclopropane carboxylic acid.
(2) background technology
The chemistry of cilastatin is called (+)-(Z)-7-[(2R)-(2-amino-2-carboxy ethyl) sulphur]-2-[(1S)-(2,2-dimethyl-cyclopropane carboxamide base)]-2-heptenoic acid, is that first is applied to clinical kidney dehydrogenation Dipeptidase inhibitor.The recombiner that cilastatin and imipenum (imipenem) are processed with 1: 1 ratio is safe can
; Be carbapenem antibiotic; The existing extremely strong broad spectrum antibiotic activity of this medicine; The beta-lactam enzyme inhibition is arranged again; Being specially adapted to the polyinfection of multiple bacterium co-infection and aerophil and anerobes, is present clinical application one of microbiotic the most widely.The preparation of cilastatin relates to S-(+)-2; 2-dinethyl cyclopropane carboxylic acid (methane amide), 7-bromine (chlorine)-2-oxo enanthic acid and three synthetic intermediates of L-halfcystine; Wherein have optically active S-(+)-2, the 2-dinethyl cyclopropane carboxylic acid is one of key intermediate of synthetic cilastatin medicine.In addition, 2,2-dinethyl cyclopropane carboxylic acid verivate also is the key intermediate of synthetic pyrethroid insecticides, purposes is very extensively.2, the synthetic and disassemble technique of 2-dinethyl cyclopropane carboxylic acid received people's attention in recent years day by day.
Adopt chemical method to prepare S-(+)-2, the synthesis step of 2-dimethyl-cyclopropane carboxamide (or formic acid) is more, and reaction conditions is relatively harsher; Yield is on the low side, needs in the reaction process to use a large amount of organic solvents, and environment is polluted; Or use expensive chiral catalyst (Wang Qinwei; Etal.Tetrahedron:Asymmetry, 1998,9:3971-3977).Adopt microorganism catalysis to prepare S-(+)-2, the 2-dinethyl cyclopropane carboxylic acid has reaction conditions gentleness, selectivity height and advantages of environment protection.
At present, the biocatalysis that document has been reported is synthesized S-(+)-2, and it is the biosynthesizing route of raw material that the method for 2-dinethyl cyclopropane carboxylic acid (or methane amide) adopts with the nitrile substrate more, for example:
(Journal of Molecular Catalysis B:Enzymatic 18 2002:267-272.) screens bacterial classification Rhodococcus sp.AJ270 to Wang Mei-Xiang etc. from soil, hydrolyzable 2; 2-dimethylcyclopropane formonitrile HCN obtains S-(+)-2, the 2-dinethyl cyclopropane carboxylic acid, and reaction conditions is following: the pH 7.0 potassium phosphate buffer 50ml of 0.1mol/L; 2 of 1mmol, 2-dimethylcyclopropane formonitrile HCN is a substrate, 30 ℃ are reacted 3h down; Product e.e. value is 82%, and productive rate is 39%.When reacting 4.5h under 20 ℃, the e.e. value is 88%, and productive rate is 44%.
(Enzyme and Microbial Technology such as Yeom; 2007 (41): 842-848.) utilize rhodococcus Rhodococcus erythropolis ATCC 25544; With 2; 2-dimethylcyclopropane formonitrile HCN is a substrate, obtains S-(+)-2 through the microorganism cells hydrolysis, the 2-dinethyl cyclopropane carboxylic acid.Reaction conditions is following: pH 7.0 potassium phosphate buffers of 50mmol/L, and 10% (v/v) methyl alcohol, concentration of substrate is 100mmol/L, and 20 ℃ of following reaction 64h after product e.e. values reach 81.8%, and productive rate is 45%.
(Zheng Yuguo etc. such as Zheng Yuguo; Chinese invention patent; Application number: 200510061680.9) adopt Rhodococcuss equi CCTCC M 205114 to unite with the two bacterial classifications of Delftia tsuruhatensis CCTCC M205115 and transform racemize 2,2-dimethylcyclopropane formonitrile HCN prepares S-(+)-2, the 2-dimethyl-cyclopropane carboxamide; Productive rate is more than 47%, and the e.e. value is greater than 98%.
The substrate of using in the above-mentioned document 2,2-dimethylcyclopropane formonitrile HCN need to use the prussiate of severe toxicity in building-up process, have influence on the environmental benefit of this route to a certain extent.And the present invention is the mikrobe novel bacterial that utilization obtains from row filter--the ester hydrolase that rhodococcus (Rhodococcus sp.) ZJPH1003 cell contains; With racemize 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester is a substrate; Prepare optical purity S-(+)-2 through the biology fractionation, the 2-dinethyl cyclopropane carboxylic acid is " green " novel preparation method that is different from above-mentioned bibliographical information.Although adopt lypase Novozyme 435 also alternative hydrolysis racemizes 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester synthesizes S-(+)-2,2-dinethyl cyclopropane carboxylic acid (Wang Pu etc., catalysis journal; 2010; 31 (6): 651-655), but the price of commodity lypase is higher, has also limited its industrial applications to a certain extent.
(3) summary of the invention
The object of the invention provides a strain and can be used for microorganism catalysis asymmetric hydrolysis 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester prepares S-(+)-2; The new bacterial strain of the mikrobe of 2-dinethyl cyclopropane carboxylic acid---rhodococcus (Rhodococcus sp.) ZJPH1003; And this bacterial classification prepares S-(+)-2 at the catalysis asymmetric hydrolysis, the application in the 2-dinethyl cyclopropane carboxylic acid.
The technical scheme that the present invention adopts is:
Rhodococcus (Rhodococcus sp.) ZJPH1003 is preserved in Chinese typical culture collection center, the address: China, Wuhan, Wuhan University, postcode: 430072; Deposit number: CCTCC NO:M 2010371, preservation date: on December 29th, 2010.
Compare with existing similar bacterial strain, bacterial classification of the present invention is prone to cultivate, and the preparation cost of biological catalyst is low, and the substrate conversion reaction times shortens than the lipase-catalyzed process of commodity greatly.
The bacterial classification source: rhodococcus (Rhodococcus sp.) ZJPH1003 bacterial strain system separation screening from pick up from farmland, area, Wenzhou, Zhejiang soil sample obtains.
The characteristic of this novel bacterial is following:
Colonial morphology: cultivate 48h for 30 ℃, bacterium colony is rounded, and surface wettability is smooth, somewhat raised.Bacterium colony is incarnadine, and is opaque, glossy, and color deepens with the increase of culture presevation time gradually.
Cellular form: cell is spherical, and some presents rod-short, can arrange or be gathered into clump single, in pairs.
Physiological and biochemical property: Gram-positive, the spore of not sprouting does not move, no pod membrane, obligate is aerobic.In the automatic assessing instrument detected result of Biolog carbon source; Utilizable carbon source has: dextrin, D-turanose, α-D-lactose, N-acetyl-D-amino semi-lactosi, alpha-D-glucose, D-seminose, D-fructose, L-Fucose, L-rhamnosyl, D-arabitol, D-G-6-P salt, D-aspartic acid, glycyl-L-proline(Pro), L-Histidine, pectin, D-galacturonic acid, maltonic acid, glucuronamide, cement acid, quinic acid, D-methyl lactate, L-lactic acid, Hydrocerol A, α-Tong Jiwuersuan, L MALIC ACID, bromosuccinic acid, polysorbate40, beta-hydroxy-D, L-butyric acid, etheric acid, propionic acid, acetate.
Unavailable carbon source has: D-SANMALT-S; The D-trehalose; The D-cellobiose; Gentiobiose; Sucrose; Stachyose; The D-raffinose; The D-melibiose; Beta-methyl-D-glucoside; The D-saligenin; N-acetyl-D-glycosamine; N-acetyl-β-D-mannosamine; N-acetylneuraminic amine saccharic acid; The D-semi-lactosi; The 3-methyl glucoside; The D-Fucose; Inosine; The D-sorbyl alcohol; D-N.F,USP MANNITOL; Inositol; Glycerine; D-fructose-6-phosphate salt; The D-Serine; Gelatin; The L-L-Ala; The L-l-arginine; The L-aspartic acid; L-L-glutamic acid; The L-Pyrrolidonecarboxylic acid; The L-Serine; The L-galactonolactone; The D-glucuronic acid; The D-saccharic acid; The P-hydroxyl phenylacetic acid; The methylacetone hydrochlorate; The D-oxysuccinic acid; Gamma-amino-butyric acid; The Alpha-hydroxy butyric acid; α-alpha-ketobutyric acid; Formic acid.
The 16S rDNA sequence characteristic of bacterial classification: total DNA is a template with the cell that extracts, and utilizes the 16S rDNA gene of universal primer P1 and P2 amplification bacterial strain, again the PCR product is carried out agarose gel electrophoresis and cuts glue purification.Confirm that through order-checking the 16S rDNA gene order of said rhodococcus ZJPH1003 is following again:
GTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTACAGGGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGAAATGGGTCTAATACCGGATACGACCAAAGGCTGCATGGTTTTTGGTGGAAAGGTTTACTGGTGCAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTT?TCAGCAGGGACGAAGCGCAAGTGACGGTACCTGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAAACCCGCAGCTCAACTGCGGGCTTGCAGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATACCGGACCGCCGTAGAGATACGGTTTCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTAATGGTGGGGACTCGCAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGGTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG。
This sequence has been submitted GenBank (the GenBank accession number is No.HQ840650) to; The 16SrRNA sequence of ZJPH1003 bacterial strain is carried out homology comparison (BLAST) on NCBI website (http://www.ncbi.nlm.nih.gov), the result shows: the part bacterial strain sequence homology of ZJPH1003 bacterial strain and Rhod (Rhodococcus sp.) is higher.The sequence homology of ZJPH1003 bacterial strain and Rhodococcus sp.BFXJ-2 bacterial strain (the GenBank accession number is No.EU017404) reaches 100%.
According to physio-biochemical characteristics and binding molecule biological assay, this bacterial strain is accredited as rhodococcus (Rhodococcus sp.) ZJPH1003.
Adopting Box-Behnken rotation center combination experiment design method that glycerol concentration, peptone concentration and three of NaCl concentration are influenced significant factor to the product enzyme is optimized; The optimization substratum that obtains rhodococcus (Rhodococcus sp.) ZJPH1003 bacterial strain consists of: glycerine 5～30g/L; Peptone 10～30g/L; NaCl 1.0～4.5g/L, MgSO
47H
2O 0.3～0.6g/L, K
2HPO
40.5～4g/L, KH
2PO
40.5～2g/L, pH 6.0～7.5.
The culture condition of rhodococcus (Rhodococcus sp.) ZJPH1003 is: initial pH 6.0～7.5, shake bottled liquid measure 80～100mL/250mL Erlenmeyer flask, 30 ℃ of culture temperature, shaking speed 180～220rpm, inoculum size 4～12%, incubation time 36～48h.
Microbial strains involved in the present invention is to obtain through following method screening:
1) enrichment culture: get and pick up from soil sample 2～3g in all parts of the country and join the 250mL that the 50mL enrichment medium is housed and shake in the bottle; 30 ℃, 200rpm cultivates 3～5d; After treating that nutrient solution becomes muddiness; Get the 5mL nutrient solution and be forwarded in the fresh enrichment medium, continue to cultivate 3～5d, so repeat enrichment culture 3～5 times.With 2,2-dinethyl cyclopropane carboxylic acid ethyl ester is a sole carbon source in the enrichment medium.The enrichment culture based formulas is following: 2, and 2-dinethyl cyclopropane carboxylic acid ethyl ester 50mmol/L, (NH
4)
2SO
44g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 0.5g/L, MgSO
47H
2O0.5g/L, pH 7.0.
2) the dull and stereotyped cultivation: enrichment culture liquid is through gradient dilution to 10
-6～10
-8, be applied on the separating plate, cultivate 3～5d for 30 ℃, plate culture medium consists of the agar that adds 15-20g/L in the enrichment medium.
3) slant culture: picking list bacterium colony carries out slant culture in the bacterium colony of from flat board, growing, and cultivates 2～3d after plentiful lawn occurs for 30 ℃, places 4 ℃ of refrigerators to preserve, and is subsequent use.The slant culture based formulas is following: glucose 10g/L, peptone 5g/L, yeast extract paste 3g/L, (NH
4)
2SO
44g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 0.5g/L, MgSO
47H
2O 0.5g/L, agar 20g/L, pH 7.0.
4) seed culture: choose a ring thalline and insert the 250mL that the 100mL seed culture medium is housed and shake the bottle from cultivating sophisticated inclined-plane, 30 ℃, 200r/min cultivated 24 hours.The seed culture based formulas is following: glucose 10g/L, peptone 5g/L, yeast extract paste 3g/L, (NH
4)
2SO
44g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 0.5g/L, MgSO
47H
2O 0.5g/L, pH 7.0.
5) fermentation culture: the inoculum size with 10% is transferred to the 250mL that the 100mL fermention medium is housed with seed liquor and shakes in the bottle, and 30 ℃, 200r/min cultivated 48 hours.Fermentative medium formula is with the seed substratum.
6) bioconversion reaction: the centrifugal wet thallus that obtains is suspended in the phosphate buffered saline buffer, adds a certain amount ofly 2,2-dinethyl cyclopropane carboxylic acid ethyl ester is put in 30 ℃ of shaking tables and is transformed certain hour.After transforming end, conversion fluid is used the equal-volume ethyl acetate extraction, after centrifugal, obtains supernatant, adopts the content of chiral gas chromatography analysis purposes product and residual substrate, and the optical purity of product.
7) analyzing and testing: the product in the reaction, extraction liquid and the concentration of residual substrate adopt gas chromatographic analysis, use inner mark method ration.Internal standard substance is a dodecyl.Getting the 1mL extraction liquid adds 1 μ L dodecyl and analyzes.GC conditions: day island proper Tianjin GC-2014 gas chromatograph; U.S. Varian CP-Chirasil-Dex chiral capillary gas chromatography post (25m * 0.25mm * 0.25 μ m).Carrier gas is a high pure nitrogen, and flow is 2mL/min; Sample size 1 μ L, splitting ratio is 15: 1; Detector and injector temperature are 250 ℃; 80～180 ℃ of chromatogram column temperatures; Heat-up rate: 8 ℃/min; Detector is FID.Gas chromatogram is seen Fig. 1～3.
The invention still further relates to said rhodococcus (Rhodococcus sp.) ZJPH1003 and prepare S-(+)-2 at the microorganism catalysis asymmetric hydrolysis, the application in the 2-dinethyl cyclopropane carboxylic acid.
Concrete; Said being applied as: with 2,2-dinethyl cyclopropane carboxylic acid ethyl ester is a substrate, is the enzyme source with rhodococcus ZJPH1003 wet thallus cell; Under 20～40 ℃; In pH is 5.29～9.18 damping fluid, carried out conversion reaction 12～48 hours, reaction finishes afterreaction liquid and obtains S-(+)-2 through separation and purification, the 2-dinethyl cyclopropane carboxylic acid.
In the said damping fluid transformation system, substrate 2, the starting point concentration of 2-dinethyl cyclopropane carboxylic acid ethyl ester is 10～50mmol/L damping fluid, the consumption of rhodococcus ZJPH1003 wet thallus cell is counted 300～500g/L damping fluid with weight in wet base.
Said damping fluid is for the conventional damping fluid that is used for cell response, and is preferred like phosphate buffered saline buffer, citrate buffer, Tris-HCl damping fluid etc., carries out in Sodium phosphate, dibasic-potassium phosphate buffer of the said pH of being reflected at 6.98.
Said wet thallus cell can separate obtain after by strain fermentation according to ordinary method; Preferably; Said rhodococcus ZJPH1003 wet thallus cell is prepared by following method: rhodococcus ZJPH1003 is seeded to the fermention medium that is applicable to rhodococcus; 30 ℃, 200rpm shaking table were cultivated 48 hours, and fermented liquid is collected and obtained said wet thallus cell through centrifugal, washing precipitation.
The said fermention medium of rhodococcus that is applicable to is used for the substratum that rhodococcus is cultivated for this area routine, among the present invention, as optimized technical scheme; Said fermention medium final concentration is formed as follows: glycerine 5～30g/L; Peptone 10～30g/L, NaCl 1.0～4.5g/L, MgSO
47H
2O 0.3～0.6g/L, K
2HPO
40.5～4g/L, KH
2PO
40.5～2g/L, pH 6.0～7.5.
Concrete, said application method is following:
(1) rhodococcus ZJPH1003 is seeded to fermention medium, 30 ℃, 200rpm shaking culture 48 hours, fermented liquid are collected and are obtained the wet thallus cell through centrifugal, washing precipitation; Said fermention medium final concentration is formed as follows: glycerine 15g/L, peptone 25g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 1.5g/L, MgSO
47H
2O 0.5g/L, pH 7.0;
(2) getting pH is Sodium phosphate, dibasic-potassium phosphate buffer of 6.98, adds step (1) gained wet thallus cell, and said wet thallus cell concn is counted the 400g/L damping fluid with weight in wet base; Add substrate 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester to concentration of substrate is the 30mmol/L damping fluid, 30 ℃ of shaking table 200rpm oscillatory reaction 24 hours, and reaction is centrifugal with reaction solution after finishing; Get supernatant; Add equal volume of ethyl acetate twice, in organic phase, obtain S-(+)-2, the 2-dinethyl cyclopropane carboxylic acid.
The present invention screens from soil sample and has obtained the new microbial transformation bacterial classification of a strain, has proposed production S-(+)-2, a kind of novel method of 2-dinethyl cyclopropane carboxylic acid; Promptly utilize ester hydrolase to produce bacterium hydrolysis 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester obtains S-(+)-2, the 2-dinethyl cyclopropane carboxylic acid, and this method is not seen as yet has bibliographical information; Thereby be equipped with the crucial chiral intermediate S-(+)-2 of cilastatin medicine in research microorganism catalysis legal system, 2-dinethyl cyclopropane carboxylic acid aspect provides beneficial reference.
Beneficial effect of the present invention is mainly reflected in: provide a strain to can be used for microorganism catalysis asymmetric hydrolysis 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester prepares S-(+)-2, and the mikrobe novel bacterial of 2-dinethyl cyclopropane carboxylic acid adopts this novel bacterial catalytic preparation S-(+)-2; The 2-dinethyl cyclopropane carboxylic acid has the route novelty; The biological catalyst preparation cost is low, and catalytic efficiency (is high, advantages of environment protection; Through adopting rhodococcus (Rhodococcus sp.) ZJPH1003 cell is the biocatalysis of catalyzer, when concentration of substrate is 30mmol/L, and purpose product S-(+)-2, the e.e. of 2-dinethyl cyclopropane carboxylic acid reaches 82.5%, and productive rate reaches 41.3%.
(4) description of drawings
Fig. 1 is substrate standard substance gas chromatograms;
Fig. 2 is product standard substance gas chromatograms;
Fig. 3 is a rhodococcus ZJPH1003 bacterial strain biocatalytic reaction 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 detection method of product
The GC method is measured the optical purity and the productive rate of title product: the product in the reaction, extraction liquid and the concentration of residual substrate adopt gas chromatographic analysis, use inner mark method ration.Internal standard substance is a dodecyl.Getting the 1mL extraction liquid adds 1 μ L dodecyl and analyzes.GC conditions: day island proper Tianjin GC-2014 gas chromatograph; U.S. Varian CP-Chirasil-Dex chiral capillary gas chromatography post (25m * 0.25mm * 0.25 μ m).Carrier gas is a high pure nitrogen, and flow is 2mL/min; Sample size 1 μ L, splitting ratio is 15: 1; Detector and injector temperature are 250 ℃; 80～180 ℃ of chromatogram column temperatures; Heat-up rate: 8 ℃/min; Detector is FID.
Calculate substrate and the concentration of product in the reaction solution respectively with relative correction factor.And then obtain the productive rate (Yield) of reaction.Calculating formula is:
Productive rate=C
i/ C
0* 100%
C in the formula
0, C
iThe volumetric molar concentration of product when being respectively the volumetric molar concentration of the initial substrate of reaction and reacting end.
(enantiomeric excess e.e.) representes the optical purity of product by enantiomeric excess value.
e.e.＝(C
S-C
R)/(C
S+C
R)×100％
C in the formula
SAnd C
RBe respectively S type and R type 2, the volumetric molar concentration of 2-dinethyl cyclopropane carboxylic acid.
Embodiment 2: the acquisition of wet thallus cell
Seed culture based formulas: glucose 10g/L, peptone 5g/L, yeast extract paste 3g/L, (NH
4)
2SO
44g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 0.5g/L, MgSO
47H
2O0.5g/L, solvent are water, and pH 7.0.
Fermentative medium formula: glycerine 15g/L, peptone 25g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 1.5g/L, MgSO
47H
2O 0.5g/L, solvent are water, and pH 7.0.
Choose a ring thalline (CCTCC NO:M 2010371) and insert the 250mL that the 100mL seed culture medium is housed and shake the bottle from cultivating sophisticated inclined-plane; 30 ℃; 200rpm cultivates and got seed liquor in 24 hours; With the inoculum size of volume ratio 10% seed liquor is transferred to the 250mL that the 100mL fermention medium is housed again and shakes in the bottle, 30 ℃, 200rpm cultivated 48 hours.It is centrifugal and with the saline water washing once cultivate to finish secondary fermentation liquid, collects the wet thallus cell, subsequent use.
Embodiment 3:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 10mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 4.65mmol/L, optical purity e.e. value 80.1%, productive rate 46.5%.
Embodiment 4:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 20mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 8.57mmol/L, optical purity e.e. value 82.4%, productive rate 42.9%.
Embodiment 5:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 11.53mmol/L, optical purity e.e. value 83.5%, productive rate 38.4%.
Embodiment 6:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 40mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, anti-24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 12.86mmol/L, optical purity e.e. value 84.7%, productive rate 32.2%.
Embodiment 7:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 50mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 13.09mmol/L, optical purity e.e. value 85.1%, productive rate 26.2%.
Embodiment 8:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution (pH 5.29), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 7.38mmol/L, optical purity e.e. value 84.9%, productive rate 24.6%.
Embodiment 9:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution (pH 5.91), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 30 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 8.28mmol/L, optical purity e.e. value 84.2%, productive rate 27.6%.
Embodiment 10:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-phosphate sodium dihydrogen buffer solution (pH 6.47), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 9.57mmol/L, optical purity e.e. value 83.8%, productive rate 31.9%.
Embodiment 11:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 24h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 11.19mmol/L, optical purity e.e. value 83.4%, productive rate 37.3%.
Embodiment 12:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 12h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 7.03mmol/L, optical purity e.e. value 84.5%, productive rate 23.4%.
Embodiment 13:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 20 ℃ as substrate, reacts 20h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 9.72mmol/L, optical purity e.e. value 83.8%, productive rate 32.4%.
Embodiment 14:
The wet thallus of embodiment 2 gained is suspended in 10mL Sodium phosphate, dibasic-potassium phosphate buffer (pH 6.98), and wet thallus is 400g/L in weight in wet base concentration; Add 2 of 30mmol/L, 2-dinethyl cyclopropane carboxylic acid ethyl ester places 30 ℃ as substrate, reacts 28h in the shaking table of 200rpm.Adopt the detection method of embodiment 1, product S-(+)-2, the concentration of 2-dinethyl cyclopropane carboxylic acid is 12.39mmol/L, optical purity e.e. value 82.5%, productive rate 41.3%.
SEQUENCE?LISTING
< 110>Zhejiang Polytechnical University
< 120>rhodococcus ZJPH1003 and the preparation S-(+)-2, the application in the 2-dinethyl cyclopropane carboxylic acid
<130>
<160> 1
<170> PatentIn?version?3.4
<210> 1
<211> 1320
<212> DNA
<213> Rhodococcus?sp.
<400> 1
gtcgaacgat?gaagcccagc?ttgctgggtg?gattagtggc?gaacgggtga?gtacaggggg 60
tgatctgccc?tgcacttcgg?gataagcctg?ggaaatgggt?ctaataccgg?atacgaccaa 120
aggctgcatg?gtttttggtg?gaaaggttta?ctggtgcagg?atgagcccgc?ggcctatcag 180
cttgttggtg?gggtaatggc?ctaccaaggc?gacgacgggt?agccggcctg?agagggcgac 240
cggccacact?gggactgaga?cacggcccag?actcctacgg?gaggcagcag?tggggaatat 300
tgcacaatgg?gcgaaagcct?gatgcagcga?cgccgcgtga?gggatgacgg?ccttcgggtt 360
gtaaacctct?ttcagcaggg?acgaagcgca?agtgacggta?cctgcagaag?aagcaccggc 420
caactacgtg?ccagcagccg?cggtaatacg?tagggtgcga?gcgttgtccg?gaattactgg 480
gcgtaaagag?ctcgtaggcg?gtttgtcgcg?tcgtctgtga?aaacccgcag?ctcaactgcg 540
ggcttgcagg?cgatacgggc?agacttgagt?actgcagggg?agactggaat?tcctggtgta 600
gcggtgaaat?gcgcagatat?caggaggaac?accggtggcg?aaggcgggtc?tctgggcagt 660
aactgacgct?gaggagcgaa?agcgtgggta?gcgaacagga?ttagataccc?tggtagtcca 720
cgccgtaaac?ggtgggcgct?aggtgtgggt?ttccttccac?gggatccgtg?ccgtagctaa 780
cgcattaagc?gccccgcctg?gggagtacgg?ccgcaaggct?aaaactcaaa?ggaattgacg 840
ggggcccgca?caagcggcgg?agcatgtgga?ttaattcgat?gcaacgcgaa?gaaccttacc 900
tgggtttgac?atataccgga?ccgccgtaga?gatacggttt?cccttgtggt?cggtatacag 960
gtggtgcatg?gctgtcgtca?gctcgtgtcg?tgagatgttg?ggttaagtcc?cgcaacgagc 1020
gcaacccttg?tcctgtgttg?ccagcacgta?atggtgggga?ctcgcaggag?actgccgggg 1080
tcaactcgga?ggaaggtggg?gacgacgtca?agtcatcatg?ccccttatgt?ccagggcttc 1140
acacatgcta?caatggccgg?tacagagggc?tgcgataccg?tgaggtggag?cgaatccctt 1200
aaagccggtc?tcagttcgga?tcggggtctg?caactcgacc?ccgtgaagtc?ggagtcgcta 1260
gtaatcgcag?atcagcaacg?gtgcggtgaa?tacgttcccg?ggccttgtac?acaccgcccg 1320
Claims (7)
1. rhodococcus (Rhodococcus sp.) ZJPH1003 is preserved in Chinese typical culture collection center, the address: China, Wuhan, Wuhan University, postcode: 430072; Deposit number: CCTCC NO:M 2010371, preservation date: on December 29th, 2010.
2. rhodococcus (Rhodococcus sp.) CCTCC NO:M 2010371 prepares S-(+)-2 at the microorganism catalysis asymmetric hydrolysis; Application in the 2-dinethyl cyclopropane carboxylic acid; Said being applied as: with 2,2-dinethyl cyclopropane carboxylic acid ethyl ester is a substrate, is the enzyme source with rhodococcus CCTCC NO:M 2010371 wet thallus cells; Under 20～40 ℃; In pH is 5.29～9.18 damping fluid, carried out conversion reaction 12～48 hours, reaction finishes afterreaction liquid and obtains S-(+)-2 through separation and purification, the 2-dinethyl cyclopropane carboxylic acid.
3. application as claimed in claim 2; It is characterized in that in the said damping fluid transformation system; Substrate 2; The starting point concentration of 2-dinethyl cyclopropane carboxylic acid ethyl ester is 10～50mmol/L damping fluid, and the consumption of rhodococcus CCTCC NO:M 2010371 wet thallus cells is counted 300～500g/L damping fluid with weight in wet base.
4. application as claimed in claim 2 is characterized in that carrying out in Sodium phosphate, dibasic-potassium phosphate buffer of the said pH of being reflected at 6.98.
5. application as claimed in claim 2; It is characterized in that said rhodococcus CCTCC NO:M2010371 wet thallus cell is prepared by following method: rhodococcus CCTCC NO:M2010371 is seeded to the fermention medium that is applicable to rhodococcus; 30 ℃, 200rpm shaking table were cultivated 48 hours; Fermented liquid is collected and is obtained said wet thallus cell through centrifugal, washing precipitation.
6. application as claimed in claim 5 is characterized in that said fermention medium final concentration composition as follows: glycerine 5～30g/L, peptone 10～30g/L, NaCl 1.0～4.5g/L, MgSO
47H
2O0.3～0.6g/L, K
2HPO
40.5～4g/L, KH
2PO
40.5～2g/L, pH 6.0～7.5.
7. application as claimed in claim 2 is characterized in that said being applied as:
(1) rhodococcus CCTCC NO:M 2010371 is seeded to fermention medium, 30 ℃, 200rpm shaking culture 48 hours, fermented liquid are collected and are obtained the wet thallus cell through centrifugal, washing precipitation; Said fermention medium final concentration is formed as follows: glycerine 15g/L, peptone 25g/L, K
2HPO
42g/L, KH
2PO
41g/L, NaCl 1.5g/L, MgSO
47H
2O0.5g/L, pH 7.0;
(2) getting pH is Sodium phosphate, dibasic-potassium phosphate buffer of 6.98, adds step (1) gained wet thallus cell, and said wet thallus cell concn is counted the 400g/L damping fluid with weight in wet base; Add substrate 2; 2-dinethyl cyclopropane carboxylic acid ethyl ester to concentration of substrate is the 30mmol/L damping fluid, 30 ℃ of shaking table 200rpm oscillatory reaction 24 hours, and reaction is centrifugal with reaction solution after finishing; Get supernatant; Add equal volume of ethyl acetate twice, in organic phase, obtain S-(+)-2, the 2-dinethyl cyclopropane carboxylic acid.
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| 王普等.脂肪酶Novozyme435选择性催化2，2-二甲基环丙烷甲酸乙酯合成S-(+)-2，2-二甲基环丙烷甲酸.《催化学报》.2010,第31卷(第6期),651-655. * |
| 郑裕国等.腈转化酶在精细化学品生产中的应用.《生物工程学报》.2009,第25卷(第12期),1795-1807. * |
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