CN104232508A - Bacillus cereus ZJB-11071 and application thereof - Google Patents
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Abstract
The invention provides Bacillus cereus ZJB-11071 and an application thereof in carrying out biological catalytic synthesis of epoxy chloropropane and a chiral medical intermediate. The strain is collected in the China Center for Type Culture Collection, the address is Wuhan university, Wuhan, China 430072, the collection number is CCTCC No:M 2013314, and the collection date is July 5, 2013. The Bacillus cereus ZJB-11071 provided by the invention achieves the transformation rate of 42.3% on 1,3-dichloro-2-propanol, achieves the transformation rate of 21.1% on 2,3-dichloro-2-propanol, achieves the transformation rate of 95% on 1,3-dibromo-2-propanol and achieves the transformation rate of 24.6% on S(-)-4-chlorine-3-ethyl hydroxybutyrate.
Description
(1) technical field
The present invention relates to bacterial strain---bacillus cereus (Bacillus cereus) ZJB-11071, and the application in biocatalysis synthesize epoxide and chirality pharmaceutical intermediate compound that dehalogenase is produced in a strain.
(2) background technology
Heppel in 1948 etc. first describe the methylene dichloride that can make produced by microorganism and are hydrolyzed into the dehalogenase of formaldehyde, and after this dissimilar dehalogenase is found in succession.Nineteen sixty Janssen is halogen family plasma diffusing W,Mo enzyme out being made to be referred to as dehalogenase (Dehalogenase) from halogen family organic compound.So far, people screen the microorganism of multiple halohydrin of can degrading to Flavobacterium (Flavobaterium sp.) bacterial strain of surviving as sole carbon source with 2,3-dibromo-propanols from Late Cambrian such as nineteen sixty-eight Castro in succession.Comprising the agrobacterium radiobacter be separated from fresh water throw out (Agrobacterium radiobacter) AD1 and Arthrobacter (Arthrobacter sp.) AD2 and coryneform bacteria (Corynebacterium sp.) N-1074 etc. that obtains from soil.Although although the approach of their degraded Organohalogen compounds exists notable difference, halohydrin dehalogenase is as one of key enzyme, and the fracture of catalysis carbon-halogen bond is present in all pathways metabolisms.
(3) summary of the invention
The object of this invention is to provide a kind of bacterial strain that can produce halide alcohol dehalogenase---bacillus cereus (Bacillus cereus) ZJB-11071, and the application in biocatalysis synthesize epoxide and chirality pharmaceutical intermediate compound.
The technical solution used in the present invention is:
Bacillus cereus (Bacillus cereus) ZJB-11071, is preserved in China typical culture collection center, address: China. Wuhan. and Wuhan University, 430072, deposit number CCTCC No:M2013314, preservation date on July 5th, 2013.This bacterial strain is separated by Bioengineering Research Institute of Zhejiang Polytechnical University and obtains from soil, has the performance catalyzing and synthesizing epoxide and chirality pharmaceutical intermediate compound (R (-) 4-cyano-3-hydroxy ethyl butyrate) after testing.
The invention still further relates to described bacillus cereus ZJB-11071 and prepare application in epoxide at microorganism catalysis halohydrin, described is applied as: with the wet thallus of bacillus cereus ZJB-11071 after fermentation culture for catalyzer, take halohydrin as substrate, be in the damping fluid of 7.0 ~ 7.5 in pH value, 35 DEG C, carry out conversion reaction under 150rpm condition, after conversion reaction terminates, conversion reaction liquid is extracted with ethyl acetate, centrifugal, get upper organic phase and be mixed solution containing epoxide, by mixed solution separation and purification, obtain described epoxide.
The consumption of described catalyzer is in wet thallus quality, and final concentration is 30 ~ 80g/L, preferred 50g/L, and the starting point concentration of described substrate is 20 ~ 50mmol/L.
Described halohydrin is 1,3-bis-chloro-2-propyl alcohol, 2,3-bis-chloro-2-propyl alcohol or the bromo-2-propyl alcohol of 1,3-bis-.
The invention still further relates to the application of described bacillus cereus ZJB-11071 in biocatalysis synthesis of chiral pharmaceutical intermediate, described is applied as: with the wet thallus of bacillus cereus ZJB-11071 after fermentation culture for catalyzer, with S (-)-4-chloro-3-hydroxyl ethyl butyrate for substrate, be in the damping fluid of 7.0 ~ 7.5 in pH value, conversion reaction is carried out at 35 DEG C, after conversion reaction terminates, conversion reaction liquid is extracted with ethyl acetate, centrifugal, get upper organic phase, by mixed solution separation and purification, obtain R (-)-4-cyano-3-hydroxy ethyl butyrate, the consumption of described catalyzer is in wet thallus quality, and final concentration is 50g/L, and the starting point concentration of described substrate is 20 ~ 50mmol/L.
Wet thallus of the present invention is prepared as follows:
(1) slant culture
Bacillus cereus ZJB-11071 is seeded to slant medium, cultivates 12h at 30 DEG C, obtain inclined-plane thalline;
Described slant medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, agar 20.0g/L, and solvent is water, and pH value is 7.0;
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 24h at 30 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, and solvent is water, and pH value is 7.0;
(3) fermentation culture
Be seeded in fermention medium by seed liquor with the inoculum size of volumetric concentration 1%, 30 DEG C, after 150rpm shaking culture 60h, centrifugal 5min under 12000g, collects wet thallus;
Described fermention medium final concentration consists of: lactose 8g/L, peptone 6g/L, Na
2hPO
43.2g/L, K
2hPO
41.5g/L, Na
2eDTA0.06g/L, MnSO
40.002g/L, MgSO
40.001g/L, ZnSO
40.002g/L, FeSO
40.01g/L, solvent is water, and pH value is 6.8.
Common, before carrying out next step operation after the bacterial strain of preservation takes out from refrigerator, need to carry out activation culture, conventionally, selection LB substratum carries out slant culture, culture temperature 30 DEG C, incubation time 2 days usually.
Beneficial effect of the present invention is mainly reflected in: the invention provides a strain new strains--bacillus cereus (Bacillus cereus) ZJB-11071, and the application in biocatalysis epoxide and chirality pharmaceutical intermediate compound, bacillus cereus ZJB-11071 is to 1,3-bis-chloro-2-propyl alcohol transformation efficiency reaches 42.3%, to 2,3-bis-chloro-2-propyl alcohol transformation efficiency reaches 21.1%, to 1,3-bis-bromo-2-propyl alcohol transformation efficiency reaches 95%, 24.6% is reached to S (-)-4-chloro-3-hydroxyl ethyl butyrate transformation efficiency, there is important application prospect.
(4) accompanying drawing illustrates:
Fig. 1 is the gas chromatogram of 1,3-DCP and ECH.
(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 screening of bacillus cereus ZJB-11071
The present invention, from soil sampling in all parts of the country, is total to soil sampling 80 parts.The concrete grammar of screening: take 1g soil sample and be placed in 50mL enrichment medium, in 30 DEG C, 150r/min shaking culture 2-3 days.Getting 1mL pregnant solution adds in the fresh enrichment medium of 50mL, carries out separation and purification after so repeating 3 times.
Bromothymol blue is selected to detect the bacterium colony of substrate of can degrading as indicator.Its principle is: can discharge proton while chlorinated organics degraded, thus the pH value of substratum local is changed.Indicator Bromothymol blue color change interval is 5.8-7.6 (yellow-blue), and the periphery of bacterial colonies of chlorinated organics therefore can be utilized to present yellow, and the dull and stereotyped color of unavailable then periphery of bacterial colonies is still dark green.
By pregnant solution sterilized water stepwise dilution 10 gradients, choose 10
-6, 10
-7, 10
-8, 10
-9, 10
-10the diluent of five gradients is respectively got 0.1mL and is uniformly coated on plate screening substratum, 30 DEG C of constant temperature culture 48h.The dull and stereotyped yellow single colony inoculation of picking instruction is on slant medium, and the constant incubator putting into 30 DEG C is cultivated, when to grow to colony diameter size be about 3-4mm, in 4 DEG C of preservations.
By the inoculation that is stored on inclined-plane in seed culture medium, cultivate 24h at 30 DEG C.Seed liquor is seeded in fermention medium with the inoculum size of volumetric concentration 1%, 30 DEG C, 150rpm shaking culture 60h.Centrifugal 5min under 12000g, collects wet thallus, and the phosphoric acid buffer (50mM, pH7.0) adding certain volume makes 0.5g/mL bacteria suspension for transforming.Conversion condition is as follows: phosphate buffered saline buffer (50mM, pH7.0) 0.5g/mL bacteria suspension (consumption of bacteria suspension counts 50g/L reaction system with wet thallus quality) 10ml is added in 10ml, 1, (3-DCP0.03mol final concentration 30mM), be placed in 30 DEG C of shaking baths and transform 24h, get 1mL conversion fluid, vapor detection analysis after extraction into ethyl acetate.Finally obtain the wild strain that a strain catalysis activity is higher, and be bacterial strain ZJB-11071 by this Strain Designation.
Gas phase analysis condition is: the gas chromatograph of employing is U.S. Agilent Agilent-7890 type, is equipped with fid detector.Chromatographiccondition: chromatographic column is HP-5 capillary column; Column temperature is 80 DEG C and keeps 4min10 DEG C/min to be warming up to 100 DEG C; Front injector temperature is 230 DEG C; Detector FID temperature is 250 DEG C; Carrier gas (N
2) flow velocity is 54.0mL/min.Under this condition, standard specimen goes out peak situation as shown in Figure 1, and the appearance time of 1,3-DCP and ECH is respectively 5.1min and 3.7min.
Described enrichment medium final concentration consists of (g/L): Na
2hPO
45.37, KH
2pO
41.36, (NH
4)
2sO
41.25, Na
2eDTA0.06, MnSO
40.002, MgSO
40.001, ZnSO
40.002, FeSO
40.01, solvent is water, and pH value is 6.8.
Described plate screening substratum is (g/L): K
2hPO
45.37, (NH
4)
2sO
41.25, KH
2pO
41.36, yeast extract 0.2, agar 20, solvent is water, and pH value is 6.8.121 DEG C of 20min sterilizings.Add Bromothymol blue 0.06g when being cooled to 50-60 DEG C, 1,3-DCP2mL, after utilizing NaOH to be adjusted to dark green, be down flat plate.
Described slant medium final concentration consists of (g/L): NaCl10.0, yeast powder 5.0, peptone 10.0, agar 20.0, and solvent is water, and pH value is 7.0.
Described fermention medium final concentration consists of (g/L): lactose 8, peptone 6, Na
2hPO
43.2, K
2hPO
41.5, Na
2eDTA0.06, MnSO
40.002, MgSO
40.001, ZnSO
40.002, FeSO
40.01, solvent is water, and pH value is 6.8.
Embodiment 2: the qualification of bacterial strain ZJB-11071
The embodiment of the present invention 1 screens the bacterial strain ZJB-11071 that obtains on slant medium, and cultivate after 24 hours for 37 DEG C and form circular or subcircular, quality is soft, and surface is more coarse, flat, and edge is irregular, glossiness oyster white bacterium colony, diameter 5-7mm.Gramstaining is observed: purple rod-short.Produce gemma, gemma circle or cylindricality, middle life or near middle raw, sporangiocyst is without obviously expanding.
By Biolog (GENIII) automatic microbe verification system, 94 kinds of phenotype tests are carried out to bacterial strain ZJB-11071, comprised 71 kinds of utilization of carbon source situations and 23 kinds of chemosensitivities detections.Analyze the Metabolic Fingerprinting of bacterial strain through Biolog readout instrument, result shows that bacterial strain ZJB-11071 with 22 kinds of chemical substances, sensitive reaction can occur, comparatively large to the utilization ratio of 40 kinds of carbon sources, and lower or can not utilize completely to the utilization of other carbon sources of 31 kinds.Biolog system provides 36h qualification result, as shown in Table 1 and Table 2, determines that bacterial strain ZJB-11071 is bacillus cereus (Bacillus cereus).Simultaneously with the STb gene of bacterial strain ZJB-11071 for template, utilize primer: the 16S rDNA gene of P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3' amplification bacterial strain, after gene product is connected with carrier T, the raw work in Shanghai is entrusted to increase and order-checking to this bacterium 16S rDNA, after obtaining the 16S rDNA sequence of this bacterial strain, NCBI website is retrieved with BLAST the 16S rDNA gene order of related strain in GenBank, and carries out sequence analysis.Based on the qualification of form, physiological and biochemical property and the aspect such as 16S rDNA sequence and phylogenetic analysis, finally determine that this bacterium is bacillus cereus, called after bacillus cereus (Bacillus cereus) ZJB-11071.
Table 1 bacterial strain is to the Utilization ability of 71 kinds of carbon sources on Biolog GEN III plate
Table1Utilization?of71carbon-substrates?by?the?strain?using?Biolog?GENⅢ?microplate
Notes:+,positive；-,negative；B,borderline
Table 2 bacterial strain is to the chemosensitivity of 23 kinds of chemical substances on Biolog GEN III plate
Table2Sensitivity?of23chemical-substrates?by?the?strain?using?Biolog?GENⅢ?microplate
Notes:+,positive；-,negative；B,borderline
After utilizing primer P1 and P2 to carry out pcr amplification, confirm that this fragment physical length is 1544bp through order-checking.The 16S rDNA sequence of bacterial strain ZJB-110711 is for shown in SEQ ID NO:1.
Nucleotides sequence shown in described SEQ ID NO:1 is classified as:
Any nucleotide sequence carrying out the replacement of one or more Nucleotide, disappearance to nucleotide sequence shown in SEQ ID NO:1 or insert that process obtains, as long as itself and this Nucleotide has the homology of more than 90%, all belongs to protection scope of the present invention.
Embodiment 3: the preparation of wet thallus
(1) slant culture
Bacillus cereus ZJB-11071 is seeded to slant medium, cultivates 12h at 30 DEG C, obtain inclined-plane thalline.
Described slant medium final concentration consists of (g/L): NaCl10.0, yeast powder 5.0, peptone 10.0, agar 20.0, and solvent is water, and pH value is 7.0.
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 24h at 30 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, and solvent is water, and pH value is 7.0;
(3) fermentation culture
Be seeded in fermention medium by seed liquor with the inoculum size of volumetric concentration 1%, 30 DEG C, after 150rpm shaking culture 60h, centrifugal 5min under 12000g, collects wet thallus.
Described fermention medium final concentration consists of (g/L): lactose 8, peptone 6, Na
2hPO
43.2, K
2hPO
41.5, Na
2eDTA0.06, MnSO
40.002, MgSO
40.001, ZnSO
40.002, FeSO
40.01, solvent is water, and pH value is 6.8.
Embodiment 4: be the bioconversion reaction of substrate with the chloro-2-propyl alcohol of 1,3-bis-
At 10mL phosphoric acid buffer (50mM, pH7.0) wet thallus 0.5g prepared by embodiment 3 method is added in, substrate 1,3-DCP (1, the chloro-2-propyl alcohol of 3-bis-) final concentration is 20mM, put into shaking bath 35 DEG C, under 150rpm condition, transform 6h (the sulfuric acid termination enzyme that reaction terminates rear 2M is lived).Get 700 μ L conversion fluids in EP pipe, and with 700 μ L extraction into ethyl acetate, leave standstill the centrifugal 2min of 12000r/min after 10min, get upper organic phase and carry out vapor detection.Under similarity condition, not add wet thallus as blank.Result shows that the transformation efficiency of bacillus cereus ZJB-11071 to the chloro-2-propyl alcohol of 1,3-bis-is 42.3%.
The testing conditions of the chloro-2-propyl alcohol (1,3-DCP) of 1,3-bis-and epoxy chloropropane (ECH): the gas chromatograph of employing is U.S. Agilent Agilent-7890 type, is equipped with fid detector.Chromatographiccondition: chromatographic column is HP-5 capillary column; Column temperature is 80 DEG C and keeps 4min, and 10 DEG C/min is warming up to 100 DEG C; Front injector temperature is 230 DEG C; Detector FID temperature is 250 DEG C; Carrier gas (N
2) flow velocity is 54.0mL/min.Under this condition 1,3-DCP and the appearance time of ECH be respectively 5.1min and 3.7min.
Embodiment 5: the bioconversion reaction being substrate with the chloro-2-propyl alcohol (2,3-DCP) of 2,3-bis-
At 10mL phosphoric acid buffer (50mM, pH7.0) add wet thallus 0.5g prepared by embodiment 3 method in, substrate 2,3-DCP final concentration is 20mM, put into shaking bath 35 DEG C, under 150rpm condition, transform 6h (the sulfuric acid termination enzyme that reaction terminates rear 2M is lived).Get 700 μ L conversion fluids in EP pipe, and with 700 μ L extraction into ethyl acetate, leave standstill the centrifugal 2min of 12000r/min after 10min, get upper organic phase and carry out vapor detection.Under similarity condition, not add wet thallus as blank.Result shows that the transformation efficiency of bacillus cereus ZJB-11071 to the chloro-2-propyl alcohol of 2,3-bis-is 21.1%.
The testing conditions of the chloro-2-propyl alcohol of 2,3-bis-and epoxy chloropropane: gas chromatograph is Agilent7890 type, is equipped with fid detector.Chromatographiccondition: chromatographic column: HP-5 capillary column; Column temperature: 150 DEG C keep 7min; Front injector temperature: 230 DEG C; Detector: FID temperature 250 DEG C; Carrier gas (N
2) flow velocity: 54.0mL/min; The appearance time of 2,3-bis-trimethylewne chlorohydrin 3-and epoxy chloropropane is respectively 2.4min and 2.6min.
Embodiment 6: be the bioconversion reaction of substrate with S (-)-4-chloro-3-hydroxyl ethyl butyrate
Add 10mL phosphoric acid buffer (50mM in a kettle., pH7.0), wet thallus 0.5g prepared by embodiment 3 method, the final concentration of S (-)-4-chloro-3-hydroxyl ethyl butyrate is 50mM, in reaction process, stream adds NaCN maintenance pH7.2 ~ 7.5, temperature of reaction is 35 DEG C, rotating speed is 300r/min, transform after 2 hours and sample, sample is after ethyl acetate 1:1 ratio extraction by volume, GC is utilized to detect the content analyzing S (-)-4-chloro-3-hydroxyl ethyl butyrate and R (-) 4-cyano-3-hydroxy ethyl butyrate, result shows that the transformation efficiency of bacillus cereus ZJB-11071 to S (-)-4-chloro-3-hydroxyl ethyl butyrate is 24.6%.
The testing conditions of S (-)-4-chloro-3-hydroxyl ethyl butyrate and R (-) 4-cyano-3-hydroxy ethyl butyrate: gas chromatograph is Agilent7890 type, is equipped with fid detector.Chromatographiccondition: HP-5 capillary column; Column temperature: 165 DEG C keep 4.5min; Injector temperature: 230 DEG C; Detector: FID temperature 250 DEG C; Carrier gas (N2) flow velocity: 54.0mL/min; The appearance time of S (-)-4-chloro-3-hydroxyl ethyl butyrate and R (-) 4-cyano-3-hydroxy ethyl butyrate is respectively 2.9min and 3.4min.
Embodiment 7: be the bioconversion reaction of substrate with the bromo-2-propyl alcohol of 1,3-bis-
At 10mL phosphoric acid buffer (50mM, pH7.0) wet thallus 0.5g prepared by embodiment 3 method is added in, substrate 1, the final concentration of the bromo-2-propyl alcohol of 3-bis-is 20mM, put into shaking bath 35 DEG C, under 150rpm condition, transform 6h (the sulfuric acid termination enzyme that reaction terminates rear 2M is lived).Get 700 μ L conversion fluids in EP pipe, and with 700 μ L extraction into ethyl acetate, leave standstill the centrifugal 2min of 12000r/min after 10min, get upper organic phase and carry out vapor detection.Not add wet thallus as blank under similarity condition.Result shows that the transformation efficiency of bacillus cereus ZJB-11071 to the bromo-2-propyl alcohol of 1,3-bis-is 95%.
The testing conditions of the bromo-2-propyl alcohol of 1,3-bis-and epoxy bromopropane: gas chromatograph is Agilent7890 type, is equipped with fid detector.Chromatographiccondition: chromatographic column: HP-5 capillary column; Column temperature: 110 DEG C keep 7min; Injector temperature: 230 DEG C; Detector: FID temperature 250 DEG C; Carrier gas (N
2) flow velocity: 54.0mL/min; The appearance time of bromo-2 propyl alcohol of 1,3-bis-and epoxy bromopropane is respectively 5.3min and 3.0min.
Reached a conclusion by above embodiment: bacillus cereus ZJB-11071 of the present invention can produce halide alcohol dehalogenase, and then catalysis halohydrin generates corresponding epoxide, R (-)-4-cyano-3-hydroxy ethyl butyrate can also be prepared by catalysis S (-)-4-chloro-3-hydroxyl ethyl butyrate simultaneously.
Claims (8)
1. bacillus cereus (Bacillus cereus) ZJB-11071, is preserved in China typical culture collection center, address: Wuhan, China Wuhan University, 430072, deposit number CCTCC No:M2013314, preservation date on July 5th, 2013.
2. bacillus cereus ZJB-11071 as claimed in claim 1 generates the application in epoxide at biocatalysis halohydrin, it is characterized in that described being applied as: with the wet thallus of bacillus cereus ZJB-11071 after fermentation culture for catalyzer, take halohydrin as substrate, be in the damping fluid of 7.0 ~ 7.5 in pH value, conversion reaction is carried out at 35 DEG C, after conversion reaction terminates, conversion reaction liquid is extracted with ethyl acetate, centrifugal, get upper organic phase and be mixed solution containing epoxide, by mixed solution separation and purification, obtain described epoxide.
3. apply as claimed in claim 2, it is characterized in that the consumption of described catalyzer is in wet thallus quality, final concentration is 30 ~ 80g/L, and the starting point concentration of described substrate is 20 ~ 50mmol/L.
4. apply as claimed in claim 2, it is characterized in that described halohydrin is 1,3-bis-chloro-2-propyl alcohol, 2,3-bis-chloro-2-propyl alcohol or the bromo-2-propyl alcohol of 1,3-bis-.
5. apply as claimed in claim 2, it is characterized in that described wet thallus is prepared as follows:
(1) slant culture
Bacillus cereus ZJB-11071 is seeded to slant medium, cultivates 12h at 30 DEG C, obtain inclined-plane thalline;
Described slant medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, agar 20.0g/L, and solvent is water, and pH value is 7.0;
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 24h at 30 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, and solvent is water, and pH value is 7.0;
(3) fermentation culture
Be seeded in fermention medium by seed liquor with the inoculum size of volumetric concentration 1%, 30 DEG C, after 150rpm shaking culture 60h, centrifugal 5min under 12000g, collects wet thallus;
Described fermention medium final concentration consists of: lactose 8g/L, peptone 6g/L, Na
2hPO
43.2g/L, K
2hPO
41.5g/L, Na
2eDTA0.06g/L, MnSO
40.002g/L, MgSO
40.001g/L, ZnSO
40.002g/L, FeSO
40.01g/L, solvent is water, and pH value is 6.8.
6. the application of bacillus cereus ZJB-11071 in biocatalysis synthesis of chiral pharmaceutical intermediate as claimed in claim 1, it is characterized in that described being applied as: with the wet thallus of bacillus cereus ZJB-11071 after fermentation culture for catalyzer, with S (-)-4-chloro-3-hydroxyl ethyl butyrate for substrate, be in the damping fluid of 7.0 ~ 7.5 in pH value, conversion reaction is carried out at 35 DEG C, after conversion reaction terminates, conversion reaction liquid is extracted with ethyl acetate, centrifugal, get upper organic phase, by mixed solution separation and purification, obtain R (-)-4-cyano-3-hydroxy ethyl butyrate.
7. apply as claimed in claim 6, it is characterized in that the consumption of described catalyzer is in wet thallus quality, final concentration is 30 ~ 80g/L, and the starting point concentration of described substrate is 20 ~ 50mmol/L.
8. apply as claimed in claim 6, it is characterized in that described wet thallus is prepared as follows:
(1) slant culture
Bacillus cereus ZJB-11071 is seeded to slant medium, cultivates 12h at 30 DEG C, obtain inclined-plane thalline;
Described slant medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, agar 20.0g/L, and solvent is water, and pH value is 7.0;
(2) seed culture
Be seeded to seed culture medium from inclined-plane thalline picking one transfering loop thalline, cultivate 24h at 30 DEG C, obtain seed liquor; Described seed culture medium final concentration consists of: NaCl10.0g/L, yeast powder 5.0g/L, peptone 10.0g/L, and solvent is water, and pH value is 7.0;
(3) fermentation culture
Be seeded in fermention medium by seed liquor with the inoculum size of volumetric concentration 1%, 30 DEG C, after 150rpm shaking culture 60h, centrifugal 5min under 12000g, collects wet thallus;
Described fermention medium final concentration consists of: lactose 8g/L, peptone 6g/L, Na
2hPO
43.2g/L, K
2hPO
41.5g/L, Na
2eDTA0.06g/L, MnSO
40.002g/L, MgSO
40.001g/L, ZnSO
40.002g/L, FeSO
40.01g/L, solvent is water, and pH value is 6.8.
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| CN104774828A (en) * | 2015-03-26 | 2015-07-15 | 浙江工业大学 | Recombinant halohydrin dehalogenase, encoding gene, vector, engineering bacteria, and applications of recombinant halohydrin dehalogenase |
| CN110527646A (en) * | 2019-08-20 | 2019-12-03 | 浙江工业大学 | Tropical bacillus WZZ018 and its application |
| CN115011520A (en) * | 2022-06-14 | 2022-09-06 | 南京工业大学 | Bacillus cereus and application thereof in carbonyl reduction |
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| CN104774828A (en) * | 2015-03-26 | 2015-07-15 | 浙江工业大学 | Recombinant halohydrin dehalogenase, encoding gene, vector, engineering bacteria, and applications of recombinant halohydrin dehalogenase |
| CN104774828B (en) * | 2015-03-26 | 2019-02-01 | 浙江工业大学 | Recombinate halide alcohol dehalogenase, encoding gene, carrier, engineering bacteria and its application |
| CN110527646A (en) * | 2019-08-20 | 2019-12-03 | 浙江工业大学 | Tropical bacillus WZZ018 and its application |
| CN110527646B (en) * | 2019-08-20 | 2021-05-11 | 浙江工业大学 | Tropical bacillus WZZ018 and application thereof |
| CN115011520A (en) * | 2022-06-14 | 2022-09-06 | 南京工业大学 | Bacillus cereus and application thereof in carbonyl reduction |
| CN115011520B (en) * | 2022-06-14 | 2023-05-12 | 南京工业大学 | Bacillus cereus and application thereof in carbonyl reduction |
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| CN104232508B (en) | 2016-08-17 |
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