CN101113423B - Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion - Google Patents
Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion Download PDFInfo
- Publication number
- CN101113423B CN101113423B CN2007101180641A CN200710118064A CN101113423B CN 101113423 B CN101113423 B CN 101113423B CN 2007101180641 A CN2007101180641 A CN 2007101180641A CN 200710118064 A CN200710118064 A CN 200710118064A CN 101113423 B CN101113423 B CN 101113423B
- Authority
- CN
- China
- Prior art keywords
- gamma
- lactam
- microbacterium
- under
- culture
- 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.)
- Expired - Fee Related
Links
Images
Abstract
The present invention discloses a microbacterium hydrocarbonoxydans (L29-9) with a preservation number of CGMCC No.2085 and a method for transforming and producing chiral pharmaceutical intermediates (1R, 4S)-2-azabicyclo-(2.2.1)-heptane-5- alkene-3-ketone (hereinafter called (-)gamma-lactam) through the microbacterium hydrocarbonoxydans strain, and the method comprises the steps: (1) filtration of the strains is carried out; (2) the strains are added to a culture medium that contains N- acetyl phenethyl amino acid or carbon source, necessary nitrogen source and nutrients are added at the same time and the pH value of the fermented fluid is adjusted through alkaline substances so as to produce a lactamase, then the (1R, 4S)-2-azabicyclo-(2.2.1)-heptane-5- alkene-3-ketone and (1S, 4R)-2-azabicyclo-(2.2.1)-heptane-5- alkene-3-ketone are obtained by biotransformation racemization of 2-azabicyclo-(2.2.1)-heptane-5- alkene-3-ketone.
Description
Technical field
The present invention relates to a kind of microbacterium and use this bacterial classification produce chiral drug intermediate (1R, 4S)-method of 2-azabicyclo-[2.2.1]-heptane-5-alkene-3-ketone.
Background technology
(1R, 4S)-2-azabicyclo-[2.2.1]-heptane-5-alkene-3-ketone is a kind of very useful pharmaceutical intermediate, it has two kinds of isomer i.e. (1R, 4S) configuration and (1S, 4R) configuration.Many carbocyclic ring purine such as Abacavir and carbovir and pyrimidine nucleoside acid-like substance have anti-HIV effect, and (1R, 4S) compound of configuration is the raw material of synthetic above medicine.Screening obtains can stereo selective hydrolysis racemization 2-azabicyclo-[2.2.1]-heptane-5-alkene-3-ketone and obtain its (1R, 4S) the bacterium producing multi enzyme preparation of configuration of compound, at pharmaceutical industry actual application value is arranged, especially synthetic significant to anti-AIDS drug.
People such as Steven nineteen ninety is at " J.CHEM.SOC.CHEM.COMMUN. (16): 1120～1121.Chemoenzymic synthesis of (-)-carbovir utilizing a whole cell catalyzed resolution of2-Azabicyclo[2.2.1] hept-5-en-3-ones " in the literary composition, utilize two kinds of different bacterium ENZA-1 (Rhedococcusequi NCIB 40231) and ENZA-20 (Pseudomonas solanacearum NCIB 40249) intact cell biology
Transform the research of hydrolysis (+/-) gamma-lactam, produced the enantiomorph of two kinds of configurations respectively.
Summary of the invention
One of technical issues that need to address of the present invention are to disclose a kind of bacteria culture that can transform production (-) gamma-lactam, promptly a kind of microbacterium (Microbacterium hydrocarbonoxydans) (L29-9), preserving number is: CGMCC No.2085.
Two of the technical issues that need to address of the present invention are to disclose a kind of method for culturing bacteria.
Three of the technical issues that need to address of the present invention are to disclose a kind of method of producing (-) gamma-lactam that transforms with the bacterium intact cell, make its highly selective hydrolysis (+) gamma-lactam, enantiomeric excess value reaches more than 99.5%, to obtain highly purified (-) gamma-lactam.
It is a kind of microbacterium (Microbacteriumhydrocarbonoxydans) that the present invention is used for transforming the microorganism that produces (-) gamma-lactam.
This bacterial classification has following character:
1. form physiological and biochemical property:
Morphological specificity: bacillus, bacterium colony are light yellow, and be moistening, thickness, and circle, neat in edge is opaque.
Physiological and biochemical property: gram positive bacterium, produce the gamma-lactam lytic enzyme.
2. the substratum of Cai Yonging:
Plate screening substratum: Na
2HPO
412H
2O 0.7%; KH
2PO
40.2%; MgSO
47H
2O 0.02%; CaCl
22H
2O 0.001%; FeSO
47H
2O 0.0007%; ZnSO
40.000001%; NH
4Cl 0.2%; N-acetylphenylalanine 0.3%; Agar powder 2% (being mass/volume per-cent) is dissolved in deionized water, is 7.0 with the NaOH adjust pH, the 105kPa 30min that sterilizes.
Substratum is preserved on the inclined-plane: yeast powder 0.5%; Tryptones 1%; NaCl 1%; N-acetylphenylalanine 0.3%; Agar powder 2% (being mass/volume per-cent) is dissolved in deionized water, is 7.0 with the NaOH adjust pH, the 105kPa 30min that sterilizes.Liquid produces enzyme substratum: Na
2HPO
412H
2O 0.7%; KH
2PO
40.2%; MgSO
47H
2O 0.02%; CaCl
22H
2O 0.001%; FeSO
47H
2O 0.0008%; N-acetylphenylalanine 0.3%; Carbon source 0.5%; Nitrogenous source 0.5% (being mass/volume per-cent) is dissolved in deionized water, is 7.0 with the NaOH adjust pH, the 105kPa 30min that sterilizes.Liquid fermentation medium: Na
2HPO
412H
2O 0.7%; KH
2PO
40.2%;
MgSO
47H
2O 0.02%; CaCl
22H
2O 0.001%; FeSO
47H
2O 0.0008%; N-acetylphenylalanine 0.3%; Carbon source 0.5%; Nitrogenous source 0.5% (being mass/volume per-cent) is dissolved in deionized water, is 7.0 with the alkaline matter adjust pH, the 105kPa 30min that sterilizes.
Carbon source: a kind of in glucose, sucrose, wood sugar, citric acid, starch or the lactose.
Nitrogenous source: a kind of in extractum carnis, Tryptones, yeast powder or the corn steep liquor.
3. culture condition:
Culture temperature: 25～50 ℃, temperature preferably: 25～40 ℃.
Training method: carry out fermentation culture under the aerobic conditions.
The intact cell that utilization of the present invention contains the gamma-lactam lytic enzyme transforms the method for (/-) gamma-lactam preparation (-) gamma-lactam, and its sequence of steps is as follows:
(1) bacterial screening: the environment soil sample of gathering is coated the plate screening substratum after with the sterilized water gradient dilution, and 30 ℃ of aerobic conditions were cultivated 4 days down, chose single bacterium colony and were kept at the inclined-plane and preserve on the substratum cultivation 2 days and be stored in 4 ℃ of refrigerators.To preserve under the bacterial strain aseptic condition and be connected in the liquid fermentation medium with transfering loop, under 30 ℃ of conditions, shaking table shaking culture 48 hours, with under 8000 rev/mins of conditions of bacterium liquid of cultivating centrifugal 5 minutes, the collecting precipitation thalline, and, obtain wet thallus with the phosphate buffered saline buffer washing.
Wet thallus and 2/L (+/-) gamma-lactam solution is mixed, and wet thallus concentration is 1% mass/volume per-cent, at 30 ℃, and under pH 7.0 conditions, 200 rev/mins of vibrations 12 hours; Centrifugal 3 minutes with 10000 rev/mins, the supernatant liquor that obtains detects with chirality HPLC, the bacterial strain that productive rate reaches more than 38% and the ee value reaches more than 77% that screening obtains (-) gamma-lactam carries out preservation, and called after microbacterium (Microbacterium hydrocarbonoxydans) (L29-9), and preserving number is: CGMCCNo.2085.Preservation date on June 28th, 2007, depositary institution is CGMCC.
(2) slant culture: the microbacterium that screening is obtained is inoculated on the inclined-plane preservation substratum, cultivates 20～50 hours for 25～40 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the liquid fermentation medium with transfering loop under the aseptic condition, under 25～40 ℃ of conditions, shaking table shaking culture 20～50 hours makes seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation produces in the enzyme substratum in liquid, and under 25～40 ℃ of aerobic conditions, shaking table shaking culture 20～50 hours makes fermented liquid;
(5) collect thalline: got under 8000～10000 rev/mins of conditions of fermented liquid of step (4) centrifugal 3～5 minutes, the collecting precipitation thalline, and, obtain wet thallus as biological catalyst with the phosphate buffered saline buffer washing;
(6) bio-transformation experiment: the biological catalyst in the step (5) and 0.5～5g/L (+/-) gamma-lactam solution is mixed; Wet thallus concentration is 0.1～1% mass/volume per-cent; At 25～50 ℃, under pH 6.0～8.0 conditions, 200～220 rev/mins vibrated 1～12 hour;
(7) sample separation: with 5000～10000 rev/mins centrifugal 3～5 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure the content of (+) gamma-lactam and (-) gamma-lactam, calculate the productive rate that the ee value reaches (-) gamma-lactam.
In aforesaid method, chirality HPLC adopts CHIRALPAK AS-H 250 * 4.6mm, moving phase: acetonitrile: Virahol (80: 20).Measure by chirality HPLC and to obtain the ee value that gamma-lactam that microbacterium transforms racemization obtains (-) gamma-lactam and be up to 99.8%, the productive rate of (-) gamma-lactam is up to 42.5%.
Description of drawings
Fig. 1 is the chirality HPLC collection of illustrative plates of racemize gamma-lactam.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Fig. 2 is the method according to the embodiment of the invention 1, the chirality HPLC collection of illustrative plates of the gamma-lactam after the conversion.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Fig. 3 is the method according to the embodiment of the invention 2, the chirality HPLC collection of illustrative plates of the gamma-lactam after the conversion.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Fig. 4 is the method according to the embodiment of the invention 3, the chirality HPLC collection of illustrative plates of the gamma-lactam after the conversion.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Fig. 5 is the method according to the embodiment of the invention 4, the chirality HPLC collection of illustrative plates of the gamma-lactam after the conversion.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Fig. 6 is the method according to the embodiment of the invention 4, the chirality HPLC collection of illustrative plates of the gamma-lactam after the conversion.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Fig. 7 is the method according to the embodiment of the invention 4, the chirality HPLC collection of illustrative plates of the gamma-lactam after the conversion.No. 1 peak is a solvent ethyl acetate, and No. 2 peaks are (+) gamma-lactam, and No. 3 peaks are (-) gamma-lactam.
Embodiment
Bacterial screening: will coat the plate screening substratum after with the sterilized water gradient dilution from the some local environment soil samples of gathering in Ningxia, Tibet, Beijing, 30 ℃ of aerobic conditions were cultivated 4 days down, chose single bacterium colony and were kept at the inclined-plane and preserve and cultivate 2 and be stored in 4 ℃ of refrigerators on the substratum.
To preserve under the bacterial strain aseptic condition and be connected in the liquid fermentation medium with transfering loop, under 30 ℃ of conditions, shaking table shaking culture 40 hours, with under 8000 rev/mins of conditions of bacterium liquid of cultivating centrifugal 5 minutes, the collecting precipitation thalline, and, obtain wet thallus with the phosphate buffered saline buffer washing.Wet thallus and 2g/L (+/-) gamma-lactam solution is mixed, and wet thallus concentration is 0.5% mass/volume per-cent, at 30 ℃, and under pH 7.0 conditions, 200 rev/mins of vibrations 12 hours; Centrifugal 3 minutes with 10000 rev/mins, the supernatant liquor that obtains detects with chirality HPLC, screening obtains productive rate and the high bacterial strain of ee value carries out preservation, and called after microbacterium (Microbacterium hydrocarbonoxydans) (L29-9), and preserving number is: CGMCCNo.2085.
Selected bacterial classification is the microbacterium that screening obtains in following examples.
Embodiment 1: the method for (+/-) gamma-lactam preparation (-) gamma-lactam that utilizes the intact cell of microbacterium to transform
(1) bacterial classification is selected: select microbacterium (Microbacterium hydrocarbonoxydans) CGMCC No.2085 for use;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 48 hours for 30 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the 5ml liquid fermentation medium with transfering loop under the aseptic condition, under 30 ℃ of conditions, shaking table shaking culture 24 hours makes seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in the 50ml liquid fermentation medium, and under 30 ℃ of conditions, shaking table shaking culture 40 hours makes fermented liquid;
(5) collect thalline: got under 8000 rev/mins of conditions of fermented liquid of step (4) centrifugal 5 minutes, the collecting precipitation thalline, and, promptly make biological catalyst with the phosphate buffered saline buffer washing;
(6) transformation experiment: the biological catalyst in the step (5) and 2g/L (+/-) gamma-lactam solution is mixed, wet thallus concentration 0.5% mass/volume per-cent, at 30 ℃, under pH 7.0 conditions, 200 rev/mins of vibrations 2 hours;
(7) sample separation: with 10000 rev/mins centrifugal 3 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure the content of (+) gamma-lactam and (-) gamma-lactam, obtaining the ee value is 99.8%, productive rate 42.5%.
Embodiment 2: the method for (+/-) gamma-lactam preparation (-) gamma-lactam that utilizes the intact cell of microbacterium to transform
(1) bacterial classification is selected: select microbacterium (Microbacterium hydrocarbonoxydans) CGMCC No.2085 for use;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 50 hours for 25 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the 5ml liquid fermentation medium with transfering loop under the aseptic condition, under 25 ℃ of conditions, shaking table shaking culture 20 hours makes seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in the 50ml liquid fermentation medium, and under 25 ℃ of conditions, shaking table shaking culture 50 hours makes fermented liquid;
(5) collect thalline: got under 10000 rev/mins of conditions of fermented liquid of step (4) centrifugal 3 minutes, the collecting precipitation thalline, and, promptly make biological catalyst with the phosphate buffered saline buffer washing;
(6) transformation experiment: the biological catalyst in the step (5) and 0.5g/L (/-) gamma-lactam solution is mixed, wet thallus concentration 0.1% mass/volume per-cent, at 25 ℃, under the pH8.0 condition, 200 rev/mins of vibrations 12 hours;
(7) sample separation: with 10000 rev/mins centrifugal 3 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure the content of (+) gamma-lactam and (-) gamma-lactam, obtaining the ee value is 97.2%, productive rate 20.8%.
Embodiment 3: the method for (+/-) gamma-lactam preparation (-) gamma-lactam that utilizes the intact cell of microbacterium to transform
(1) bacterial classification is selected: select microbacterium (Microbacterium hydrocarbonoxydans) CGMCC No.2085 for use;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 20 hours for 40 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the 5ml liquid fermentation medium with transfering loop under the aseptic condition, under 40 ℃ of conditions, shaking table shaking culture 20 hours makes seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in the 50ml liquid fermentation medium, and under 40 ℃ of conditions, shaking table shaking culture 20 hours makes fermented liquid;
(5) collect thalline: got under 10000 rev/mins of conditions of fermented liquid of step (4) centrifugal 3 minutes, the collecting precipitation thalline, and, promptly make biological catalyst with the phosphate buffered saline buffer washing;
(6) transformation experiment: the biological catalyst in the step (5) and 5g/L (+/-) gamma-lactam solution is mixed, wet thallus concentration 0.5% mass/volume per-cent, at 40 ℃, under pH 6.0 conditions, 200 rev/mins of vibrations 1 hour;
(7) sample separation: with 8000 rev/mins centrifugal 5 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure the content of (+) gamma-lactam and (-) gamma-lactam, obtaining the ee value is 90.3%, productive rate 32.4%.
Embodiment 4: the method for (+/-) gamma-lactam preparation (-) gamma-lactam that utilizes the intact cell of microbacterium to transform
(1) bacterial classification is selected: select microbacterium (Microbacterium hydrocarbonoxydans) CGMCC No.2085 for use;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 48 hours for 30 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the 5ml liquid fermentation medium with transfering loop under the aseptic condition, under 30 ℃ of conditions, shaking table shaking culture 20 hours makes seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in the 50ml liquid fermentation medium, and under 30 ℃ of conditions, shaking table shaking culture 50 hours makes fermented liquid;
(5) collect thalline: got under 8000 rev/mins of conditions of fermented liquid of step (4) centrifugal 5 minutes, the collecting precipitation thalline, and, promptly make biological catalyst with the phosphate buffered saline buffer washing;
(6) transformation experiment: the biological catalyst in the step (5) and 2g/L (+/-) gamma-lactam solution is mixed, wet thallus concentration 1% mass/volume per-cent, at 30 ℃, under pH 6.0 conditions, 200 rev/mins of vibrations 2 hours;
(7) sample separation: with 10000 rev/mins centrifugal 3 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure (+) γ-Nei acyl
The content of amine and (-) gamma-lactam, obtaining the ee value is 99.5%, productive rate 40%.
Embodiment 5: the method for (+/-) gamma-lactam preparation (-) gamma-lactam that utilizes the intact cell of microbacterium to transform
(1) bacterial classification is selected: select microbacterium (Microbacterium hydrocarbonoxydans) CGMCCNo.2085 for use;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 48 hours for 35 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the 5ml liquid fermentation medium with transfering loop under the aseptic condition, under 35 ℃ of conditions, shaking table shaking culture 20 hours,
Make seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in the 50ml liquid fermentation medium, and under 35 ℃ of conditions, shaking table shaking culture 48 hours makes fermented liquid;
(5) collect thalline: got under 8000 rev/mins of conditions of fermented liquid of step (4) centrifugal 5 minutes, the collecting precipitation thalline, and, promptly make biological catalyst with the phosphate buffered saline buffer washing;
(6) transformation experiment: the biological catalyst in the step (5) and 1g/L (+/-) gamma-lactam solution is mixed, wet thallus concentration 0.5% mass/volume per-cent, at 35 ℃, under pH 6.0 conditions, 200 rev/mins of vibrations 10 hours;
(7) sample separation: with 10000 rev/mins centrifugal 3 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure the content of (+) gamma-lactam and (-) gamma-lactam, obtaining the ee value is 97.43%, productive rate 40.07%.
Embodiment 6: the method for (+/-) gamma-lactam preparation (-) gamma-lactam that utilizes the intact cell of microbacterium to transform
(1) bacterial classification is selected: select microbacterium (Microbacterium hydrocarbonoxydans) CGMCC No.2085 for use;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 48 hours for 32 ℃;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the 5ml liquid fermentation medium with transfering loop under the aseptic condition, under 32 ℃ of conditions, shaking table shaking culture 24 hours makes seed;
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in the 50ml liquid fermentation medium, and under 32 ℃ of conditions, shaking table shaking culture 40 hours makes fermented liquid;
(5) collect thalline: got under 8000 rev/mins of conditions of fermented liquid of step (4) centrifugal 5 minutes, the collecting precipitation thalline, and, promptly make biological catalyst with the phosphate buffered saline buffer washing;
(6) transformation experiment: the biological catalyst in the step (5) and 1g/L (+/-) gamma-lactam solution is mixed, wet thallus concentration 0.1% mass/volume per-cent, at 32 ℃, under the pH8.0 condition, 220 rev/mins of vibrations 12 hours;
(7) sample separation: with 5000 rev/mins centrifugal 5 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
Get 5 μ L sample feedings in the step (7), utilize chirality HPLC to measure the content of (+) gamma-lactam and (-) gamma-lactam, obtaining the ee value is 96.16%, productive rate 48.0%.
Carbon source among the above embodiment in the used liquid fermentation medium, nitrogenous source and alkaline matter see the following form:
Claims (5)
1. one kind is the method for bacterial classification production (-) gamma-lactam with the microbacterium, and its feature comprises:
(1) bacterial classification is selected: selecting microbacterium (Microbacterium hydrocarbonoxydans) preserving number for use is CGMCC No.2085;
(2) slant culture: above-mentioned bacterial classification inoculation is preserved on the substratum in the inclined-plane, cultivated 20～50 hours for 25～50 ℃; The inclined-plane is preserved substratum and is consisted of: yeast powder 0.5%; Tryptones 1%; NaCl1%; N-acetylphenylalanine 0.3%; Agar powder 2%; Above content is mass/volume per-cent, is dissolved in deionized water, is 7.0 with the NaOH adjust pH, the 105kPa 30min that sterilizes;
(3) seed culture: the bacterial strain with step (2) is cultivated, be connected in the liquid fermentation medium with transfering loop under the aseptic condition, under 25～40 ℃ of conditions, shaking table shaking culture 25～50 hours makes seed; Wherein liquid fermentation medium consists of: Na
2HPO
412H
2O 0.7%; KH
2PO
40.2%; MgSO
47H
2O 0.02%; CaCl
22H
2O 0.001%; FeSO
47H
2O 0.0008%; N-acetylphenylalanine 0.3%; Carbon source 0.5%; Nitrogenous source 0.5%; More than being mass/volume per-cent, being dissolved in deionized water, is 7.0 with the alkaline matter adjust pH, the 105kPa 30min that sterilizes.
(4) shake-flask culture: with 5% volume/volume per-cent inoculum size, inoculation is in liquid fermentation medium, and under 25～50 ℃ of aerobic conditions, shaking table shaking culture 20～50 hours makes fermented liquid;
(5) collect thalline: got under 8000～10000 rev/mins of conditions of fermented liquid of step (4) centrifugal 3～5 minutes, the collecting precipitation thalline, and, obtain wet thallus as biological catalyst with the phosphate buffered saline buffer washing;
(6) bio-transformation experiment: the biological catalyst wet thallus in the step (5) and 0.5～5g/L (+/-) gamma-lactam solution is mixed; Wet thallus concentration is 0.1～1% mass/volume per-cent; At 25～50 ℃, under pH6.0～8.0 conditions, 200～220 rev/mins vibrated 1～12 hour;
(7) sample separation: with 5000～10000 rev/mins centrifugal 3～5 minutes, biological catalyst in the separating step (6) obtains supernatant liquor and is sample;
2. method according to claim 1 is characterized in that: cultivation and invert point are 25～40 ℃.
3. method according to claim 1 is characterized in that: described alkaline matter comprises a kind of in NaOH, KOH, the ammoniacal liquor.
4. method according to claim 1 is characterized in that: described carbon source comprises a kind of in glucose, sucrose, wood sugar, citric acid, starch or the lactose.
5. method according to claim 1 is characterized in that: described nitrogenous source comprises a kind of in extractum carnis, Tryptones, yeast powder or the corn steep liquor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101180641A CN101113423B (en) | 2007-06-28 | 2007-06-28 | Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007101180641A CN101113423B (en) | 2007-06-28 | 2007-06-28 | Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101113423A CN101113423A (en) | 2008-01-30 |
| CN101113423B true CN101113423B (en) | 2011-08-17 |
Family
ID=39021954
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2007101180641A Expired - Fee Related CN101113423B (en) | 2007-06-28 | 2007-06-28 | Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101113423B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899406B (en) * | 2010-04-27 | 2012-02-22 | 北京大学 | Bacterial strain for removing polycyclic aromatic hydrocarbons and/or degrading polycyclic aromatic hydrocarbons and application thereof |
| CN101921742B (en) * | 2010-06-23 | 2012-09-12 | 中国科学院微生物研究所 | (+) gamma-lactamase with activity on splitting racemate gamma-lactam as well as coded gene and application thereof |
| CN102796720A (en) * | 2012-08-16 | 2012-11-28 | 北京化工大学 | (+)gamma-lactamase with racemate gamma-lactam resolution activity and its application |
| CN104164469B (en) * | 2014-03-06 | 2017-02-22 | 北京化工大学 | Method for using Candida antarctica lipase B to produce ticagrelor chiral medicine intermediate |
| CN104630194A (en) * | 2015-02-01 | 2015-05-20 | 北京化工大学 | (+)-gamma-lactamase from microbacterium as well as coding gene and application of (+)-gamma-lactamase |
| CN108486021B (en) * | 2018-05-24 | 2021-08-31 | 苏州中益世纪生态环境设计研究有限公司 | Microbial strain, screening method thereof and application of microbial strain in heavy metal contaminated soil treatment |
| CN110205268B (en) * | 2019-06-04 | 2020-07-28 | 江苏师范大学 | Microbacterium and application thereof in preparation of microbial flocculant by converting hydrolysate of reed straw |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807581A (en) * | 2004-01-30 | 2006-07-26 | 华北制药集团有限责任公司 | Engineered bacteria producing penicillium chrysogenum and its uses in producing 7-amido -3-deacetoxy cefaeicosanoic acid |
-
2007
- 2007-06-28 CN CN2007101180641A patent/CN101113423B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807581A (en) * | 2004-01-30 | 2006-07-26 | 华北制药集团有限责任公司 | Engineered bacteria producing penicillium chrysogenum and its uses in producing 7-amido -3-deacetoxy cefaeicosanoic acid |
Non-Patent Citations (2)
| Title |
|---|
| 李海泉，等，.产γ－内酰胺水解酶菌株的筛选及发酵条件研究.微生物学报46 4.2006,46(4),571-575，具体参见摘要、讨论，2.1.3的最后一段. |
| 李海泉,等,.产γ-内酰胺水解酶菌株的筛选及发酵条件研究.微生物学报46 4.2006,46(4),571-575,具体参见摘要、讨论,2.1.3的最后一段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101113423A (en) | 2008-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101113423B (en) | Microbacterium and method for producing chirality pharmaceutical intermediate compound by using the bacterial conversion | |
| CN104342390B (en) | A kind of Sinorhizobium meliloti strain and combinations thereof and application | |
| CN101165168A (en) | Streptomycete and method for producing vanillin by using the same to biologically transform ferulic acid | |
| US11085059B2 (en) | Methylopila sp. and use thereof in selective resolution preparation of (S)-α-ethyl-2-oxo-1-pyrrolidineacetate | |
| CN100345974C (en) | Microbiological preparation method of S-(+)-2,2-dimethyl cyclo propyl formamide | |
| CN101363014B (en) | Method for producing gamma-glutamyl transpeptidase using bacillus subtilis | |
| CN101619299B (en) | Rhodococcus ruber and method for preparing 5-cyanovaleramide by utilizing same | |
| CN102994429B (en) | Method for preparing (S)-alpha-ethyl-2-oxyen-1-pyrrolidine acetic acid ester through microorganism catalysis and bacterial strain | |
| CN102719378A (en) | Method for preparing (-) gamma-lactam by catalysis asymmetry of microorganism | |
| CN101134943B (en) | Bacillus alcaligenes and method for preparing single enantiomer amygdalic acid | |
| CN103146595B (en) | Bacillus subtilis and method for fermentation production of D- ribose | |
| CN101457214A (en) | Screen method of pseudomonas and method for converting the same to produce surfactants | |
| CN103589665B (en) | Celebrating sheng, a reed pipe wind instrument rhodococcus and the application in preparation (S)-4-chloro-3-hydroxyl ethyl butyrate thereof | |
| CN108410749B (en) | Method for preparing (-) gamma-lactam by asymmetric hydrolysis of marine low-temperature (+) gamma-lactamase | |
| CN114456980B (en) | Gamma-polyglutamic acid high-yield strain and application thereof | |
| CN101457250A (en) | Method for synthesizing betulic acid from betulin through microbial cell bioconversion | |
| CN102120977B (en) | Microbacterium chocolatum and method for preparing (4S,5R)-half ester by using same | |
| CN105112320B (en) | A kind of Burkholderia gladioli strain and its method for fermentation production of alkaline lipase | |
| CN100547067C (en) | Brevibacterium epidermidis ZJB-07021 and the preparation (S)-2, the application in the 2-dimethyl-cyclopropane carboxamide | |
| CN101240257B (en) | Micro-bacilus, mutagenesis method and application thereof in chiral medicament intermediate production | |
| CN1139546C (en) | Biological flocculant prepared with Nocardia bacteria | |
| CN102952761A (en) | Nocardia sp. capable of converting quininone into (R)-3-quinuclidinol and conversion method | |
| BG98484A (en) | Separation of arylalkanic acid | |
| JPH0714355B2 (en) | Method for preparing L-carnitine | |
| CN109749968A (en) | One plant of biocatalysis synthesizes Bei Laisi Bacillus strain and its application of (R) -1,3 butylene glycol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20110817 Termination date: 20130628 |