CN105586289A - Pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and screening and application thereof - Google Patents
Pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and screening and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms and particularly relates to pseudomonas allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam and a method using pseudomonas to split (+/-)gamma-lactam. According to the method, a microorganism strain allowing (+/-)gamma-lactam to be split to obtain (-)gamma-lactam is screened from soil and identified as pseudomonas granadensis B6; (+/-)gamma-lactam is hydrolyzed in a water-phase system selectively in a three-dimensional mode to prepare (-)gamma-lactam. Under the condition that concentration of a substrate is 300 g/L, extracting, drying, evaporating, concentrating, cooling and crystallizing are carried out to obtain the product (-)gamma-lactam, optical purity (Ee value) ranges from 95.0% to 99.9%, and chemical purity ranges from 95% to 99%. The method of catalyzing and asymmetrically hydrolyzing (+/-)gamma-lactam through organisms to prepare (-)gamma-lactam has the advantages of being high in stereoselectivity, large in reaction yield and high in product concentration.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of can split (+/-) gamma-lactam and obtain (-) γ-NeiThe pseudomonad of acid amides, this pseudomonad is Granada pseudomonad (PseudomonasgranadensisB6).
Background technology
Abacavir and Peramivir are respectively the specific medicaments for the treatment of AIDS and bird flu and first influenza. Current grindsStudy carefully result and show, the enzymology synthetic method taking optically pure (-) gamma-lactam as initial compounds, is synthetic these two kinds of spiesEasy, the most economic method of effect medicine. Find microorganism can selectively split (+/-) gamma-lactam obtain (-) γ-NeiBefore acid amides, the preparation of bibliographical information optically pure (-) gamma-lactam generally adopts the method for chemical asymmetric syntheses. UseThe shortcoming of this method is: raw material costliness, and step is loaded down with trivial details, and cost is higher, and can cause more serious environmental pollution, produces simultaneouslyIn thing, conventionally contain metal impurities. Form therewith sharp contrast, with microorganism catalyze and synthesize optically pure (-) γ-It is high that lactams has the gentle efficiency of reaction condition, the high and environment amenable feature of product purity.
Fractionation (+/-) gamma-lactam that mainly concentrates on the high enantioselectivity of screening about the research of the method obtains (-)The production bacterial strain of gamma-lactam. Domestic correlative study is less, and Li Haiquan etc., taking N-acetylphenylalanine as sole carbon source, screenGo out the bacterial strain L29-9 that a strain selectively splits (+/-) gamma-lactam and obtain (-) gamma-lactam. [Li Haiquan etc.: microorganismJournal, 46 (4): 57l-575,2006] this bacterium is through being accredited as oxygenated hydrocarbon microbacterium (MicrobacteriumHydrocarbonoxydans). Chen Honggan etc., taking N-acetylphenylalanine as sole carbon source, have filtered out a strain and have selectively split(+/-) gamma-lactam obtains the Delftiasp. bacterial strain of (-) gamma-lactam; [Chen Honggan etc.: Chinese biological engineering magazine, 32(9): 41-47,2012]. Do not utilize at present the relevant report of Granada pseudomonad screening.
Summary of the invention
In order to address the above problem, one of object of the present invention is to provide one can split (+/-) gamma-lactam and obtainsThe pseudomonad of (-) gamma-lactam.
This pseudomonad is Granada pseudomonad (PseudomonasgranadensisB6), in October, 2015Within 10th, be preserved in Chinese Typical Representative culture collection center, deposit number is: CCTCCM2015596, depositary institution address: ChinaWuhan Wuhan University.
Two of object of the present invention has been to provide described (+/-) gamma-lactam that can split and has obtained the vacation of (-) gamma-lactamThe cultural method of monad.
Three of object of the present invention has been to provide described pseudomonad fractionation (+/-) gamma-lactam and has obtained (-) γ-Nei acylThe method of amine.
The present invention is achieved through the following technical solutions:
A kind of pseudomonad that can split (+/-) gamma-lactam and obtain (-) gamma-lactam; It is at Chinese Typical Representative cultureThe deposit number at preservation center is: CCTCCM2015596.
The screening of described pseudomonad, is specially:
(1) just cultivate: get mud from lakebed, with coating on culture medium after sterilized water dilution, 30 DEG C of cultivations, 2 days;
The culture medium that described primary dcreening operation is cultivated is: NH4CL2.0～10g，KH2PO40.01～10g，Na2HPO40.01～10g，MgSO40.01～1g，CaCL20.01～0.5g, (+/-) gamma-lactam 1～10g, micro-100 μ L, agar powder10～30g; Wherein trace element calculates and comprises according to every liter of addition: CaCL2·2H2O3.6g，ZnO2.0g，CuCL·2H2O0.85g，NaMoO·2H2O4.8g，MnCL2·4H2O2.0g，FeCL3·6H2O5.4g，CoCL2·6H2O2.4g；
(2) the picking list bacterium colony separation and purification list bacterium colony of again ruling on screening and culturing base;
Picking list colony morphology characteristic is: bacterium colony circle, neat in edge, protuberance are moistening, and white is slightly yellow, bacterium colony insideBe mucus shape, Gram's staining is negative;
(3) enzymatic production is cultivated: first the single bacterium colony out of picking described in step (2) is proceeded to enzymatic production and cultivated1～3 day, described enzymatic production culture medium composition was counted with g/L: glucose 5.0g, NH4CL2.0～10g，KH2PO40.01～10g，Na2HPO40.01～10g，MgSO40.01～1g，CaCL20.01～0.5g, (+/-) gamma-lactam 2g, FeSO40.08g；
(4) the bacterium liquid of above-mentioned enzymatic production being cultivated carries out described (+/-) gamma-lactam that can split of centrifugal acquisition and obtainsThe pseudomonad of (-) gamma-lactam, for subsequent use.
Preferably, described in step (1), mud is taken from Ai Xihu.
Preferably, 20～40 DEG C of the temperature of wherein just cultivating in described step (1), pH3～10.
Energy fractionation (+/-) gamma-lactam obtains the method for (-) gamma-lactam, and employing can split (+/-) gamma-lactam and obtainTo the microbial strains of (-) gamma-lactam, in aqueous phase system, taking (+/-) gamma-lactam as substrate, carry out asymmetric hydrolysisFractionation prepares described (-) gamma-lactam.
Concrete steps are as follows:
(1) microbial strains: described energy fractionation (+/-) gamma-lactam obtains the pseudomonad of (-) gamma-lactam;
(2) first the pseudomonad described in step (1) is carried out to centrifugal acquisition wet thallus for subsequent use;
(3) preparation of fractionation system: (+/-) gamma-lactam of preparing 2g/L～600g/L with phosphate buffer;
(4) split (+/-) gamma-lactam and obtain (-) gamma-lactam: by (+/-) γ-Nei acyl preparing of step (3)Amine joins in the wet thallus of step (2) and reacts.
(5) processing and the analysis of fractionation liquid: by the liquid after gained microorganism resolution reaction in step (4), centrifugal removingRemove thalline, get supernatant and adopt and be extracted with ethyl acetate, after extract dilution, carry out chirality HPLC detection; Chromatographic column model isCHIRALPAKAS-H250 × the 4.6mm of Daicel company; Mobile phase is isopropyl alcohol/acetonitrile (v/v)=80/20; Flow velocity 0.5mL/Min; Detect wavelength 230nm.
(6) separation and Extraction of product: adopt gained thalline in dichloromethane extraction step (5), and successively through super-dry, steamingSend out concentrated, crystallisation by cooling, finally obtain described (-) gamma-lactam;
Preferably, pH3～10 of phosphate buffer in described step (3), concentration is 0.01～0.5mol/L.
Preferably, in described step (4), thalline final concentration is 0.5g/L～100g/L wet thallus, and reaction temperature is 20～60DEG C, the reaction time is 1～24h, shaking speed is 100～300rpm.
Preferably, in described step (5) at 20～80 DEG C crystallisation by cooling at evaporation and concentration, 1～4 DEG C.
Preferably, the optical purity of (-) gamma-lactam described in described step (6) (Ee value) 95.0%～100%, changesLearning purity is 95%～99%.
The fractionation with high enantioselectivity (+/-) gamma-lactam that the present invention utilizes screening to obtain obtains (-) γ-NeiThe bacterial strain Granada pseudomonad (PseudomonasgranadensisB6) of acid amides, optional water in aqueous phase systemSeparate racemic gamma-lactam and obtain (-) gamma-lactam, under 300g/L concentration of substrate condition, the light of product (-) gamma-lactamLearn purity (Ee value) and reach 100%, product obtains optically pure (-) gamma-lactam through extraction, evaporation and concentration, crystallisation by cooling, producesThing optical purity (Ee value) is 95.0%～99.9%, and chemical purity is 95%～99%. This microorganism catalysis asymmetric hydrolysisThe method of preparation (-) gamma-lactam has the feature of highly-solid selectively, high reaction yield, high production concentration.
Brief description of the drawings
Fig. 1 is that the microscope (1000X) of pseudomonad in the present invention is observed photo,
Fig. 2 is the flat-plate bacterial colony form of pseudomonad of the present invention.
Detailed description of the invention
The following specific embodiments of the present invention is to make a detailed explanation.
Embodiment 1
1. can split (+/-) gamma-lactam and obtain the screening of the pseudomonad of (-) gamma-lactam:
(1) just cultivate: get mud from Ai Xi lakebed, with coating on culture medium after sterilized water dilution, 30 DEG C of cultivations, 2 daysAfter;
Described primary dcreening operation culture medium is: NH4CL2.0g，KH2PO41.5g，Na2HPO41.5g，MgSO40.2g，CaCL20.1g, (+/-) gamma-lactam 2g, micro-100 μ L, agar powder 20g; Wherein trace element calculates according to every liter of additionComprise: CaCL2·2H2O3.6g，ZnO2.0g，CuCL·2H2O0.85g，NaMoO·2H2O4.8g，MnCL2·4H2O2.0g，FeCL3·6H2O5.4g，CoCL2·6H2O2.4g；
(2) the picking list bacterium colony separation and purification list bacterium colony of again ruling on screening and culturing base;
Picking list colony morphology characteristic is: bacterium colony circle, neat in edge, protuberance are moistening, and white is slightly yellow, bacterium colony insideBe mucus shape, Gram's staining is negative;
(3) enzymatic production is cultivated: first the single bacterium colony out of picking described in step (2) is proceeded to enzymatic production and cultivated1～3 day, described enzymatic production culture medium composition was counted with g/L: glucose 5g, NH4CL2.0g，KH2PO42g，Na2HPO47g，MgSO40.4g，CaCL20.01g, (+/-) gamma-lactam 2g, FeSO40.08g; Again by centrifugal above-mentioned zymotic fluid acquisitionWet thallus is for subsequent use;
(4) the bacterium liquid of above-mentioned enzymatic production being cultivated carries out described (+/-) gamma-lactam that can split of centrifugal acquisition and obtainsThe pseudomonad of (-) gamma-lactam, for subsequent use.
2. fractionation (+/-) gamma-lactam obtains the method for (-) gamma-lactam:
(1) microbial strains: above-mentioned pseudomonad;
(2) first the pseudomonad described in step (1) is carried out to centrifugal acquisition wet thallus for subsequent use;
(3) preparation of fractionation system: with (+/-) gamma-lactam of phosphate buffer preparation 2g/L; Phosphate bufferPH7, concentration is 0.5mol/L.
(4) split (+/-) gamma-lactam and obtain (-) gamma-lactam: by (+/-) γ-Nei acyl preparing of step (3)Amine joins in the wet thallus of step (2) and reacts; Thalline final concentration is 60g/L wet thallus, and reaction temperature is 30 DEG C, anti-Between seasonable, be 5h, shaking speed is 200rpm.
(5) processing and the analysis of fractionation liquid: by the liquid after gained microorganism resolution reaction in step (4), centrifugal removingRemove thalline, get supernatant and adopt and be extracted with ethyl acetate, after extract dilution, carry out chirality HPLC detection; Chromatographic column model isCHIRALPAKAS-H250 × the 4.6mm of Daicel company; Mobile phase is isopropyl alcohol/acetonitrile (v/v)=80/20; Flow velocity 0.5mL/Min; Detect wavelength 230nm.
(6) separation and Extraction of product: adopt gained thalline in dichloromethane extraction step (5), and successively through super-dry,Crystallisation by cooling at evaporation and concentration at 40 DEG C, 2 DEG C, finally obtains described (-) gamma-lactam; Its purity 95.0%～100%,Chemical purity is 95%～99%.
Embodiment 2
With embodiment 1, difference is:
1. can split (+/-) gamma-lactam and obtain the screening of the pseudomonad of (-) gamma-lactam:
Described primary dcreening operation culture medium is: NH4CL10g，KH2PO40.01g，Na2HPO40.01g，MgSO40.01g，CaCL20.01g, (+/-) gamma-lactam 1g.
Described pseudomonad carries out microorganism enzymatic production to be cultivated 1 day, 40 DEG C of cultivation temperature, pH3, described microorganismEnzymatic production culture medium composition is counted with g/L: glucose 5.0g, NH4CL10g，KH2PO40.01g，Na2HPO40.01g，MgSO40.01g，CaCL20.01g, (+/-) gamma-lactam 2g, FeSO40.08g; Again that centrifugal above-mentioned zymotic fluid acquisition is wetThalline is for subsequent use.
2. fractionation (+/-) gamma-lactam obtains the method for (-) gamma-lactam:
(3) preparation of fractionation system: with (+/-) gamma-lactam of phosphate buffer preparation 600g/L; Phosphate-bufferedThe pH3 of liquid, concentration is 0.01mol/L.
(4) split (+/-) gamma-lactam and obtain (-) gamma-lactam: by (+/-) γ-Nei acyl preparing of step (3)Amine joins in the wet thallus of step (2) and reacts; Thalline final concentration is 0.5g/L wet thallus, and reaction temperature is 20 DEG C,Reaction time is 1h, and shaking speed is 100rpm.
(6) crystallisation by cooling at evaporation and concentration, 1 DEG C at 20 DEG C.
Embodiment 3
With embodiment 1, difference is:
1. can split (+/-) gamma-lactam and obtain the screening of the pseudomonad of (-) gamma-lactam:
Described primary dcreening operation culture medium is: NH4CL2.0g，KH2PO410g，Na2HPO410g，MgSO4～1g，CaCL20.01g, (+/-) gamma-lactam 1g.
Described pseudomonad carries out microorganism enzymatic production to be cultivated 1～3 day, 20 DEG C of cultivation temperature, pH10, described micro-lifeThing enzymatic production culture medium composition is counted with g/L: glucose 5.0g, NH4CL10g，KH2PO410g，Na2HPO40.01g，MgSO40.01g，CaCL20.5g, (+/-) gamma-lactam 2g, FeSO40.08g; Again by wet centrifugal above-mentioned zymotic fluid acquisition bacteriumBody is for subsequent use;
2. fractionation (+/-) gamma-lactam obtains the method for (-) gamma-lactam:
(3) preparation of fractionation system: with (+/-) gamma-lactam of phosphate buffer preparation 2g/L; Phosphate bufferPH10, concentration is 0.5mol/L.
(4) split (+/-) gamma-lactam and obtain (-) gamma-lactam: by (+/-) γ-Nei acyl preparing of step (3)Amine joins in the wet thallus of step (2) and reacts; Thalline final concentration is 100g/L wet thallus, and reaction temperature is 60 DEG C,Reaction time is 24h, and shaking speed is 300rpm.
(6) crystallisation by cooling at evaporation and concentration, 4 DEG C at 80 DEG C.
Embodiment 4
With embodiment 1, difference is:
1. can split (+/-) gamma-lactam and obtain the screening of the pseudomonad of (-) gamma-lactam:
Described just culture medium is: NH4CL5g，KH2PO41g，Na2HPO45g，MgSO42g，CaCL20.2g，(+/-)Gamma-lactam 2g.
Described pseudomonad carries out microorganism enzymatic production to be cultivated 2 days, 35 DEG C of cultivation temperature, and pH5, described microorganism sends outFerment culture medium composition is counted with g/L: glucose 5.0g, NH4CL5g，KH2PO45g，Na2HPO45g，MgSO40.5g，CaCL20.1g, (+/-) gamma-lactam 2g, FeSO40.08g; Again by for subsequent use centrifugal above-mentioned zymotic fluid acquisition wet thallus.
2. fractionation (+/-) gamma-lactam obtains the method for (-) gamma-lactam:
(3) preparation of fractionation system: with (+/-) gamma-lactam of phosphate buffer preparation 20g/L; Phosphate-bufferedThe pH5 of liquid, concentration is 0.2mol/L.
(4) split (+/-) gamma-lactam and obtain (-) gamma-lactam: by (+/-) γ-Nei acyl preparing of step (3)Amine joins in the wet thallus of step (2) and reacts; Thalline final concentration is 20g/L wet thallus, and reaction temperature is 50 DEG C, anti-Between seasonable, be 7h, shaking speed is 250rpm.
(6) crystallisation by cooling at evaporation and concentration, 3 DEG C at 68 DEG C.
Above-described embodiment, is preferred embodiment of the present invention, is not used for limiting the scope of the present invention, therefore all with thisThe equivalence that feature described in invention claim and principle are done changes or modifies, and all should be included in the claims in the present invention scopeWithin.
Related experiment:
One, the impact of different carbon sources on enzymatic production
Culture medium is with 2g/lNH4CL is nitrogenous source, adds respectively following carbon source by 5g/L concentration: lactose, starch, cottonSon sugar, glucose, beef extract, citric acid, sucrose, natrium citricum, wood sugar and glycerine. 30 DEG C, 220r/min are cultivated the thalline of 48hSplit transformation experiment. Investigating different carbon sources produces bacterial strain pseudomonad (PseudomonasgranadensisB6) fermentationThe impact of enzyme, product optical purity and substrate conversion efficiency.
The impact of table 1 carbon source on enzymatic production
As can be seen from Table 1: carbon source is larger on the biomass impact of thalline, and Ee value and conversion ratio are had to considerable influence.Wherein culture medium taking lactose, starch, gossypose sucrose, natrium citricum and glycerine during as carbon source thalline do not grow, and withGlucose, beef extract and citric acid are that carbon source biomass is more approaching, and the biomass taking wood sugar as carbon source is much smaller. Wherein with PortugalThe Transformed E e value of the thalline after the culture medium that grape sugar is carbon source is cultivated reaches 100%, and conversion ratio reaches 75.2%.
Two, the impact of different nitrogen sources on enzymatic production
Culture medium, taking 5g/l glucose as carbon source, adds respectively following nitrogenous source by 2g/l: beef extract, peptone, urineElement, ammonium sulfate, sell ammonium, diammonium hydrogen phosphate, potassium nitrate, ammonium oxalate, diammonium hydrogen citrate and carbonic hydroammonium. 30 DEG C, 220r/The thalline that min cultivates 48h splits transformation experiment. Investigate different nitrogen sources to bacterial strain pseudomonad (PseudomonasGranadensisB6) impact of enzymatic production, product optical purity and substrate conversion efficiency. The results are shown in Table 2.
The impact of table 2 nitrogenous source on enzymatic production
Nitrogenous source is larger on the biomass impact of thalline as can be seen from Table 2, and Ee value and conversion ratio are had to considerable influence. ItsMiddle culture medium is with ammonium sulfate, and when sell ammonium and carbonic hydroammonium are nitrogenous source, thalline is not grown. Culture medium with beef extract,When peptone, urea, diammonium hydrogen phosphate, potassium nitrate, ammonium oxalate and diammonium hydrogen citrate are nitrogenous source, Ee value all reaches more than 95%,When wherein culture medium is taking peptone, urea, potassium nitrate, ammonium oxalate and diammonium hydrogen citrate as nitrogenous source, Ee value has all reached100%, conversion ratio is also higher.
Three, the impact of cultivation temperature on enzymatic production
Temperature is a key factor that affects Growth of Cells and enzymatic production, has investigated different fermentations temperature to false unit cellThe impact that the thalline output of bacterium (PseudomonasgranadensisB6) bacterial strain and enzyme are lived, the results are shown in Table 3.
The impact of table 3 cultivation temperature on enzymatic production
Table 3 can find out that temperature all has a certain impact to biomass and enzyme work, in the time that thalline cultivation temperature is 30 DEG C, rawThing amount and enzyme are lived the highest, thus with 30 DEG C for the suitableeest fermentation temperature.
Four, the impact of the initial pH of culture medium on enzymatic production
The activity of Growth of Cells and the various enzymes of thalline all can be subject to the regulating action of the initial pH of culture medium. At the beginning of investigatingThe impact of beginning pH on pseudomonad (PseudomonasgranadensisB6) strain enzyme-producing, is adjusted to difference by fermentation mediumPH, split the experiment of (+/-) gamma-lactam to cultivating the thalline obtaining.
The impact of the initial pH of table 4 culture medium on thalli growth and product enzyme
The results are shown in Table 4, the initial pH of culture medium is to pseudomonad (Pseudomonas as can be seen from the tableGranadensisB6) biomass and conversion ratio impact are not very large, can grow from the culture medium thalline of pH3.0-10.0,And Ee value and conversion ratio are all good.
Five, conversion temperature splits the impact of (+/-) gamma-lactam generation (-) gamma-lactam on bacterial strain
The impact that conversion temperature splits (+/-) gamma-lactam generation (-) gamma-lactam to bacterial strain the results are shown in Table 5.
Table 5 conversion temperature produces the impact of (-) gamma-lactam on splitting (+/-) gamma-lactam
As can be seen from Table 5,30 DEG C is optimum conversion temperature, and along with the rising gradually of temperature, Ee value and conversion ratio are allDecline gradually.
Six, the pH value of the buffer solution of preparation conversion of substrate (+/-) gamma-lactam produces splitting (+/-) gamma-lactamThe impact of (-) gamma-lactam
The pH value of the buffer solution of preparation conversion of substrate (+/-) gamma-lactam produces (-) to splitting (+/-) gamma-lactamThe impact of gamma-lactam the results are shown in Table 6.
The pH value of table 6 buffer solution produces the impact of (-) gamma-lactam on splitting (+/-) gamma-lactam
As seen from Table 6: the optimal pH of the buffer solution of preparation conversion of substrate (+/-) gamma-lactam is 8, the slightly bar of meta-alkalescenceUnder part, catalytic effect is best. Too high or too low pH is unfavorable to transforming. In the time of pH < 6, Ee value and conversion ratio are very very low.
Seven, the separation and Extraction of product
Pseudomonad (PseudomonasgranadensisB6)
Wet thallus concentration is 40g/L, and concentration of substrate is 300g/L, and reaction system is 2L, in the reactor of 5L, carries out notSymmetrical hydrolysis resolution reaction. 30 DEG C of temperature, pH8.0, the 200rpm of Hydrolysis Resolution reaction. Turning after microorganism catalysis reactionChange liquid, the centrifugal thalline of removing, the dichloromethane extraction of use 1L 3 times, 3 extracts mix uses anhydrous sodium sulfate drying.Extract is vacuumized to rotary evaporation, and evaporating temperature is 30 DEG C, adds n-hexane crystallisation by cooling temperature and be 4 DEG C by the crystalline substance obtainingBody is dissolved in ethyl acetate, and the optical purity (Ee value) that chirality HPLC measures (-) gamma-lactam is greater than 99.5%, product chemical pureDegree is greater than 99%.
Sequence number
Applicant: institute of microbiology of Jiangxi Prov. Academy of Science
Denomination of invention: a kind of can split (+/-) gamma-lactam obtain the pseudomonad of (-) gamma-lactam and screen withApplication
16SrRNA gene order:
CAGACTACACATGCAGTCGAGCGGATGAAGGGAGCTTGCTCCTGAATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGTTGTAGATTAATACTCTGCAATTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTTGAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTACCACGGTTTATT
Claims (10)
1. the pseudomonad that can split (+/-) gamma-lactam and obtain (-) gamma-lactam, is characterized in that, described false singleBorn of the same parents bacterium is Granada pseudomonad (PseudomonasgranadensisB6), is preserved in China on October 10th, 2015Typical case's culture collection center, deposit number is: CCTCCM2015596.
2. the screening of pseudomonad described in claim 1, is characterized in that: is specially,
(1) just cultivate: get mud from lakebed, with coating on culture medium after sterilized water dilution, 30 DEG C of cultivations, 2 days;
The culture medium that described primary dcreening operation is cultivated is: NH4CL2.0～10g，KH2PO40.01～10g，Na2HPO40.01～10g，MgSO40.01～1g，CaCL20.01～0.5g, (+/-) gamma-lactam 1～10g, micro-100 μ L, agar powder 10～30g; Wherein trace element calculates and comprises according to every liter of addition: CaCL2·2H2O3.6g，ZnO2.0g，CuCL·2H2O0.85g，NaMoO·2H2O4.8g，MnCL2·4H2O2.0g，FeCL3·6H2O5.4g，CoCL2·6H2O2.4g；
(2) the picking list bacterium colony separation and purification list bacterium colony of again ruling on screening and culturing base;
Picking list colony morphology characteristic is: bacterium colony circle, neat in edge, protuberance are moistening, and white is slightly yellow, and bacterium colony inside is glutinousAqueous, Gram's staining is negative;
(3) enzymatic production is cultivated: first the single bacterium colony out of picking described in step (2) is proceeded to enzymatic production and cultivated 1～3My god, described enzymatic production culture medium composition is counted with g/L: glucose 5.0g, NH4CL2.0～10g，KH2PO40.01～10g，Na2HPO40.01～10g，MgSO40.01～1g，CaCL20.01～0.5g, (+/-) gamma-lactam 2g, FeSO40.08g；
(4) the bacterium liquid of above-mentioned enzymatic production being cultivated carry out centrifugal acquisition described can split (+/-) gamma-lactam obtain (-) γ-The pseudomonad of lactams, for subsequent use.
3. the screening of pseudomonad described in claim 2, is characterized in that: described in step (1), mud is taken from Ai Xihu.
4. the screening of pseudomonad described in claim 2, is characterized in that, the temperature of wherein just cultivating in described step (1)Spend 20～40 DEG C, pH3～10.
5. with an application for pseudomonad described in any one in claim 1-4, it is characterized in that, adopt described false unit cellBacterium, in aqueous phase system, taking (+/-) gamma-lactam as substrate, carries out asymmetric hydrolysis fractionation and prepares described (-) γ-NeiAcid amides.
6. the application of pseudomonad described in claim 5, is characterized in that, described taking (+/-) gamma-lactam as substrate,Carry out asymmetric hydrolysis and split that to prepare the concrete steps of described (-) gamma-lactam as follows:
(1) microbial strains: the pseudomonad in claim 1-5 described in any one;
(2) first the pseudomonad described in step (1) is carried out to centrifugal acquisition wet thallus for subsequent use;
(3) preparation of fractionation system: (+/-) gamma-lactam of preparing 2g/L～600g/L with phosphate buffer;
(4) split (+/-) gamma-lactam and obtain (-) gamma-lactam: (+/-) gamma-lactam preparing of step (3) is addedEnter to reacting in the wet thallus of step (2);
(5) split processing and the analysis of liquid: by the liquid after gained microorganism resolution reaction in step (4), centrifugally remove bacteriumBody, gets supernatant and adopts and be extracted with ethyl acetate, and after extract dilution, carries out chirality HPLC detection;
(6) separation and Extraction of product: adopt gained thalline in dichloromethane extraction step (5), and successively through super-dry, evaporate denseContracting, crystallisation by cooling, finally obtain described (-) gamma-lactam.
7. the application of pseudomonad described in claim 6, is characterized in that, the middle phosphate buffer of described step (3)PH3～10, concentration is 0.01～0.5mol/L.
8. the application of pseudomonad described in claim 6, is characterized in that, in described step (4), thalline final concentration is0.5g/L～100g/L wet thallus, reaction temperature is 20～60 DEG C, and the reaction time is 1～24h, and shaking speed is 100～300rpm。
9. the application of pseudomonad described in claim 6, is characterized in that, in described step (5), at 20～80 DEG C, steamsSend out concentrated, crystallisation by cooling at 1～4 DEG C.
10. the application of pseudomonad described in claim 6, is characterized in that, (-) γ described in described step (6)-Nei acylThe optical purity of amine is 95.0%～100%, and chemical purity is 95%～99%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136154A (en) * | 2018-09-29 | 2019-01-04 | 河南省农业科学院植物保护研究所 | One plant can prevent and treat tobacco black shank and root black rot and Granada pseudomonad and its application with growth-promoting functions simultaneously |
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| CN108410749B (en) * | 2018-01-29 | 2021-06-29 | 大连大学 | Method for preparing (-) gamma-lactam by asymmetric hydrolysis of marine low-temperature (+) gamma-lactamase |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109136154B (en) * | 2018-09-29 | 2021-06-04 | 河南省农业科学院植物保护研究所 | Pseudomonas glanadensis capable of preventing and treating tobacco black shank and root black rot simultaneously and having growth promoting effect and application thereof |
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