CN1673344A - Method for screening levo-phosphonomycin bioconversion strain with cis-propene phosphoric acid as substrate - Google Patents
Method for screening levo-phosphonomycin bioconversion strain with cis-propene phosphoric acid as substrate Download PDFInfo
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- CN1673344A CN1673344A CN 200510045889 CN200510045889A CN1673344A CN 1673344 A CN1673344 A CN 1673344A CN 200510045889 CN200510045889 CN 200510045889 CN 200510045889 A CN200510045889 A CN 200510045889A CN 1673344 A CN1673344 A CN 1673344A
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- phosphoric acid
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- phosphonomycin
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- propene phosphoric
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Abstract
The present invention relates to biological conversion/ biological catalysis technology and microbe strain screening technology, and is especially the screening police and method of levorotary phosphonomycin bioconverting strain with maleic phosphoric acid as substrate. The screening process includes two steps of initial screening and re-screening. In the initial screening, the selective culture medium has maleic phosphoric acid as only carbon source so as to eliminating most of irrelative microbes. In the re-screening, the strain obtained in the initial screening and the solid culture medium with levorotary phosphonomycin bioconverting strain and maleic phosphoric acid substrate are adopted to sort out the phosphonomycin bioconverting strain. The present invention has greatly raised screening efficiency.
Description
Technical field
The present invention relates to bio-transformation/biocatalysis technology, microorganism strains screening field, being specially a kind of is the screening strategy and the method for the phosphonomycin bioconversion strain of substrate with the cis-propene phosphoric acid.
Background technology
Phosphonomycin [(-)-suitable-(1R, 2S)-1, the 2-phosphate epoxypropyl, English name: Fosfomycin is called for short FOM] be a kind of Broad spectrum antibiotics.The chemical structure of phosphonomycin is simple and totally different in most of common antibiotics, characteristics such as it has, and toxicity is low, no antigen, has a broad antifungal spectrum, many, the difficult formation resistances of indication.In recent years result of study has been found some new effects of phosphonomycin again, for example: with other antibiotic synergy, alleviate other antibiotic toxic side effects, drug combination is treated critical infection, immuno-potentiation.Therefore, phosphonomycin is called as the new antibiotic of 21st century.
At present, chemical synthesis is mainly adopted in phosphonomycin production, promptly is raw material with the propiolic alcohol, through technologies such as over-churning, hydrolysis, hydrogenation, epoxidation, fractionation, salifies and finally obtain phosphonomycin.But the production yield of chemical synthesis process is less than 20%, and this is because what form in epoxidation process is the phosphonomycin raceme, i.e. the mixture of 50% phosphonomycin and 50% dextrorotation phosphonomycin.Raceme is carried out chemistry split, isolated phosphonomycin is medicable finished product, and isolated dextrorotation phosphonomycin then falls as waste discharge.This not only causes production cost to improve and the wasting of resources, has also formed the serious environmental pollution problem simultaneously.For example, the organophosphorus wastewater discharge of certain pharmacy corporation is 20 ton per days, and the COD value is up to 200000～300000mg/L.Therefore, realizing the asymmetric synthesis of phosphonomycin or the utilization again of dextrorotation phosphonomycin, is an active demand that reduces phosphonomycin production cost, the raising output value and profits tax, minimizing discharge of wastewater.
In recent years, people recognize that gradually bio-transformation (Biotransformation)/biocatalysis (Biocatalysis) will become the gordian technique of producing chipal compounds.Because most of biological catalysts (microbial cells or enzyme) itself are exactly chiral catalyst, they often have strict demand to the substrate configuration, can optionally act in the enantiomorph, and inoperative to another.Bio-transformation or the synthetic height stereoselectivity of utilizing enzymatic reaction just change into the single optical activity product with racemoid, precursor or the latent chipal compounds of chemosynthesis.At present, 89% of the biotransformation product is chirality.The advantage of biotransformation is that the chirality specificity is strong, reaction conditions is gentle (20 ℃～50 ℃, pH neutrality), stereoselectivity is strong, side reaction is few, yield is high, product optical purity height, and is low in the pollution of the environment.
Phosphonomycin can (cis-propenylphosphonic acid cPA) be used for synthesizing through microbial transformation by cis-propene phosphoric acid.At present, have been found that fungi, more than ten bacterial strain in actinomycetes and the bacterium has the ability of catalysis cis-propene phosphoric acid asymmetric synthesis phosphonomycin, and these bacterial strains comprise Penicillinspinulosum, Cellvibrio gilvus, Acetobacter aceti, Achromobacter turbidus, Brecibacterium acetylicum, Derxia gummosa, Pseudomonas purpurrescens, Pseudomonas syringae, Flavobacterium esteroaromaticum, Pseudomonas putida, Alcaligenesfaecalis, Corynebacterium sp., Streptomyces albidoflavus, Bacillus firmus etc.But because these bacterial strains are all very low to the tolerance of substrate and product, their conversion capability all can not reach the industrial demand that is applied to far away.Can therefore, screen the bacterial strain (or enzyme) with high-performance bio conversion capability be the key that successfully be applied to asymmetric biosynthesizing/transformation technology the phosphonomycin production practice.
Summary of the invention
The purpose of this invention is to provide a kind of high efficiency be the phosphonomycin biotransformation strain screening strategy and the method for substrate with the cis-propene phosphoric acid.
Technical scheme of the present invention is:
A kind of is the phosphonomycin biotransformation strain screening method of substrate with the cis-propene phosphoric acid, comprise primary dcreening operation and sieve two steps again, and be starting strain with the bacterial strain that is obtained in the primary dcreening operation process in the sieve process again; Adopting with the cis-propene phosphoric acid in primary dcreening operation is the selective medium of sole carbon source, and it can be the bacterial strain that substrate is grown with the cis-propene phosphoric acid that this selective medium selectivity is turned out those; The multiple sieve substratum that adopts is to be the phosphonomycin bioconversion strain solid medium of substrate with the cis-propene phosphoric acid.
Described primary dcreening operation step is as follows:
1) cis-propene phosphoric acid sole carbon source selective medium
Percentage ratio meter by weight, cis-propene phosphoric acid sole carbon source selective medium has following prescription: (NH
4)
2SO
40.3%, KCl 0.1%, and NaCl 0.2%, MgSO
47H
2O 0.02%, KH
2PO
40.05%, agar powder 2%, VITAMIN complex liquid 0.1%, cis-propene phosphoric acid 0.3～2.0%, surplus is a distilled water, with the said components uniform mixing, transfers pH 7.2-7.4, sterilizes 20 minutes for 121 ℃, and is standby;
2) primary dcreening operation process
Draw soil supension, be added on the cis-propene phosphoric acid sole carbon source selective medium flat board, coating evenly is inverted in 37 ℃ and cultivated 5～7 days;
3) primary dcreening operation obtains the separation and purification and the preservation bacterial classification of bacterial strain
Percentage ratio meter by weight, the separation and purification culture medium prescription is as follows: (NH
4)
2SO
40.3%, KCl 0.1%, and NaCl 0.2%, MgSO
47H
2O 0.02%, KH
2PO
40.05%, agar powder 2%, VITAMIN complex liquid 0.1%, yeast extract paste 0.1%, cis-propene phosphoric acid 0.3%, surplus is a distilled water, with the said components uniform mixing, transfers pH7.2-7.4, sterilizes 20 minutes for 121 ℃;
The bacterium colony that picking is grown on cis-propene phosphoric acid sole carbon source selective medium, streak inoculation is carried out purifying on above-mentioned separation and purification medium slant, and then is inoculated in and carries out preservation on the same medium inclined-plane.
The described step of sieving again is as follows:
1) with the cis-propene phosphoric acid is the phosphonomycin bioconversion strain solid medium of substrate
Percentage ratio meter by weight, the cis-propene phosphoric acid in the multiple sieve are that the phosphonomycin bioconversion strain solid medium of substrate has following prescription: cis-propene phosphoric acid 0.3～1.0%, extractum carnis 0.5%, peptone 1%, NaCl0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.05%, agar 1.8%, surplus is a distilled water, with the said components uniform mixing, transfers pH 7.2-7.4, sterilizes 30 minutes for 115 ℃, and is standby;
2) preparation of agar block
Above-mentioned substratum is melted, leave standstill solidify after, get agar block with punch tool, the bacterial strain to be detected that obtains in its surface seeding primary dcreening operation step places 30 ℃ then, cultivates 4-5 days under the 75%-85% relative humidity.
The calibrating that obtains phosphonomycin conversion bacterial strain through multiple sieve comprises the steps:
1) bioassay substratum and dull and stereotyped preparation thereof
Substratum I composition: percentage ratio meter by weight, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a distilled water, with the said components uniform mixing, transfers pH 7.0-7.2; The medium ii composition: percentage ratio meter by weight, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, surplus is a distilled water, with the said components uniform mixing, transfers pH7.0-7.2; Substratum I, medium ii are all 121 ℃ of sterilizations 20 minutes; Pour the substratum I that melts into the biological assay dish, make lower floor's flat board; Again the medium ii of melting is cooled to 55 ℃, adds the indicator bacteria suspension, pour the biological assay dish behind the mixing into, make upper panel;
2) phosphonomycin transforms the bacterial strain biological assay
Cultured single bacterium colony agar block is placed under the ultraviolet lamp sterilization 20 minutes, agar block is inverted on the biological assay dish again, cultivated 16 hours for 37 ℃, observe and also to measure antibacterial circle diameter, screen bacterial strain according to the having or not of inhibition zone and the size of antibacterial circle diameter with phosphonomycin bio-transformation ability.
The invention has the beneficial effects as follows:
The invention provides a kind of efficient height, the easy screening strategy of operation and corresponding concrete operation method, being used to screen with the cis-propene phosphoric acid is the phosphonomycin bioconversion strain of substrate.The main points of this screening strategy and concrete operation method are to have comprised primary dcreening operation and sieve two steps again in screening process.The primary dcreening operation process is eliminated the uncorrelated microorganism that accounts for the overwhelming majority in the sample, is that starting strain further filters out the phosphonomycin bioconversion strain with the bacterial strain that obtains in the primary dcreening operation again in the multiple sieve.Owing to eliminating in advance in the primary dcreening operation arranged, thereby significantly reduced multiple sieve workload, the whole efficiency of screening operation is greatly improved.The weak point of this screening strategy and method thereof is to exist in the primary dcreening operation step possibility that eliminates the potential purpose microorganism of part.
Embodiment
Screening strategy of the present invention is divided into two steps to screening process, i.e. primary dcreening operation and sieve step again.The primary dcreening operation step is eliminated the uncorrelated microorganism that accounts for the overwhelming majority in the sample, be starting strain then in the sieve process again with the bacterial strain that obtains in the primary dcreening operation process, further filtering out the purpose bacterial strain, promptly is the phosphonomycin bioconversion strain of substrate with the cis-propene phosphoric acid.
Comprise primary dcreening operation and sieve two steps again with the corresponding specific operation process of this screening strategy.Adopted in primary dcreening operation with the cis-propene phosphoric acid is the selective medium of sole carbon source, it can be the bacterial strain that substrate is grown with the cis-propene phosphoric acid that this selective medium can selectivity be turned out those, can not be the bacterial strain of substrate growth in a large number with the cis-propene phosphoric acid thereby exclude.Because eliminated a large amount of uncorrelated bacterial strains in the primary dcreening operation step, it is few that the bacterial strain quantity that enters multiple sieve step is fallen greatly, alleviated multiple sieve workload.The bacterial strain that obtains in the primary dcreening operation step through sieving process again, can be the bacterial strain that substrate generates phosphonomycin with the cis-propene phosphoric acid thereby determine again.Sieving the substratum that adopts in the step again is to be the phosphonomycin bioconversion strain solid medium of substrate with the cis-propene phosphoric acid.
Concrete operations step of the present invention is as follows:
One, preparation soil supension:
Take by weighing pedotheque 5g, put into the triangular flask that fills the 100mL stroke-physiological saline solution and have the 250mL of granulated glass sphere, put shaking table vibration 30min, pedotheque is uniformly dispersed in water, be made for soil supension.
Two, primary dcreening operation:
1. cis-propene phosphoric acid sole carbon source selective medium
Cis-propene phosphoric acid sole carbon source selective medium prescription is as follows: percentage ratio meter by weight, (NH
4)
2SO
40.3%, KCl 0.1%, and NaCl 0.2%, MgSO
47H
2O 0.02%, KH
2PO
40.05%, agar powder 2%, VITAMIN complex liquid 0.1%, cis-propene phosphoric acid 1.0%, surplus is a distilled water.PH 7.2-7.4 sterilized 20 minutes for 121 ℃.Wherein, VITAMIN complex liquid composed as follows: percentage ratio meter by weight, folic acid 0.002 ‰, vitamin H 0.002 ‰, vitamins B
60.01 ‰, vitamins B
10.005 ‰, calcium pantothenate 0.005 ‰, vitamins B
20.005 ‰, vitamins B
120.0001 ‰, surplus is a distilled water.The VITAMIN complex liquid must be through 0.2 μ m membrane filtration degerming, and adds with aseptic technique behind medium sterilization.
2. primary dcreening operation process:
Draw the 0.1mL soil supension, be added on the cis-propene phosphoric acid sole carbon source selective medium flat board, coating evenly.Being inverted in 37 ℃ cultivated 5～7 days.Observe the colony growth situation every day.
3. primary dcreening operation obtains the separation and purification and the preservation bacterial classification of bacterial strain
The separation and purification culture medium prescription is as follows: percentage ratio meter by weight, (NH
4)
2SO
40.3%, KCl 0.1%, and NaCl 0.2%, MgSO
47H
2O 0.02%, KH
2PO
40.05%, agar powder 2%, pH 7.2-7.4, VITAMIN complex liquid 0.1%, yeast extract paste 0.1%, cis-propene phosphoric acid 0.3%, surplus is a distilled water.Sterilized 20 minutes for 121 ℃.
The bacterium colony that picking is grown on cis-propene phosphoric acid sole carbon source selective medium, streak inoculation is carried out purifying on above-mentioned separation and purification medium slant, and then is inoculated in and carries out preservation on the same medium inclined-plane.
Two, multiple sieve:
1. with the cis-propene phosphoric acid phosphonomycin bioconversion strain solid medium of substrate
Culture medium prescription: percentage ratio meter by weight, cis-propene phosphoric acid 0.3%, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.05%, agar 1.8%, surplus is a distilled water.Sterilized 30 minutes for 115 ℃.
The preparation of agar block: above-mentioned substratum is melted, in each diameter 90mm plate, pour 25mL into, leave standstill and solidify.After solidifying, get agar block (diameter 6mm, high 8mm) with punch tool, the bacterial strain to be detected that obtains in its surface seeding primary dcreening operation step places 30 ℃ then, cultivates 4-5 days under the 75%-85% relative humidity.
2. phosphonomycin transforms the calibrating of bacterial strain
Bioassay substratum and dull and stereotyped preparation thereof:
Substratum I composition: percentage ratio meter by weight, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a distilled water, pH 7.0-7.2; The medium ii composition: percentage ratio meter by weight, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, surplus is a distilled water, pH7.0-7.2; Substratum I, medium ii are all 121 ℃ of sterilizations 20 minutes.The substratum I that 200mL is melted pour into the biological assay dish (20 * 30cm), make lower floor's flat board; The medium ii that 100mL is melted is cooled to 55 ℃ again, adds indicator bacteria suspension 1mL, pours the biological assay dish into behind the mixing rapidly, makes upper panel.
Phosphonomycin transforms the bacterial strain biological assay:
Cultured single bacterium colony agar block is placed under the ultraviolet lamp (30W, 40cm) sterilization is 20 minutes, agar block is inverted on the biological assay dish that has been coated with indicator again, cultivates 16hr for 37 ℃.Observe and measure antibacterial circle diameter.Screen bacterial strain according to having or not of inhibition zone with phosphonomycin bio-transformation ability with the size of antibacterial circle diameter.
Described indicator is intestinal bacteria (E.coli), and the indicator cultivation is formulated as follows with suspension:
The indicator substratum is formed: percentage ratio meter by weight, and extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a tap water.After the heating for dissolving, transfer pH 7.0-7.2.The eggplant type of packing into bottle 90mL, 121 ℃ of moist heat sterilizations are after 20 minutes, and the pendulum inclined-plane is standby.
Indicator is cultivated: cultivate 24hr for 37 ℃, lawn is translucent to white.
The preparation of indicator suspension: the physiological saline of 100mL sterilization is poured in the eggplant type bottle, scraped with transfering loop and wash lawn, will scrape in the empty conical flask that washing lotion pours sterilization into again, measuring absorbancy (O.D. value) is 0.54-0.57.
Embodiment:
Make soil supension with gathering from five kinds of pedotheques of different loci, through the primary dcreening operation step, obtained 180 surplus the strain bacterial isolates, these bacterial strains are again through sieving step again, find wherein have 30 surplus the strain bacterium tangible inhibition zone is arranged.Through the thin-layer chromatography check, proved that these bacterial strains are that phosphonomycin presents inhibition zone by transforming cis-propene phosphoric acid.
Claims (4)
1, a kind of is the phosphonomycin biotransformation strain screening method of substrate with the cis-propene phosphoric acid, it is characterized in that: comprising primary dcreening operation and sieve two steps again, is starting strain with the bacterial strain that is obtained in the primary dcreening operation process in the sieve process again; Adopting with the cis-propene phosphoric acid in primary dcreening operation is the selective medium of sole carbon source, and it can be the bacterial strain that substrate is grown with the cis-propene phosphoric acid that this selective medium selectivity is turned out those; The multiple sieve substratum that adopts is to be the phosphonomycin bioconversion strain solid medium of substrate with the cis-propene phosphoric acid.
2, in accordance with the method for claim 1, it is characterized in that described primary dcreening operation step is as follows:
1) cis-propene phosphoric acid sole carbon source selective medium
Percentage ratio meter by weight, cis-propene phosphoric acid sole carbon source selective medium has following prescription: (NH
4)
2SO
40.3%, KCl 0.1%, and NaCl 0.2%, MgSO
47H
2O 0.02%, KH
2PO
40.05%, agar powder 2%, VITAMIN complex liquid 0.1%, cis-propene phosphoric acid 0.3～2.0%, surplus is a distilled water, with the said components uniform mixing, transfers pH 7.2-7.4, sterilizes 20 minutes for 121 ℃, and is standby;
2) primary dcreening operation process
Draw soil supension, be added on the cis-propene phosphoric acid sole carbon source selective medium flat board, coating evenly is inverted in 37 ℃ and cultivated 5～7 days;
3) primary dcreening operation obtains the separation and purification and the preservation bacterial classification of bacterial strain
Percentage ratio meter by weight, the separation and purification culture medium prescription is as follows: (NH
4)
2SO
40.3%, KCl 0.1%, and NaCl 0.2%, MgSO
47H
2O 0.02%, KH
2PO
40.05%, agar powder 2%, VITAMIN complex liquid 0.1%, yeast extract paste 0.1%, cis-propene phosphoric acid 0.3%, surplus is a distilled water, with the said components uniform mixing, transfers pH7.2-7.4, sterilizes 20 minutes for 121 ℃;
The bacterium colony that picking is grown on cis-propene phosphoric acid sole carbon source selective medium, streak inoculation is carried out purifying on above-mentioned separation and purification medium slant, and then is inoculated in and carries out preservation on the same medium inclined-plane.
3, in accordance with the method for claim 1, it is characterized in that the described step of sieving again is as follows:
1) with the cis-propene phosphoric acid is the phosphonomycin bioconversion strain solid medium of substrate
Percentage ratio meter by weight, the cis-propene phosphoric acid in the multiple sieve are that the phosphonomycin bioconversion strain solid medium of substrate has following prescription: cis-propene phosphoric acid 0.3～1.0%, extractum carnis 0.5%, peptone 1%, NaCl0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.05%, agar 1.8%, surplus is a distilled water, with the said components uniform mixing, transfers pH 7.2-7.4, sterilizes 30 minutes for 115 ℃, and is standby;
2) preparation of agar block
Above-mentioned substratum is melted, leave standstill solidify after, get agar block with punch tool, the bacterial strain to be detected that obtains in its surface seeding primary dcreening operation step places 30 ℃ then, cultivates 4-5 days under the 75%-85% relative humidity.
4, in accordance with the method for claim 3, it is characterized in that the calibrating that obtains phosphonomycin conversion bacterial strain through multiple sieve comprises the steps:
1) bioassay substratum and dull and stereotyped preparation thereof
Substratum I composition: percentage ratio meter by weight, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a distilled water, with the said components uniform mixing, transfers pH 7.0-7.2; The medium ii composition: percentage ratio meter by weight, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, surplus is a distilled water, with the said components uniform mixing, transfers pH7.0-7.2; Substratum I, medium ii are all 121 ℃ of sterilizations 20 minutes; Pour the substratum I that melts into the biological assay dish, make lower floor's flat board; Again the medium ii of melting is cooled to 55 ℃, adds the indicator bacteria suspension, pour the biological assay dish behind the mixing into, make upper panel;
2) phosphonomycin transforms the bacterial strain biological assay
Cultured single bacterium colony agar block is placed under the ultraviolet lamp sterilization 20 minutes, agar block is inverted on the biological assay dish again, cultivated 16 hours for 37 ℃, observe and also to measure antibacterial circle diameter, screen bacterial strain according to the having or not of inhibition zone and the size of antibacterial circle diameter with phosphonomycin bio-transformation ability.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510045889 CN1283777C (en) | 2005-02-18 | 2005-02-18 | Method for screening levo-phosphonomycin bioconversion strain with cis-propene phosphoric acid as substrate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510045889 CN1283777C (en) | 2005-02-18 | 2005-02-18 | Method for screening levo-phosphonomycin bioconversion strain with cis-propene phosphoric acid as substrate |
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| Publication Number | Publication Date |
|---|---|
| CN1673344A true CN1673344A (en) | 2005-09-28 |
| CN1283777C CN1283777C (en) | 2006-11-08 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1876830B (en) * | 2006-04-26 | 2012-11-14 | 中国科学院沈阳应用生态研究所 | Levo-phosphonomycin biotransformation strain screening method using dextro-phosphonomycin as substrate |
-
2005
- 2005-02-18 CN CN 200510045889 patent/CN1283777C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1876830B (en) * | 2006-04-26 | 2012-11-14 | 中国科学院沈阳应用生态研究所 | Levo-phosphonomycin biotransformation strain screening method using dextro-phosphonomycin as substrate |
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Effective date of registration: 20090717 Address after: No. 72, Wenhua Road, Shenhe District, Liaoning, Shenyang: 110016 Co-patentee after: Dongbei Pharmaceutical Factory Patentee after: Shenyang Institute of Applied Ecology, Chinese Academy of Sciences Address before: No. 72, Wenhua Road, Shenhe District, Liaoning, Shenyang: 110016 Patentee before: Shenyang Application Ecelogy Inst., Chinese Academy of Sciences |
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