CN104099398A - Method for screening clavulanic acid high-yield strains by threonine tolerant model - Google Patents
Method for screening clavulanic acid high-yield strains by threonine tolerant model Download PDFInfo
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Abstract
The invention discloses a method for screening clavulanic acid high-yield strains by a threonine tolerant model. The method comprises the steps of primary screening, re-screening and fermentation medium test, etc. The screening method provided by the invention is simple to operate and is practicable, can effectively improve the probability of acquiring positive mutant strains, and provides high-yield strains for fermentation production of clavulanic acid, thereby improving the fermentation production level of clavulanic acid. The involved medium raw materials have wide sources and are low in cost, thus being very suitable for industrial production needs.
Description
Technical field
The invention belongs to biomedicine field, be specifically related to the method for a seed amino acid tolerance model discrimination Clavulanic Acid High-Producing Strains strain.
Background technology
Due to being widely used of penicillin, some bacterium has produced resistance to it.Penicillins and Cephalosporins all contain a common β-lactam antibitics.In the time that use has the microbiotic of this structure, part pathogenic bacteria produces resistance by producing β-lactamase.Clavulanic acid (clavulanic acid, CA) claim again clavulanic acid, is the efficient beta-lactamase inhibitor of one that clavuligerus (Streptomyces clavuligerus) produces.Clavulanic acid can improve the susceptibility of above-mentioned pathogenic bacteria to β-lactam antibitics greatly.The anti-microbial activity of clavulanic acid itself is very weak, but it is brute force, wide spectrum and irreversible beta-lactamase inhibitor.No matter the β-lactamase that can produce with the gram-positive microorganism of resistance and Gram-negative bacteria in vitro or in vivo generates firm irreversible binding substances, thereby suppress the Decomposition of drug tolerant bacteria to β-lactam antibitics, recover the anti-microbial activity of penicillins and the drug tolerant bacteria of cephalosporins to a lot of generation β-lactamases.
Summary of the invention
The invention provides the method for a kind of Threonine tolerance model discrimination Clavulanic Acid High-Producing Strains strain, the method can improve the probability that obtains positive mutating strain effectively, for clavulanic acid fermentative production provides excellent species, thereby improves clavulanic acid fermentative production level.
Utilizing clavulanic acid to produce bacterium may join in isolation medium as the analog of the material of precursor or precursor (as amino acid etc.), observe bacterial strain to amino acid whose tolerance situation, determine its minimum tolerance dose, the amino acid of selecting starting strain can not grow under high density, as screening model.
Bacterial classification after mutagenesis, screen with set up screening model, model to starting strain because can not enduring high-concentration amino acid growing, if can grow after mutagenesis, illustrate and can tolerate the amino acid of this concentration, necessarily there is variation in its pathways metabolism and metabolism stream, just likely screening obtains high productive mutant.
The amino acid analysis of fermented liquid shows, Threonine, L-glutamic acid, arginine, aspartic acid, L-Ala, Serine, glycine, α-amino-isovaleric acid, Histidine, tryptophane, tyrosine, proline(Pro), Methionin, aspartic acid, l-asparagine, leucine, Isoleucine (not sulfur-containing amino acid) can be utilized by clavuligerus, and methionine(Met), Gelucystine, halfcystine are unfavorable to clavulanic acid biosynthesizing containing S amino acid.Utilizing clavulanic acid to produce bacterium may join and in isolation medium, screen discovery as the analog of the material of precursor or precursor (as sulfur-containing amino acid not): the probability of its generation positive mutating strain of mutant strain that can tolerate Threonine is the highest.Therefore select Threonine to carry out the screening of superior strain as screening model.Observe the tolerance situation of bacterial strain to Threonine (observe on flat board have or not colony growth), determine its minimum tolerance dose.The following method of definite employing of its tolerance dose: the monospore suspension after mutagenesis is coated on respectively on the isolation medium that contains different concns Threonine and is cultivated after suitably diluting, and starting strain in contrast product is cultivated simultaneously.Observe the growing state of bacterium colony on different concns flat board, if on the flat board of certain concentration without colony growth, just can determine that this concentration is the minimum inhibitory concentration of Threonine.The Threonine of selecting starting strain can not grow under high density, as screening model.
The present invention is exactly the principle based on above and the test carried out.
The method of a kind of Threonine tolerance model discrimination Clavulanic Acid High-Producing Strains strain involved in the present invention, comprises primary dcreening operation, multiple sieve and fermention medium test.
(1) primary dcreening operation: coat and contain on the isolation medium that Threonine dosis tolerata is 50g/l after the monospore suspension of mutagenic treatment is suitably diluted, in 28 DEG C of incubators, cultivate 5-7 days, select on single colony inoculation slant medium of resistance to Threonine, cultivate 5-7 days for 28 DEG C, inclined-plane is inoculated in seed culture medium after producing spore, after 28 DEG C of cultivation 32h, be inoculated on fermention medium, after 28 DEG C of cultivation 47h, measure and tire, pick out the high bacterial strain of tiring;
(2) multiple sieve: will tire higher inoculation in seed culture medium, cultivate after 32h for 28 DEG C, select inoculation that mycelial growth is good in fermention medium, cultivate after 47h for 28 DEG C and measure and tire;
(3) fermention medium test: the highest bacterial strain carries out fermention medium test by tiring.Seed culture medium is packed in 3 750ml triangular flasks into every bottle of 100ml.After sterilizing, dig piece kind and enter in seed culture medium, cultivate 32h for 28 DEG C, plant respectively and in the fermention medium that contains different concns Threonine, carry out fermentation test by well-grown and without the seed of miscellaneous bacteria.Shaking speed is 220rpm, after 28 DEG C of cultivation 47h, measures and tires.
(1) in step, screening and separating medium component used and content are: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, Threonine 50 g/L, agar 12 g/L, pH7.0.
(1) in step, slant culture based component used and content are: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, agar 12 g/L, pH7.0.
(1), in (2) and (3) step, seed culture based component and content used is: starch 12 g/L, analysis for soybean powder 30 g/L, glycerine 5 g/L, yeast powder 1 g/L, peptone 1 g/L, potassium primary phosphate 1 g/L, saltpetre 1 g/L, pH7.2.
(1), in (2) and (3) step, fermentation culture based component and content used is: starch 30 g/L, analysis for soybean powder 35 g/L, yeast powder 10 g/L, peptone 1 g/L, Sodium Glutamate 0.5 g/L, Threonine is respectively tetra-kinds of consumptions of 0.5,1.0,1.5,2.0 g/L, glycerine 10 g/L, saltpetre 1 g/L, potassium primary phosphate 0.8 g/L, pH7.6.
Screening method provided by the present invention is easy to operation, and related culture medium raw material source is wide, and cost is low, is applicable to very much industrial needs.Can effectively improve in the present invention the probability that obtains positive mutating strain, for clavulanic acid fermentative production provides superior strain, thereby improve clavulanic acid fermentative production level.
Embodiment
Embodiment 1
Mutagenesis: clavuligerus (Streptomyces clavuligerus) monospore suspension is passed through to UV mutagenesis.
Primary dcreening operation: according to following formulated isolation medium 2L: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, Threonine 50-100 g/L, agar 12 g/L, pH7.0.Prepare 50 of sterile petri dish, after medium sterilization, pour into and in culture dish, make flat board.Monospore suspension is made to physiological saline in ripe starting strain inclined-plane, and through vibration, granulated glass sphere disperses, and monospore suspension is collected in sterile filtration, and in sterile petri dish, ultraviolet (power 30W, lamp is apart from 30-40cm) is irradiated 1-5min.Monospore suspension after mutagenic treatment is suitably coated to flat board above after dilution, in 28 DEG C of incubators, cultivate 5-7 days.According to following formulated slant medium 2.4L: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, agar 12 g/L.pH7.0。Prepare 120, test tube, bevel is for subsequent use.In high density Threonine flat board, select growth and produce inoculation that spore is good on inclined-plane containing, in 28 DEG C of incubators, cultivate 5-7 days.After inclined-plane maturation, digging piece kind enters in seed culture medium.Seed culture based component and content are: starch 12 g/L, analysis for soybean powder 30 g/L, glycerine 5 g/L, yeast powder 1 g/L, peptone 1 g/L, potassium primary phosphate 1 g/L, saltpetre 1 g/L, pH7.2.After 28 DEG C of cultivation 32h, plant into fermention medium, after 28 DEG C of cultivation 47h, measure and tire, pick out 20 of the higher bacterial strains of tiring and carry out multiple sieve.
Multiple sieve: to seed culture medium, shaking speed is 220rpm by 20 the higher inoculation of tiring of picking out, plants in fermention medium after 28 DEG C of cultivation 32h, and shaking speed is 220rpm, after 28 DEG C of cultivation 47h, measures and tires.Pick out 1 of the highest bacterial strain of tiring, preserve, for subsequent use.
Fermention medium test: the highest bacterial strain carries out fermention medium test by tiring.Seed culture medium is packed in 3 750ml triangular flasks into every bottle of 100ml.After sterilizing, dig piece kind and enter in seed culture medium, cultivate 32h for 28 DEG C, plant respectively and in the fermention medium that contains different concns Threonine, carry out fermentation test by well-grown and without the seed of miscellaneous bacteria.According to following formulated fermention medium 1 .5L: starch 30 g/L, analysis for soybean powder 35 g/L, yeast powder 10 g/L, peptone 1 g/L, Sodium Glutamate 0.5 g/L, Threonine is respectively 0.5,1.0,1.5, tetra-kinds of dosage of 2.0g/L, glycerine 10 g/L, saltpetre 1 g/L, potassium primary phosphate 0.8 g/L, pH7.6.Inoculum size 16%(V/V).Shaking speed is 220rpm, after 28 DEG C of cultivation 47h, measures and tires, and corresponding measurement result is in table 1.HPLC method: methyl alcohol: water: 10%TBA=150:375:10, employing octadecylsilane chemically bonded silica is weighting agent, detection wavelength is 220nm.The substratum that contains different Threonine concentration is comprehensively compared, filter out best fermentating formula.
Table 1 measuring result
| Threonine concentration g/L in formula | The clavulanic acid ug/ml that tires |
| 0.5 | 4321 |
| 1.0 | 4509 |
| 1.5 | 5003 |
| 2.0 | 4687 |
From table 1, can know that the concentration of Threonine in formula is in the time of 1.0-2.0 g/L, clavulanic acid is tired higher, and when 1.5 g/L, clavulanic acid is tired the highest.
Embodiment 2
Mutagenesis: clavuligerus (Streptomyces clavuligerus) monospore suspension is passed through to EMS mutagenesis.
Primary dcreening operation: according to following formulated isolation medium 2L: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, Threonine 50-100 g/L, agar 12 g/L, pH7.0.Prepare 60 of sterile petri dish, after medium sterilization, pour into and in culture dish, make flat board.Monospore suspension is made to potassium phosphate buffer in ripe starting strain inclined-plane, add alkylating agent EMS, final concentration is 1%-5%, and oscillation treatment 25-45min adds Sulfothiorine termination reaction, vibration 10min.Monospore suspension after mutagenic treatment is suitably coated to flat board above after dilution, in 28 DEG C of incubators, cultivate 5-7 days.According to following formulated slant medium 3L: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, agar 12 g/L, pH7.0.Prepare 150, test tube, bevel is for subsequent use.In high density Threonine flat board, select growth and produce the good inoculation inclined-plane of spore containing, in 28 DEG C of incubators, cultivating 5-7 days.After inclined-plane maturation, dig piece kind and enter seed culture medium.Seed culture based component and content are: starch 12 g/L, analysis for soybean powder 30 g/L, glycerine 5 g/L, yeast powder 1 g/L, peptone 1 g/L, potassium primary phosphate 1 g/L, saltpetre 1 g/L, pH7.2.After 28 DEG C of cultivation 32h, plant into fermentation flask, after 28 DEG C of cultivation 47h, measure and tire, pick out 25 of the higher bacterial strains of tiring and carry out multiple sieve.
Multiple sieve: 25 the higher inoculation of tiring of picking out, to seed culture medium, are prepared to seed culture medium 400ml: starch 12 g/L, analysis for soybean powder 30 g/L according to the following formulation, glycerine 5 g/L, yeast powder 1 g/L, peptone 1 g/L, potassium primary phosphate 1 g/L, saltpetre 1 g/L, pH7.2.Shaking speed is 220rpm, after 28 DEG C of cultivation 32h, plants into fermention medium, and shaking speed is 220rpm, after 28 DEG C of cultivation 47h, measures and tires.Pick out 1 of the highest bacterial strain of tiring, preserve, for subsequent use.
Fermention medium test: the highest bacterial strain carries out fermention medium test by tiring.Seed culture medium is packed in 3 750ml triangular flasks into every bottle of 100ml.After sterilizing, dig piece kind and enter seed culture medium, cultivate 32h for 28 DEG C, plant respectively and in the fermention medium that contains different concns Threonine, carry out fermentation test by well-grown and without the seed of miscellaneous bacteria.According to following formulated fermentation culture based 1.5 L: starch 30 g/L, analysis for soybean powder 35 g/L, yeast powder 10 g/L, peptone 1 g/L, Sodium Glutamate 0.5 g/L, Threonine is respectively 0.5,1.0,1.5, tetra-kinds of dosage of 2.0g/L, glycerine 10 g/L, saltpetre 1 g/L, potassium primary phosphate 0.8 g/L, pH7.6.Inoculum size 16%(V/V).Shaking speed is 220rpm, after 28 DEG C of cultivation 47h, measures and tires, and corresponding measurement result is in table 2.HPLC method: methyl alcohol: water: 10%TBA=150:375:10, employing octadecylsilane chemically bonded silica is weighting agent, detection wavelength is 220nm.The substratum that contains different Threonine concentration is comprehensively compared, filter out best fermentating formula.
Table 2 measuring result
| Threonine concentration g/L in formula | The clavulanic acid ug/ml that tires |
| 0.5 | 4265 |
| 1.0 | 4616 |
| 1.5 | 5113 |
| 2.0 | 4702 |
From table 2, can know that the concentration of Threonine in formula is in the time of 1.0-2.0 g/L, clavulanic acid is tired higher, and when 1.5 g/L, clavulanic acid is tired the highest.
Claims (7)
1. a method that adopts the strain of Threonine tolerance model discrimination Clavulanic Acid High-Producing Strains, comprises the steps:
(1) primary dcreening operation: coat and contain on the isolation medium that Threonine dosis tolerata is 50g/l after the monospore suspension of mutagenic treatment is suitably diluted, in 28 DEG C of incubators, cultivate 5-7 days, select single colony inoculation of resistance to Threonine on slant medium, cultivate 5-7 days for 28 DEG C, inclined-plane is inoculated in seed culture medium after producing spore, after 28 DEG C of cultivation 32h, be inoculated in fermention medium, after 28 DEG C of cultivation 47h, measure and tire, pick out the high bacterial strain of tiring;
(2) multiple sieve: will tire higher inoculation in seed culture medium, cultivate after 32h for 28 DEG C, select inoculation that mycelial growth is good in fermention medium, cultivate after 47h for 28 DEG C and measure and tire;
(3) fermention medium test: higher bacterial strain carries out fermention medium test by tiring, after sterilizing, digging piece kind enters in seed culture medium, cultivate 32h for 28 DEG C, plant respectively and in the fermention medium that contains different concns Threonine, carry out fermentation test by well-grown and without the seed of miscellaneous bacteria, inoculum size is 16%(volume percent), after 28 DEG C of cultivation 47h, measure and tire.
2. method according to claim 1, in (1) step described in it is characterized in that, separation and Culture based component used and content are: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, Threonine 50 g/L, agar 12 g/L, pH7.0.
3. method according to claim 1, in (1) step described in it is characterized in that, slant culture based component used and content are: Zulkovsky starch 20 g/L, anhydrous magnesium sulfate 0.244 g/L, saltpetre 1g/L, ferrous sulfate 0.01 g/L, sodium-chlor 0.5 g/L, dipotassium hydrogen phosphate 0.5 g/L, agar 12 g/L, pH7.0.
4. method according to claim 1, it is characterized in that in described (1), (2) and (3) step, seed culture based component and content used is: starch 12 g/L, analysis for soybean powder 30 g/L, glycerine 5 g/L, yeast powder 1 g/L, peptone 1 g/L, potassium primary phosphate 1 g/L, saltpetre 1 g/L, pH7.2.
5. method according to claim 1, it is characterized in that in described (1), (2) and (3) step, fermentation culture based component and content used is: starch 30 g/L, analysis for soybean powder 35 g/L, yeast powder 10 g/L, peptone 1 g/L, Sodium Glutamate 0.5 g/L, Threonine 0.5-2.0 g/L, glycerine 10 g/L, saltpetre 1 g/L, potassium primary phosphate 0.8 g/L, pH7.6.
6. method according to claim 5, the amount that it is characterized in that Threonine in fermention medium is 1.0-2.0 g/L.
7. according to the method described in claim 5 or 6, the amount that it is characterized in that Threonine in fermention medium is 1.5 g/L.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774834A (en) * | 2015-04-14 | 2015-07-15 | 中国医药集团总公司四川抗菌素工业研究所 | Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model |
| CN111621434A (en) * | 2020-04-20 | 2020-09-04 | 中国医药集团总公司四川抗菌素工业研究所 | Streptomyces clavuligerus, application, fermentation medium and preparation method of clavulanic acid |
-
2013
- 2013-04-01 CN CN201310111024.XA patent/CN104099398A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 王明林: "克拉维酸生产菌的研究", 《中国优秀博硕士学位论文全文数据库（硕士） 工程科技I辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104774834A (en) * | 2015-04-14 | 2015-07-15 | 中国医药集团总公司四川抗菌素工业研究所 | Method for screening daptomycin high-producing strain by adopting sodium glutamate tolerance model |
| CN111621434A (en) * | 2020-04-20 | 2020-09-04 | 中国医药集团总公司四川抗菌素工业研究所 | Streptomyces clavuligerus, application, fermentation medium and preparation method of clavulanic acid |
| CN111621434B (en) * | 2020-04-20 | 2022-05-06 | 成都大学 | Streptomyces clavuligerus, application, fermentation medium and preparation method of clavulanic acid |
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Application publication date: 20141015 |