CN105200092A - Method for adjusting fermentation yield of rifamycin SV - Google Patents
Method for adjusting fermentation yield of rifamycin SV Download PDFInfo
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- CN105200092A CN105200092A CN201510670154.6A CN201510670154A CN105200092A CN 105200092 A CN105200092 A CN 105200092A CN 201510670154 A CN201510670154 A CN 201510670154A CN 105200092 A CN105200092 A CN 105200092A
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Abstract
The invention discloses a novel material supplementing method for increasing the fermentation yield of rifamycin SV. A three-stage fermentation tank with the Amycolatopsis mediterranei fermentation culture cycle ranging from 60 h to 90 h is supplemented with fosfomycin sodium with the final concentration of 3-50 mg/L in a sterile manner, and the titer of the rifamycin SV is increased by 9.1%-14.8% after culture is finished. The novel method has the characteristics that operation is simple, the yield increase effect of the rifamycin SV is stable and the like.
Description
Technical field
The present invention relates to a kind of method regulating fermentation yield of rifamycin SV
.the invention belongs to bio-pharmaceuticals field of fermentation engineering, particularly relate to a kind of by the regulating and controlling microbial C N metabolism flow direction, enhancing Rifamycin Sodium precursor (3-amino-5-hydroxy-benzoic acid) synthesis, promote the supply of precursor homogeneity, improve the method for Rifamycin Sodium combined coefficient.
Background technology
As large microbiotic bulk drug of tuberculotherapy, improve rifomycin fermentation combined coefficient and enjoy manufacturing enterprise and research unit to pay close attention to always.At present, more to the research improving rifomycin fermentation titer both at home and abroad, comprise: the aspect such as molecular biology information characteristics, fermention medium optimization producing bacterial classification, as documents 1: add soya-bean oil and L-Ala in 10L tank level and can improve Rifamycin Sodium and to tire 12.3%(food and fermentation industries, Shang Chunliang, 2011,37(5): 56-60); Documents 2: Rifamycin Sodium fermentation 1,000,000,000 can be made to improve 5.4%(patent of invention ZL201210176175.9 at 50 tons of large tank levels interpolation soya-bean oil and oxalic acid); Documents 3: add L-Ala, soya-bean oil and fosfomycin sodium/fosfomycin trometamol and Rifamycin Sodium can be made to tire average improve 17.0%(application for a patent for invention numbers 201210176177.8).But, documents 1 only relates to soya-bean oil and L-Ala exists interaction, under the optimization concentration determined, Rifamycin Sodium is tired raising 12.3%, and preliminary analysis has been carried out on glycolytic pathway carbon metablism, the organic acid combined coefficient and the impact of synthesizing of tiring of producing bacterial classification, do not relate to the impact that the azo-cycle precursor (3-amino-5-hydroxy-benzoic acid) of die aromatischen Aminosaeuren (tryptophane) combined coefficient on Rifamycin Sodium synthesizes, documents 2 relates to committed step---the activity of the reaction of succinodehydrogenase catalysis in the dual function adjustment tricarboxylic acid cycle of soya-bean oil and oxalic acid, by promoting Rifamycin Sodium precursor---the anabolism efficiency of methylmalonyl CoA improves the combined coefficient of Rifamycin Sodium, does not also relate to the impact that the azo-cycle precursor (3-amino-5-hydroxy-benzoic acid) of die aromatischen Aminosaeuren (tryptophane) combined coefficient on Rifamycin Sodium synthesizes, the effect that fosfomycin sodium/fosfomycin trometamol plays is not mentioned in documents 3, the effect of increasing production brought of application the method also cannot conclude it is the exercising result of fosfomycin sodium/fosfomycin trometamol, contrast prior art (documents 1) is only known: soya-bean oil and L-Ala have the effect regulating Rifamycin Sodium fermentation, namely the summary of the invention of Rifamycin Sodium fermentation is regulated not prove fully about fosfomycin sodium/fosfomycin trometamol, insufficient disclosure, and the document does not relate to the impact that the azo-cycle precursor (3-amino-5-hydroxy-benzoic acid) of die aromatischen Aminosaeuren (tryptophane) combined coefficient on Rifamycin Sodium synthesize yet.
One of die aromatischen Aminosaeuren (tyrosine, tryptophane and phenylalanine) biosynthetic rate-limiting enzyme is 5-enol pyruvylshikimate synthetic enzyme (substrate is phosphoenolpyruvic acid and 3-phosphoric acid-shikimic acid).Tryptophane significantly can suppress the combined coefficient of the core azo-cycle precursor 3-amino-5-hydroxy-benzoic acid of Rifamycin Sodium.Therefore, by suppressing the activity of 5-enol pyruvylshikimate synthetic enzyme, die aromatischen Aminosaeuren can be reduced in the fermentation synthesis of middle and later periods and accumulation, particularly may reduce the restraining effect that tryptophane synthesizes Rifamycin Sodium precursor 3-amino-5-hydroxy-benzoic acid.The small molecules similar to substrate (as the phosphoenolpyruvic acid) molecular structure of 5-enol pyruvylshikimate synthetic enzyme may have the inhibit feature to this enzyme.
Fosfomycin sodium is the microbiotic of Clinical practice, and mainly through suppressing the synthesis of responsive pathogen cell wall to treat the treatment of respiratory tract infection, skin soft-tissue infection, intestinal tract infections etc., adult's intravenous drip dosage is: 4 ~ 12g on the one.Do not act on the report of the New function of Amycolatopsis mediterranei metabolism as the metabolic regulation factor about fosfomycin sodium, that is: by the report suppressing 5-enol pyruvylshikimate synthase activity to reduce die aromatischen Aminosaeuren combined coefficient, to improve Rifamycin Sodium azo-cycle precursor 3-amino-5-hydroxy-benzoic acid precursor combined coefficient, promote the New function of fermentation yield of rifamycin SV.
goal of the invention
The object of the present invention is to provide a kind of novel method regulating fermentation yield of rifamycin SV, its substance is not using the synthesis of fosfomycin sodium as the usually anti-bacteria cell walls of the antibiosis on conventional meaning, thus kill the effect of microorganism, but phosphonomycin is reduced as the metabolic regulation factor restraining effect that aromatic ring amino acid (tryptophane) synthesizes Rifamycin Sodium azo-cycle precursor (3-amino-5-hydroxy-benzoic acid), and the biosynthesizing of final high efficiency promotion Rifamycin Sodium.
To be that the fosfomycin sodium of 3-50mg/L is aseptic by final concentration fill in the culture of the Amycolatopsis mediterranei of fermentation culture 60-90h in the present invention, and at the end of cultivation, Rifamycin Sodium is tired and improved 9.1%-14.8%.Described invention has the advantages that material is easily purchased, operation steps is simple, controllability is high, be applicable to industrial fermentation production.
technical scheme
A kind of novel method regulating fermentation yield of rifamycin SV that the present invention proposes, comprises the following steps:
(1) preparation of substratum
1. seed culture medium
Glucose 1.5%, soybean cake powder 1.0%, peptone 1.0%, saltpetre 0.05%, calcium carbonate 0.2%, Zulkovsky starch 1.5%, pH7.0.For subsequent use through 0.1MPa vapor sterilization 30min;
2. fermention medium
Glucose 6-10%, fish meal 0.3-0.55%, soybean cake powder 0.5-0.65%, peptone 0.4-0.7%, saltpetre 0.6-0.8%, calcium carbonate 0.5%, potassium primary phosphate 0.02%, pH nature.For subsequent use through 0.1MPa vapor sterilization 20min;
(2) actication of culture
Go bail for hide Amycolatopsis mediterranei (
a.mediterranei) bacterial classification access seed culture medium, 48h cultivated by shaking table, and culture condition is 28 DEG C, 220r/min;
(3) collection of strain expanded culture and somatic cells
Received by above-mentioned bacterial classification in fermentation shake flask substratum, liquid amount is 50-100mL/250mL triangular flask, and inoculum size is 3%-5%, and in 27 DEG C-28 DEG C, 150-200r/min shaking culture 46-50h, shake-flask seed substratum is closed bottle by rear aseptic technique;
(4) secondary seed is cultivated and three grade fermemtation
To close bottle seed flame protection access secondary seed tank, culture temperature 28 DEG C, rotating speed is 160-200r/min, and air flow control is 0.8vvm, and tank pressure controls as 0.03-0.04MPa, and after cultivating 48h, culture transferring is to three grade fermemtation tank;
(5) three grade fermemtation tank controls
Culture temperature 28 DEG C, rotating speed 0-72h is 220r/min, 72-96h be 230r/min, 96h is 240r/min to putting tank.Air flow control: 0-48h is 0.4vvm, 48-72h is 0.6vvm, and 72 is 0.8vvm to putting tank.Tank pressure controls: 0-48h is 0.02MPa, 48-72h be 0.03MPa, 72-96h be 0.04MPa, 96h is 0.05MPa to putting tank;
(6) three grade fermemtation culture cycle is when 60-90h, asepticly fills into the fosfomycin sodium that final concentration is 3-50mg/L, and other Controlling Technology is constant.
beneficial effect
Documents 3 with the addition of soya-bean oil, L-Ala and fosfomycin sodium/fosfomycin trometamol three kinds of materials and improves Rifamycin Sodium output, but do not absolutely prove their respective mechanism of action, more phosphonomycin is not reduced as the metabolic regulation factor the inhibiting content that aromatic ring amino acid (tryptophane) synthesizes Rifamycin Sodium azo-cycle precursor (3-amino-5-hydroxy-benzoic acid).Present invention employs and add less material (only adding fosfomycin sodium), just significantly suppress the synthesis of tryptophane in fermenting process, and make precursor 3-amino-5-hydroxy-benzoic acid concentration improve 2-3 doubly, aminoglutaric acid concentration reduces 27-40%, illustrating that glutamine participates in the validity raising of 3-amino-5-hydroxy-benzoic acid route of synthesis, and flow into the competitive bypass of 3-amino-5-hydroxy-benzoic acid precursor synthesis---the amount that NADPH-linked glutamate synthase catalysis generates glutamate pathway reduces.Meanwhile, α-ketoglutaric acid and lysine concentration value obviously increase at fermenting process, illustrate that the carbon metablism efficiency through tricarboxylic acid cycle strengthens.
In the present invention fosfomycin sodium add more Effective Regulation, enhance Rifamycin Sodium precursor (3-amino-5-hydroxy-benzoic acid) combined coefficient, at the end of cultivation, Rifamycin Sodium is tired and is improved 9.1%-14.8%, well beyond the expection to Rifamycin Sodium facilitation effect, also simplify operating procedure, saved production cost, create unforeseeable technique effect.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, further description is done to the present invention.
Fig. 1 produces amino acid, 3-amino-5-hydroxy-benzoic acid and α-ketoglutaric acid change in strain fermentation process.
In figure, tryptophane (Trp) did not detect before 94h, went up to the 4.0mg/L of 118h rapidly afterwards, was slowly down to 3.0mg/L to 166h.Corresponding is 3-amino-5-hydroxy-benzoic acid (AHBA) change in concentration: before 94h, and AHBA concentration is between 300-400mg/L, and along with the lifting of Trp concentration, AHBA is reduced to 70-100mg/L gradually.L-glutamic acid (Glu) concentration is raised to about the 530mg/L of 158h by 270mg/L mono-tunnel of 46h.The main path producing bacterial strain synthesis Glu is NADPH-linked glutamate synthase (GOGAT) catalyzed reaction, and Glu is that AHBA synthesizes one of substrate.After Gln rises to the 330mg/L of 94h, start rapid reduction along with the lasting rising of Glu, and after 94h, Gln with AHBA Changing Pattern is identical.The accumulation of Trp after 94h inhibits the synthesis of AHBA, Gln participate in AHBA route of synthesis validity reduce, then enter AHBA synthesis competitive bypass---the generation Glu metabolism of GOGAT catalysis, thus Glu negative is about AHBA biosynthesizing.
α-ketoglutaric acid (α-ketoglutarate) and oxaloacetic acid (OAA) are the intermediate products that tricarboxylic acid (TCA) circulates, and can derive Methionin (Lys) by OAA.Thus Lys and α-ketoglutaric acid change in concentration rule can reflect the cycle efficiency of TCA.As shown in Figure 1: α-ketoglutaric acid is 35mg/L, 94h maximum value in 46h concentration is 135mg/L, is slowly down to 60mg/L to 166h; Lys is 6mg/L, 70h maximum value in 46h concentration is 19mg/L, is slowly down to 11mg/L to 166h, reflects the basic carbon metablism flow of TCA circulation.
Fig. 2 is the change of amino acid in fermenting process of the present invention, 3-amino-5-hydroxy-benzoic acid and α-ketoglutaric acid.
In figure, Trp did not detect before 94h, went up afterwards to the 2.6mg/L of 118h, was slowly down to 2.3mg/L to 166h.Maximum value decreases than Fig. 1 35%, Trp concentration also to only have Fig. 1 55%-76% at 118-166h.Illustrate that fosfomycin sodium serves the regulating and controlling effect suppressing Trp synthesis really.Corresponding is AHBA change in concentration: before 94h, and AHBA concentration is between 300-418mg/L, and a 94-134h high position maintains between 300-400mg/L, and is only 100-160mg/L after Fig. 1 cycle 90h.Adding fosfomycin sodium makes AHBA concentration be 2-3 times that contrasts in this interval.Confirming fosfomycin sodium by suppressing Trp synthesis, decreasing the restraining effect that it synthesizes AHBA, effectively increasing the supply of AHBA; Glu concentration slowly carries about the 380mg/L to 70h by the 280mg/L of 46h, reduces, go up again afterwards to the 405mg/L of 134h in 70-94h, Glu concentration.But its concentration only has the 60-73% of Fig. 1 in process.Corresponding to the reduction of 70-94h, Glu, glutamine (Gln) is risen to the 485mg/L of 94h by 80mg/L, after, start slow reduction along with the slow rising of Glu, but whole process Gln concentration all improves 33-60% than Fig. 1.
The fosfomycin sodium factor by reduce Trp synthesis reduce to production bacterial strain (
a.mediterranei) suppression of synthesis AHBA, the validity that Gln participates in AHBA route of synthesis improves, and thus flows into the competitive bypass of AHBA synthesis---and the amount of the generation Glu approach of GOGAT catalysis reduces, and thus Glu combined coefficient is starkly lower than contrast.
α-ketoglutaric acid is 37mg/L, 94h maximum value in 46h concentration is 190mg/L, is slowly down to 135mg/L to 166h, and after 94h, concentration all improves 24-48% than Fig. 1; Lys is 8mg/L, 94h maximum value in 46h concentration is 28mg/L, and be slowly down to 14mg/L to 166h, in process, concentration value is obviously greater than Fig. 1's.The cycle efficiency that invention increases TCA being described, increasing via its intermediate product---succinyl CoA generates the combined coefficient of three carbon precursors.
Embodiment
embodiment 1
1, the preparation of substratum
(1) seed culture medium
Glucose 1.5g, soybean cake powder 1.0g, peptone 1.0g, saltpetre 0.05g, calcium carbonate 0.2g, Zulkovsky starch 1.5g, is settled to 100mL, pH7.0.For subsequent use through 0.1MPa autoclaving 30min;
(2) fermention medium
Glucose 6-10g, fish meal 0.3-0.55g, soybean cake powder 0.5-0.65g, peptone 0.4-0.7g saltpetre 0.6-0.8g, calcium carbonate 0.5g, potassium primary phosphate 0.02g, is settled to 100mL, pH nature.For subsequent use through 0.1MPa autoclaving 30min;
2, actication of culture
Go bail for hide Amycolatopsis mediterranei (
a.mediterranei) bacterial classification access seed culture medium, 48h cultivated by shaking table, and culture condition is 28 DEG C, 220r/min;
3, the collection of strain expanded culture and somatic cells
Received by above-mentioned bacterial classification in fermentation shake flask substratum, liquid amount is 50mL/250mL triangular flask, and inoculum size is 5%, and in 27 DEG C, 150r/min shaking culture 46h, then shake-flask seed substratum is closed bottle by aseptic technique;
4, secondary seed is cultivated and three grade fermemtation
To close bottle seed flame protection access secondary seed tank, culture temperature 28 DEG C, rotating speed is 160r/min, and air flow control is 0.8vvm, and tank pressure controls to fill into three grade fermemtation tank for 0.03MPa, 48h aseptic technique;
5, three grade fermemtation tank controls
Culture temperature 28 DEG C, rotating speed 0-72h is 220r/min, 72-96h be 230r/min, 96h is 240r/min to putting tank.Air flow control: 0-48h is 0.4vvm, 48-72h is 0.6vvm, and 72 is 0.8vvm to putting tank.Tank pressure controls: 0-48h is 0.02MPa, 48-72h be 0.03MPa, 72-96h be 0.04MPa, 96h is 0.05MPa to putting tank;
6, regulatory factor is added
Final concentration is that the fosfomycin sodium of 5mg/L carries out weighing, filters after being dissolved in sterilized water through 0.22um, by flame protection access feed supplement tank.When culture cycle is 60h, the fosfomycin sodium solution in feed supplement tank is filled into fermentor tank by aseptic technique.
embodiment 2
1, the preparation of substratum: with embodiment 1.
2, actication of culture: with embodiment 1.
3, the collection of strain expanded culture and conjunction bottle cell: with embodiment 1.
4, secondary seed is cultivated and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Final concentration is that the fosfomycin sodium of 50mg/L carries out weighing, filters after being dissolved in sterilized water through 0.22um, by flame protection access feed supplement tank.When culture cycle is 90h, the fosfomycin sodium solution in feed supplement tank is filled into fermentor tank by aseptic technique.
embodiment 3
1, the preparation of substratum: with embodiment 1.
2, actication of culture: with embodiment 1.
3, the collection of strain expanded culture and conjunction bottle cell: with embodiment 1.
4, secondary seed is cultivated and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Final concentration is that the fosfomycin sodium of 20mg/L carries out weighing, filters after being dissolved in sterilized water through 0.22um, by flame protection access feed supplement tank.When culture cycle is 70h, the fosfomycin sodium solution in feed supplement tank is filled into fermentor tank by aseptic technique.
embodiment 4
1, the preparation of substratum: with embodiment 1.
2, actication of culture: with embodiment 1.
3, the collection of strain expanded culture and conjunction bottle cell: with embodiment 1.
4, secondary seed is cultivated and three grade fermemtation: with embodiment 1.
5, three grade fermemtation tank controls: with embodiment 1.
6, regulatory factor is added
Final concentration is that the fosfomycin sodium of 35mg/L carries out weighing, filters after being dissolved in sterilized water through 0.22um, by flame protection access feed supplement tank.When culture cycle is 80h, the fosfomycin sodium solution in feed supplement tank is filled into fermentor tank by aseptic technique.
Claims (1)
1. one kind regulates the method for fermentation yield of rifamycin SV, it is characterized in that: Amycolatopsis mediterranei carries out three grade fermemtation cultivation, three grade fermemtation culture cycle is aseptic when 60-90h fills into the fosfomycin sodium that final concentration is 3-50mg/L, can significantly improve the output of Rifamycin Sodium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660598A (en) * | 2012-05-31 | 2012-09-12 | 河南省南街村（集团）有限公司 | Method for improving fermentation yield of rifamycin SV |
| CN102703541A (en) * | 2012-05-31 | 2012-10-03 | 河南省南街村（集团）有限公司 | Feeding method for improving rifamycin SV fermentation yield |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102660598A (en) * | 2012-05-31 | 2012-09-12 | 河南省南街村（集团）有限公司 | Method for improving fermentation yield of rifamycin SV |
| CN102703541A (en) * | 2012-05-31 | 2012-10-03 | 河南省南街村（集团）有限公司 | Feeding method for improving rifamycin SV fermentation yield |
Non-Patent Citations (1)
| Title |
|---|
| 邓鹏飞等: "锌离子影响地中海拟无枝酸菌合成有机酸和利福霉素SV", 《中国酿造》 * |
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Application publication date: 20151230 |