CN108384819A - A kind of culture medium and fermentation process for the tacrolimus that ferments - Google Patents
A kind of culture medium and fermentation process for the tacrolimus that ferments Download PDFInfo
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- CN108384819A CN108384819A CN201710063681.XA CN201710063681A CN108384819A CN 108384819 A CN108384819 A CN 108384819A CN 201710063681 A CN201710063681 A CN 201710063681A CN 108384819 A CN108384819 A CN 108384819A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a kind of culture mediums and fermentation process for the tacrolimus that ferments.The culture medium includes carbon source and nitrogen source, wherein the carbon source includes 5 20g/L of 30 70g/L of soluble starch, 5 20g/L of fructose and lactose, the nitrogen source includes 10 30g/L of 1 40g/L of fish meal protein peptone and yeast extract.The yield of culture medium or fermentation process fermentation tacrolimus using the present invention is high, and potency is 3 times of existing culture medium fermentation titer, reaches 1000mg/L or more.
Description
Technical field
The invention belongs to industrial microbial technology field, more particularly to a kind of culture medium for the tacrolimus that ferments and
Fermentation process.
Background technology
Tacrolimus is the tunning isolated from streptomyces, is a kind of immunosuppressor of strength, compared with ring spore
Element is 100 times strong.Clinical test proves there is fine curative effect in the transplanting such as the heart, lung, intestines, marrow, in panimmunity disease
Middle age also plays positive effect.
The acquisition of tacrolimus at present, which is mainly fermented by bacterial micro-organism (or actinomycete fermentation), to be obtained.Improve Ta Kemo
The fermentation yield of department mainly passes through the improvement of strain improvement and nutrient media components.
Wherein, it to including mainly the genetic fragments such as amplification acetyl CoA synthetase, carboxylase in terms of the transformation of bacterial strain, improves
Tolerance (Jung S, Moon S, Lee K, et al.Strain development of of the bacterial strain to target product
Streptomyces sp.for tacrolimus production using sequential adaptation.[J]
.Journal of Industrial Microbiology,2009,36(12):The modes such as 1467-71).
About the improvement of nutrient media components, prior art is mainly by adding precusor amino acids (Singh B P, Behera
B K.Regulation of tacrolimus production by altering primary source of carbons
and amino acids[J].Letters in Applied Microbiology,2009,49(2):254-9.), nipecotic acid (J,Gajzlerska W,Klimaszewska M.Enhancement of tacrolimus productivity in
Streptomyces tsukubaensis by the use of novel precursors for biosynthesis[J]
.Enzyme&Microbial Technology,2012,51(6-7):388-95), methyl oleate (Mo S J, Ban Y H,
Park J W,et al.Enhanced FK506production in Streptomyces clavuligerus,CKD1119
by engineering the supply of methylmalonyl-CoA precursor[J].Journal of
Industrial Microbiology&Biotechnology,2009,36(12):1473-1482.), vegetable oil (corn oil
(Kim H S,Park Y I.Lipase activity and tacrolimus production in Streptomyces
clavuligerusCKD 1119 mutant strains.[J].Journal of Microbiology&
Biotechnology,2007,17(10):1638-1644)：Improve lipase active), compounded carbons (dextrin, soluble starch
Deng proportioning) (Wang Ya, Huang He, Bian Jiaxin, wait tacrolimus engineering bacterias structure and initial fermentation process optimization [J] in traditional Chinese medical science
Medicine industry magazine, 2016,47 (2):152-157) and it is added substances (3 carbon chain compounds such as propionic acid, propylene glycol and propyl alcohol
(Gajzlerska W,Kurkowiak J,J.Use of three-carbon chain compounds as
biosynthesis precursors to enhance tacrolimus production in Streptomyces
tsukubaensis[J].New Biotechnology,2015,32(1):32-39)) improve the yield of tacrolimus.
And nutrient media components are improved at present, it needs to produce bacterial strain for specific tacrolimus mostly；In other words,
Different tacrolimus produces bacterial strain, such as tacrolimus producing strains streptomyces tsukubaensis (S.tsukubaensis) No.9993 is (i.e.
CGMCC No.0083) wild strain and its genetic engineering bacterium need to the different component in culture medium carry out improvement just have compared with
(structure of tacrolimus engineering bacteria and initial fermentation process optimization [J] Chinese Medicine industry are miscellaneous for the yield of high tacrolimus
Will, 2016,47 (2):152-157).The universality of culture medium is not strong, and the process of strain construction is cumbersome, input is big, it is long to take；
In addition, the formula of culture medium is complicated, of high cost at present, the inorganic salts containing heavy metal ion are mostly used.It is above-mentioned that there are problems is equal
It is unfavorable for the industrialized production of tacrolimus.
Invention content
Therefore, the technical problem to be solved by the present invention is to solve the defect of the above-mentioned prior art, a kind of new be used for is provided
The culture medium and fermentation process of fermentation tacrolimus can simplify formula, reduce metallic pollution and be suitable for production not of the same race
The streptomyces tsukubaensis bacterial strain of tacrolimus improves the fermentation unit of tacrolimus, production efficiency is improved, in favor of being suitable for industry
Metaplasia is produced.
The technical solution that the present invention solves above-mentioned technical problem is as follows：
The present invention solves one of technical solution used by above-mentioned technical problem：A kind of training for the tacrolimus that ferments
Base is supported, including carbon source and nitrogen source, the carbon source include soluble starch 30-70g/L, fructose 5-20g/L and lactose 5-20g/L；
The nitrogen source includes fish meal protein peptone 1-40g/L and yeast extract 10-30g/L.
Preferably, the carbon source is soluble starch 30-70g/L, fructose 5-20g/L and lactose 5-20g/L and/or institute
It is fish meal protein peptone 1-40g/L and yeast extract 10-30g/L to state nitrogen source.
Preferably, the culture medium further includes amino acid, the content of the amino acid is preferably 1-10g/L.The amino
Acid is that this field is conventional, preferably L-arginine.
More preferably, the culture medium further includes inorganic salts, wherein the inorganic salts are preferably (NH4)2SO4With anhydrous sulphur
Sour magnesium；(the NH4)2SO4It is respectively preferably 1-20g/L and 1-10g/L with the content of anhydrous magnesium sulfate.
Further more preferably, the culture medium further includes inorganic salts CaCO3, the CaCO3Content be preferably 0.5-
1.5g/L。
Most preferably, the culture medium further includes bubble enemy；The content of the bubble enemy is preferably 1-20g/L.
In the present invention, the pH of the culture medium is routine, can be with naturally, can also adjust, preferably pH6.2-
6.8。
The present invention solve above-mentioned technical problem used by technical solution second is that：A kind of fermentation process of tacrolimus,
It includes the following steps：
(1) seed liquor for the streptomyces tsukubaensis for producing tacrolimus is inoculated in above-mentioned fermentation medium and is carried out liquid fermentation
Culture, obtains zymotic fluid；
(2) tacrolimus is isolated from the zymotic fluid.
In the present invention, the streptomyces tsukubaensis can be the streptomyces tsukubaensis of existing various production tacrolimus, preferably
Ground is streptomyces tsukubaensis (S.tsukubaensis) No.9993, i.e. CGMCC No.0083.
The preparation of the seed liquor of fermented and cultured of the present invention and inoculation method are that this field is conventional, preferably, wherein level-one kind
Sub- incubation time is 48-72h (hour), and secondary seed incubation time is 24-48h, and secondary seed inoculum concentration is 1-2%, fermentation
Liquid inoculum concentration is 5-15%；The percentage is percent by volume.
In a preferred embodiment of the present invention, the first order seed incubation time is 60h, the secondary seed culture
Time is 48h, and the secondary seed inoculum concentration is 1%, and the zymotic fluid inoculum concentration is 10%；The percentage is
Percent by volume.
On the basis of common knowledge of the art, above-mentioned each optimum condition can be combined arbitrarily to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that：Being conducive to streptomycete high-efficiency fermenting the present invention provides one kind and producing him gram
The fermentation medium and corresponding fermentation process that do not take charge of.Comprehensive proportioning is carried out by the carbon nitrogen source to culture medium prescription, is simplified
It is formulated, reduces cost and reduce Cu in the prior art2+、Zn2+、Mn2+And Co2+The use of equal metal ions and its caused
Pollution, in addition, culture medium of the present invention can not only improve fermentation yield of the streptomyces tsukubaensis to tacrolimus, to production not of the same race
Streptomyces tsukubaensis wild mushroom and the genetic engineering bacterium effect of being improved of tacrolimus, universality are strong.In conjunction with provided by the invention
The fermentation titer of fermentation process, tacrolimus reaches as high as 1000mg/L or more, and about tacrolimus fermentation is imitated in the prior art
3 times of valence, are conducive to the industrialized production of tacrolimus.
Description of the drawings
Fig. 1 is the liquid phase test map of tacrolimus.
Fig. 2 is using the relative potency block diagram obtained after different seed culture time and inoculum concentration.
Specific implementation mode
It is further illustrated the present invention below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
Product specification selects.
Material source in the following example is：
Fish meal protein peptone, anhydrous magnesium sulfate, soluble starch, (NH4)2SO4, fructose, yeast extract, lactose and CaCO3
Purchased from Chinese medicines group；L-arginine is compiled purchased from Shanghai dimension；Bubble enemy is purchased from the anti-pharmacy in river.
Following percentages are percent by volume.
1 fermentation culture method of embodiment and standard potency it is specified
1, the orthogonal experiment of seed growth time and inoculum concentration is determined
1) preparation of seed
Aseptically, one plant of streptomyces tsukubaensis (Streptomyces tsukubaensis) CGMCC No.0083 are taken
(being purchased from CGMCC), i.e., SIPI-F001 is inoculated into slant medium, and the inclined-plane for being inoculated with strain is placed in 28 DEG C of constant incubators,
It passes on period 10-14d (day), transfer period 18-20d, depending on bacterium colony growing state, general culture 15 days obtains maturation
Spore.Then ripe spore inoculating (or is inoculated in equipped with 100ml kinds in the 250ml shaking flasks equipped with 30ml seed culture mediums
The 750ml shaking flasks of sub- culture medium) in, it is cultivated on 28 DEG C, 240rpm shaking tables.Then other conditions under the same conditions, tool
Body is according to following table 1 to the incubation time and zymotic fluid of the incubation time of first order seed, secondary seed inoculum concentration, secondary seed
Inoculum concentration test.
The orthogonal experiment of 1 seed growth time of table and inoculum concentration
2) composition of used medium
Slant medium (ISP4)：Soluble starch 10g/L, ammonium sulfate 2g/L, dipotassium hydrogen phosphate 1g/L, epsom salt
1g/L, ferrous sulfate 0.01g/L, manganese chloride 0.01g/L, zinc sulfate 0.01g/L, CaCO32 g/L, agar 20g/L, pH 7.2,
121 DEG C sterilize 25 minutes.
Seed culture medium：Soluble starch 10g/L, oral glucose 15g/L, beef extract 20g/L, ammonium sulfate 3g/L,
Yeast powder 2.5g/L, peanut meal 5g/L, CaCO32g/L, seed bottle, 7.0,121 DEG C of pH sterilize 25 minutes.
Fermentation medium forms：Soluble starch 50g/L, fish meal protein peptone 20g/L, yeast extract 20g/L, fructose 10g/
L and lactose 10g/L, (NH4)2SO410g/L, anhydrous magnesium sulfate 5g/L, L-arginine 5g/L, bubble enemy 10g/L, CaCO3 1g/
L、pH 6.5。
3) it ferments
Mature seed in zymotic fluid obtained is inoculated in the inoculum concentration in above-mentioned table 1 in fermentation medium, loading amount
30mL/250mL, in 25 DEG C, the rotary shaker culture of 250rpm 168 hours takes zymotic fluid 1ml, and centrifuging and taking supernatant is with efficiently
Liquid chromatogram (HPLC) carries out the measurement of tacrolimus content.
It is specific as follows：
Instrument：1200 efficient liquid phase of Agilent；
Analytical column：Month rising sunXB-C18 (2.1 × 150mm, 3um)；
Mobile phase：A：10% acetonitrile is containing 0.1% phosphoric acid, B：100% acetonitrile；
Column temperature：40℃；
Wavelength：200nm；
Sample size：5μl；
2 cleaning procedure of table：
| Time/min | B (%) | Flow velocity ml/min | Pressure (bar) |
| 0 | 50 | 0.3 | 250 |
| 1.0 | 50 | 0.3 | 250 |
| 13.0 | 85 | 0.3 | 250 |
| 15.0 | 85 | 0.3 | 250 |
| 16.0 | 50 | 0.3 | 250 |
| 22.0 | 50 | 0.3 | 250 |
The liquid phase test map of tacrolimus is as shown in Figure 1.
Experimental result is shown in Fig. 2, wherein the experimental group that number is 1 is control group.It has been found by contrast that the experiment for being 6 in number
Inoculum concentration and incubation time under culture streptomyces tsukubaensis obtain relative potency be up to 1082.81mg/L, tacrolimus
(TAC) yield is 6.45mg/h.
3 seed Orthogonal experiment results of table：The relative potency and TAC yields of different experiments
Therefore in following embodiments, the incubation time of first order seed is all made of 60h, and the incubation time of secondary seed is all made of
48h, secondary seed inoculum concentration are all made of 1%, and zymotic fluid inoculum concentration is all made of 10%.
Embodiment 2
1, the culture of seed
Aseptically, it takes one plant of streptomyces tsukubaensis, i.e. SIPI-F001 to be inoculated into slant medium, strain will be inoculated with
Inclined-plane be placed in 28 DEG C of constant incubators, cultivate 15 days, obtain ripe spore.Then by ripe spore inoculating in equipped with
In the 250ml shaking flasks of 30ml seed culture mediums, culture 60h obtains first order seed on 28 DEG C, 240rpm shaking tables, with 1% ratio
Example is transferred in the 250ml shaking flasks equipped with 30ml seed culture mediums, and secondary seed culture 48h is carried out under the same terms, is obtained into
Ripe seed.Seed culture medium and slant medium used are the same as embodiment 1.
2, it ferments
Mature seed in zymotic fluid obtained is inoculated in 10% inoculum concentration in following fermentation mediums.Ferment item
Part is the same as embodiment 1.
Fermentation medium forms：Soluble starch 50g/L, fish meal protein peptone 20g/L, yeast extract 20g/L, fructose 10g/
L, lactose 10g/L, (NH4)2SO410g/L and anhydrous magnesium sulfate 5g/L, pH 6.5.Efficient liquid phase measures TAC potency
823.5317mg/L TAC yields are 4.90mg/h.
Embodiment 3
Fermentation medium forms：Soluble starch 50g/L, fish meal protein peptone 20g/L, yeast extract 20g/L, fructose 10g/
L, lactose 10g/L, (NH4)2SO410g/L, anhydrous magnesium sulfate 5g/L and L-arginine 5g/L, pH 6.5.Remaining same embodiment 2.
It is 1031.169mg/L that efficient liquid phase, which measures TAC potency, and TAC yields are 6.14mg/h.
Embodiment 4
Fermentation medium forms：Soluble starch 30g/L, fructose 5g/L, lactose 5g/L, fish meal protein peptone 1g/L, yeast
Medicinal extract 10g/L, (NH4)2SO41g/L, anhydrous magnesium sulfate 1g/L, L-arginine 1g/L, bubble enemy 1g/L and CaCO30.5g/L,
PH 6.2, remaining same embodiment 2.It is 850.2672mg/L that efficient liquid phase, which measures TAC potency, and TAC yields are 5.06mg/h.
Embodiment 5
Fermentation medium forms：Soluble starch 50g/L, fructose 10g/L, lactose 10g/L, fish meal protein peptone 20g/L with
And yeast extract 20g/L, pH 6.5, remaining same embodiment 2.It is 675.2141mg/L, TAC yields that efficient liquid phase, which measures TAC potency,
For 4.024mg/h.
Embodiment 6
Soluble starch 70g/L, fructose 20g/L, lactose 20g/L, fish meal protein peptone 40g/L, yeast extract 30g/L,
(NH4)2SO420g/L, anhydrous magnesium sulfate 10g/L, L-arginine 10g/L, bubble enemy 20g/L and CaCO31.5g/L, pH6.8,
Remaining same embodiment 2.It is 809.2933mg/L that efficient liquid phase, which measures TAC potency, and TAC yields are 4.82mg/h.
Comparative example 1
Fermentation medium forms：Soluble starch 50g/L, fish meal protein peptone 20g/L, pH 6.5, remaining same embodiment 2.It is high
It is 242.6045mg/L that effect liquid phase, which measures TAC potency, and TAC yields are 1.44mg/h.
Comparative example 2
Fermentation medium forms：Soluble starch 50g/L, fish meal protein peptone 20g/L and yeast extract 20g/L, pH
6.5, remaining same embodiment 2.It is 416.73mg/L that efficient liquid phase, which measures TAC potency, and TAC yields are 2.50mg/h.
Effect example 1
It is that the comparison of t bacteria AC fermentation levels is as follows in fermentative medium formula, with document report with embodiment 1：
Bibliography：
[1]Singh B P,Behera B K.Regulation of tacrolimus production by
altering primary source of carbons and amino acids[J].Letters in Applied
Microbiology,2009,49(2):254-9.
[2]J,Gajzlerska W,Klimaszewska M.Enhancement of tacrolimus
productivity in Streptomyces tsukubaensis by the use of novel precursors for
biosynthesis[J].Enzyme&Microbial Technology,2012,51(6-7):388-95.
[3]Mo S J,Ban Y H,Park J W,et al.Enhanced FK506 production in
Streptomyces clavuligerus,CKD1119 by engineering the supply of methylmalonyl-
CoA precursor[J].Journal of Industrial Microbiology&Biotechnology,2009,36
(12):1473-1482.
[4]Kim H S,Park Y I.Lipase activity and tacrolimus production in
Streptomyces clavuligerusCKD 1119 mutant strains.[J].Journal of Microbiology&
Biotechnology,2007,17(10):1638-1644.
[5] (carbon source) Wang Ya, Huang He, Bian Jiaxin waits the structure and initial fermentation process optimization of tacrolimus engineering bacterias
[J] Chinese Journal of Pharmaceuticals, 2016,47 (2):152-157.
[6]Gajzlerska W,Kurkowiak J,J.Use of three-carbon chain
compounds as biosynthesis precursors to enhance tacrolimus production in
Streptomyces tsukubaensis[J].New Biotechnology,2015,32(1):32-39.
[7]Jung S,Moon S,Lee K,et al.Strain development of Streptomyces
sp.for tacrolimus production using sequential adaptation.[J].Journal of
Industrial Microbiology,2009,36(12):1467-71.
Effect example 2
Control group fermentative medium formula：Dextrin 50g/L, yeast powder 10g/L, Dried Corn Steep Liquor Powder 5g/L, leucine 3g/L,
Piperidines -2- formic acid 1g/L, K2HPO4 1g/L、CuSO4·5H2O 0.003g/L、FeSO4·7H2O 0.0025g/L、MnCl2·
4H2O 5×10-4g/L、ZnSO4·7H2O 0.01g/L、CaCl2·2H2O 0.02g/L、CoCl2·6H2O 0.003g/L、
CaCO3 1g/L、Mg3(PO4)25g/L and bubble enemy 1g/L；PH7.2 (referring to above-mentioned bibliography [5])；Fermentation process is such as planted
Sub- preparation, inoculum concentration, cultivation temperature and incubation time etc., with above-described embodiment 2.
The present invention selects fermentative medium formula in embodiment 1.
Fermentation results are as follows：
| SIPI-F001 | SIPI-F002 | SIPI-F003 | |
| Control group TAC is generated horizontal (mg/L) | 489.0435 | 402.1252 | 670.3715 |
| Embodiment 1TAC is generated horizontal (mg/L) | 1314.749 | 699.6951 | 793.0005 |
Wherein streptomyces tsukubaensis (Streptomyces tsukubaensis) genetic engineering bacterium SIPI-F002, SIPI-
F003 may be derived from Shanghai Institute of Pharmaceutical Industry, or is made by the method that above-mentioned bibliography [5] discloses, wherein building
Streptomyces coelicolor M145 and actinoplanes N902-109 used in SIPI-F002 and SIPI-F003 may be derived from Shanghai
Medical industry research institute, carrier pLYW2 and pIJ8660 are purchased from GIBCO-BRL.Streptomyces tsukubaensis No.9993, that is, CGMCC used
No.0083。
By upper table it is found that fermentation medium of the present invention also has the generation of tacrolimus for Other Engineering bacteria strain
Different degrees of facilitation, it is not limited to which experimental strain SIPI-F001, the bacterial strain that generation same target product is planted to him are equal
With a degree of facilitation.
Claims (10)
1. a kind of culture medium for the tacrolimus that ferments, including carbon source and nitrogen source；It is characterized in that the carbon source includes solvable
Property starch 30-70g/L, fructose 5-20g/L and lactose 5-20g/L, the nitrogen source includes fish meal protein peptone 1-40g/L and yeast leaching
Cream 10-30g/L.
2. culture medium as described in claim 1, which is characterized in that the carbon source is soluble starch 30-70g/L, fructose 5-
20g/L and lactose 5-20g/L and/or the nitrogen source are fish meal protein peptone 1-40g/L and yeast extract 10-30g/L.
3. culture medium as described in claim 1, which is characterized in that the culture medium further includes amino acid, the amino acid
Content is 1-10g/L；Preferably the amino acid is L-arginine.
4. culture medium as claimed in claim 3, which is characterized in that the culture medium further includes inorganic salts.
5. culture medium as claimed in claim 4, which is characterized in that the inorganic salts are (NH4)2SO4And anhydrous magnesium sulfate；Compared with
Goodly, the described (NH4)2SO4Content with anhydrous magnesium sulfate is respectively 1-20g/L and 1-10g/L.
6. culture medium as claimed in claim 4, which is characterized in that the inorganic salts further include CaCO3, preferably, described
CaCO3Content be 0.5-1.5g/L.
7. culture medium as claimed in any one of claims 1 to 6, which is characterized in that the culture medium further includes bubble enemy；Preferably,
The content of the bubble enemy is 1-20g/L.
8. culture medium as described in claim 1, which is characterized in that the pH value of the culture medium is 6.2-6.8.
9. a kind of fermentation process of tacrolimus, which is characterized in that include the following steps：
(1) streptomyces tsukubaensis (Streptomyces tsukubaensis) of tacrolimus, preferably streptomyces tsukubaensis will be produced
The seed liquor of CGMCC No.0083 is inoculated in any one of claim 1-8 culture mediums and carries out liquid fermentation culture, obtains
Zymotic fluid；
(2) tacrolimus is isolated from the zymotic fluid.
10. fermentation process as claimed in claim 9, which is characterized in that first order seed culture in the preparation of the seed liquor
Time is 48-72h, and secondary seed incubation time is 24-48h, and secondary seed inoculum concentration is 1-2%, and zymotic fluid inoculum concentration is 5-
15%；Preferably, the first order seed incubation time be 60h, the secondary seed incubation time be 48h, described two
Grade seed inoculum concentration is 1%, and the zymotic fluid inoculum concentration is 10%；The percentage is percent by volume.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109735467A (en) * | 2019-01-30 | 2019-05-10 | 福建省微生物研究所 | A kind of streptomycete mutagenic strain of high yield tacrolimus and its application |
| CN112111482A (en) * | 2020-08-31 | 2020-12-22 | 浙江工业大学 | Method and strain for screening high-yield tacrolimus Streptomyces tsukubaensis by high-throughput mutagenesis |
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