CN106434431A - Culture medium for producing pyruvic acid through fermentation and application thereof - Google Patents
Culture medium for producing pyruvic acid through fermentation and application thereof Download PDFInfo
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- CN106434431A CN106434431A CN201610807128.8A CN201610807128A CN106434431A CN 106434431 A CN106434431 A CN 106434431A CN 201610807128 A CN201610807128 A CN 201610807128A CN 106434431 A CN106434431 A CN 106434431A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
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Abstract
The invention relates to the technical field of fermentation engineering, in particular to a culture medium for producing pyruvic acid through fermentation and application thereof. The culture medium comprises a carbon source, a nitrogen source, a phosphorus source and inorganic salt. The carbon source comprises sodium gluconate which accounts for 80-100% of the mass of the carbon source. Oxidation-state sodium gluconate is adopted for replacing glucose, the proportion of intracellular NADH/NAD+ is reduced, intracellular reducing power is balanced, heteroacid yield is reduced, and the productivity and yield of pyruvic acid are improved.
Description
Technical field
The present invention relates to fermentation engineering field, more particularly, to a kind of culture medium for fermentation production of acetone acid and its
Application.
Background technology
Acetone acid (Pyruvic acid), light yellow to yellow transparent liquid, be organism analytic metabolism intermediate product it
One, it is widely used in food additive, pesticide, medicine and other fields.Can as precursor, Enzymatic Synthesis of L-Tryptophan, L-Tyrosine,
D/L- alanine and L-Dopa etc., have in mankind's health fields such as fat-reducing, stamina intensifying, reduction cholesterol simultaneously
Application potential.The research of fermentative Production acetone acid starts from twentieth century nineties, and the country such as Japan, U.S. is in bacterial strain choosing
Educate, engineered strain the aspect such as builds, isolates and purifies and having carried out a series of work, the correlational study of China is mainly by Southern Yangtze University
Carry out with units such as Institute of Microorganism, Academia Sinica, Chinese Academy Of Sciences Process Engineering Research Institute, also achieve good entering
Exhibition.
Research both domestic and external at present concentrates on bacterial screening and structure, and most study is torulopsis glabrata
(Torulopsis glabuata) and escherichia coli (Escherichia coli) genetic engineering bacterium.Either smooth ball intends ferment
, all there is oxidoreduction force unbalance in acetone acid cumulative process in mother or escherichia coli.It is outer that people attempt expression
The nadh oxidase in source builds up solving the problems, such as NADH.From the point of view of fermentation results, alleviate reduction to a certain extent
The problem of power accumulation, improves growth rate and the Biomass of bacterial strain, improves the concentration of acetone acid simultaneously.
" Hua Zhong Agriculture University's journal, 2010,29 (4), 527-532. " Zhou Jingwen etc. has investigated higher using oxidation state
Sodium gluconate part replaces the impact that glucose ferments to polyauxotroph torulopsis glabrata.With 100g/L glucose
Matched group as sole carbon source is compared, and adds the sodium gluconate of 10g/L in the dextrose culture-medium containing 90g/L, its
Cell concentration, glucose consumption rate and Pyruvate production intensity have been respectively increased 7.96%, 13.04% and 32.81%, intracellular
NAD+ concentration improves 29.73%, NADH/NAD+ ratio and have dropped 26.89%.But this research is pointed out, gluconic acid simultaneously
Sodium can not replace glucose completely, and otherwise cell quality concentration and output of pyruvic acid all can be decreased obviously, and for multi-nutrition
Deficiency torulopsis glabrata is not quite similar with colibacillary metabolic process, so culture medium also can not be directly applied for greatly
Enterobacteria.
Chinese Academy Of Sciences Process Engineering Research Institute passes through to regulate and control fatty acid metabolism approach, strengthens glycolytic pathway, reduces TCA
The means such as intensity of circulation, build escherichia coli YP01 and YP02 obtaining high yield acetone acid.Although during the fermentation, can obtain
To higher output of pyruvic acid, but still suffer from the problem of later stage glucose utilization speed degradation of fermenting.Study card
Bright change carbon source can improve the sweat of polyauxotroph torulopsis glabrata, for escherichia coli, more suitable
The carbon source closing pyruvate fermentation will be a kind of important research direction.
Content of the invention
For the demand of the deficiencies in the prior art and reality, the present invention provides a kind of culture for fermentation production of acetone acid
Base and its application, described culture medium can reduce heteroacid yield, improves yield and the yield of acetone acid.
For reaching this purpose, the present invention employs the following technical solutions：
On the one hand, the present invention provides a kind of culture medium for fermentation production of acetone acid, and described culture medium includes carbon source, nitrogen
Source, phosphorus source and inorganic salt, described carbon source includes sodium gluconate, and the mass ratio that described sodium gluconate accounts for carbon source is 80-
100%.
In the present invention, the sodium gluconate using oxidation state replaces glucose, reduces intracellular NADH/NAD+Ratio, balance
Intracellular reducing power, reduces heteroacid yield, improves yield and the yield of acetone acid.
Preferably, described carbon source is sodium gluconate.
Preferably, described nitrogen source is any one in yeast extract, peptone, Semen Maydis pulp, molasses, ammonia, ammonium salt or carbamide
Kind or at least two mixing.
Preferably, the pH of described culture medium is 6-8, can be for example 6,6.1,6.2,6.3,6.4,6.5,6.6,6.7,
6.8th, 6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9 or 8, preferably 6.5-7.5.
Second aspect, the present invention provides a kind of method with culture medium fermentation production of acetone acid as described in relation to the first aspect,
Comprise the steps：With culture medium culturing escherichia coli as described in relation to the first aspect.
Preferably, described escherichia coli are E.coli YP01 and/or E.coli YP02.
The culture presevation number of described E.coli YP01 is CGMCC No.10691, preservation time 2016.4.07, systematic name：
Colon bacillus Escherichia coli, preservation address：China Committee for Culture Collection of Microorganisms's common micro-organismss
Center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The culture presevation number of described E.coli YP02 is CGMCC No.12463, preservation time 2016.5.18, systematic name：
Colon bacillus Escherichia coli, preservation address：China Committee for Culture Collection of Microorganisms's common micro-organismss
Center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Preferably, described colibacillary inoculum concentration is 0.1-10%, can be for example 0.1%, 0.2%, 0.3%,
0.4%th, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 1.8%, 2%, 2.5%, 3%, 3.5%, 4%,
4.5%th, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.
Preferably, the colibacillary cultivation temperature of described culture medium culturing is 35-39 DEG C, can be for example 35 DEG C, 36 DEG C,
37 DEG C, 38 DEG C or 39 DEG C, preferably 37 DEG C.
Preferably, the colibacillary incubation time of described culture medium culturing is 15-60h, can be for example 15h, 16h,
18h, 20h, 25h, 28h, 30h, 35h, 40h, 45h, 50h, 55h or 60h.
Compared with prior art, the present invention has the advantages that：
(1) present invention is directed to the metabolic characteristic of the colibacillus engineering producing acetone acid, using the gluconic acid of oxidation state
Sodium replaces glucose, reduces intracellular NADH/NAD+Ratio, balances intracellular reducing power, reduces heteroacid yield, improves the product of acetone acid
Rate and yield；
(2), in the present invention, Recombinant organism bacterial strain YP01, under aerobic condition, using minimal medium
45g/L sodium gluconate can be converted into 34g/L acetone acid, conversion ratio reaches 0.76g/g gluconic acid by fermentation 24 hours
Sodium, compares with glucose as carbon source, concentrations of pyruvate is significantly improved, the heteroacid such as α-ketoglutaric acid, fumaric acid, citric acid
Significantly reduce.
Brief description
Fig. 1 is the sweat comparison diagram with glucose and sodium gluconate as carbon source for the present invention.
Specific embodiment
For further illustrating the technological means and its effect that the present invention taken, below in conjunction with accompanying drawing and by specifically real
Apply mode to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.
Unreceipted particular technique or condition person in embodiment, according to the technology described by document in the art or condition,
Or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can be commercially available by regular channel
The conventional products obtaining.
Experimental apparatus：
Agilent (1200 type) high performance liquid chromatography；
The Aminex HPX-87H organic acid analysis column analysis of Bole company；
The culture presevation number of described E.coli YP01 is CGMCC No.10691, preservation time 2016.4.07, systematic name：
Colon bacillus Escherichia coli, preservation address：China Committee for Culture Collection of Microorganisms's common micro-organismss
Center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The culture presevation number of described E.coli YP02 is CGMCC No.12463, preservation time 2016.5.18, systematic name：
Colon bacillus Escherichia coli, preservation address：China Committee for Culture Collection of Microorganisms's common micro-organismss
Center, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Embodiment 1 Recombinant organism YP01 produces acetone acid with sodium gluconate for carbon source through fermentation
Seed culture medium and fermentation medium (in addition to sodium gluconate) containing following component：
A great number of elements (mmol/L)：(NH4)2HPO419.92, NH4H2PO47.56, KCl 2.00, MgSO4·7H2O
1.50；
Trace element (μm ol/L)：FeCl3·6H2O 8.88, CoCl2·6H2O 1.26, CuCl2·2H2O 0.88,
ZnCl22.20, Na2MoO4·2H2O 1.24, H3BO31.21, MnCl2·4H2O 2.50；
Acetone acid is produced with Recombinant organism strain YP01, comprises the following steps：
(1) seed culture：Add seed culture medium 100mL in 250mL triangular flask, add sodium gluconate 0.5wt%,
Bacterial strain YP01 is inoculated in seed culture medium according to the inoculum concentration of 1% (v/v) after cooling, in pH=by 115 DEG C of sterilizing 30min
7.0th, culture obtains seed liquor in 12 hours under conditions of 37 DEG C and 200rpm.
(2) fermentation culture：5L ferment tank culture volume is 2L, 121 DEG C of sterilizing 20min, adds high temperature sterilize
Gluconic acid mother liquid of sodium, make in fermentation tank gluconic acid na concn be 4.5wt%.By seed in step 1 according to 5% (v/v)
Inoculum concentration be inoculated in fermentation medium, under conditions of pH=7.0,37 DEG C and 400rpm culture obtain fermentation liquid within 24 hours.
Analysis method：Using high performance liquid chromatography, quantitative analyses are carried out to the composition in fermentation liquid, the Fructus Vitis viniferae in fermentation liquid
Sodium saccharate and organic acid concentration measure using the analysis of Aminex HPX-87H organic acid analysis column.Result is as shown in figure 1, matched group
It is fermentation results under equal conditions with glucose for carbon source.
As seen from Figure 1：Recombinant organism bacterial strain YP01, under aerobic condition, using inorganic salt culture
Base ferments 24 hours, 45g/L sodium gluconate can be converted into 34g/L acetone acid, conversion ratio reaches 0.76g/g gluconic acid
Sodium, compares with glucose as carbon source, concentrations of pyruvate is significantly improved, meanwhile, α-ketoglutaric acid, fumaric acid, citric acid
Significantly reduce Deng heteroacid.
Embodiment 2 Recombinant organism YP01 produces acetone acid with sodium gluconate for main carbon source through fermentation
Seed culture medium and fermentation medium (in addition to glucose and sodium gluconate) containing following component：
A great number of elements (mmol/L)：(NH4) 2H,PO4 19.92, NH4H2PO4 7.56, KCl 2.00, MgSO4 7H2O
1.50；
Trace element (μm ol/l):FeCl3 6H2O 8.88, CoCl2 6H2O 1.26, CuCl2 2H2O 0.88,
ZnCl2 2.20, Na2MoO4 2H2O 1.24, H3BO3 1.21, MnCl2 4H2O 2.50；
Acetone acid is produced with Recombinant organism strain YP01, comprises the following steps：
(1) seed culture：Add seed culture medium 100mL in 250mL triangular flask, add sodium gluconate 0.5wt%,
115 DEG C of sterilizing 30min.After cooling, bacterial strain YP01 is inoculated in seed culture medium according to the inoculum concentration of 1% (v/v), in pH=
7.0th, culture obtains seed liquor in 12 hours under conditions of 37 DEG C and 200rpm.
(2) fermentation culture：5L ferment tank culture volume is 2L, 121 DEG C of sterilizing 20min, adds high temperature sterilize
Glucose and sodium gluconate mixing mother solution, wherein the mass ratio of glucose and sodium gluconate be 1:4, make in fermentation tank
The total concentration of glucose and sodium gluconate is 4.5wt%.Seed in step 1 is inoculated according to the inoculum concentration of 5% (v/v) and sends out
Ferment culture medium, culture under conditions of pH=7.0,37 DEG C and 400rpm obtains fermentation liquid in 24 hours.
Analysis method is with embodiment 1.Recombinant organism bacterial strain YP01 with glucose and sodium gluconate is being
During carbon source, fermented 24 hours using minimal medium, 32g/L acetone acid can be generated.
Embodiment 3 Recombinant organism YP02 produces acetone acid with sodium gluconate for carbon source through fermentation
Seed culture medium and fermentation medium (in addition to sodium gluconate) containing following component：Yeast extract 10g/L, peptone
10g/L, NaCl 5g/L.
Acetone acid is produced with Recombinant organism strain YP02, comprises the following steps：
(1) seed culture：Add seed culture medium 100mL in 250mL triangular flask, add sodium gluconate 0.5wt%,
115 DEG C of sterilizing 30min.After cooling, bacterial strain YP02 is inoculated in seed culture medium according to the inoculum concentration of 1% (v/v), in pH=
7.0th, culture obtains seed liquor in 12 hours under conditions of 37 DEG C and 200rpm.
(2) fermentation culture：5L ferment tank culture volume is 2L, 121 DEG C of sterilizing 20min, adds high temperature sterilize
Gluconic acid mother liquid of sodium, make in fermentation tank gluconic acid na concn be 4.5wt%.By seed in step 1 according to 5% (v/v)
Inoculum concentration be inoculated in fermentation medium, under conditions of pH=7.0,37 DEG C and 400rpm culture obtain fermentation liquid within 24 hours.
Analysis method is with embodiment 1.Recombinant organism bacterial strain YP01 with glucose and sodium gluconate is being
During carbon source, fermented 24 hours using rich medium, generate 38g/L acetone acid.
Applicant states, the present invention illustrates the method detailed of the present invention by above-described embodiment, but the present invention not office
It is limited to above-mentioned method detailed, that is, do not mean that the present invention has to rely on above-mentioned method detailed and could implement.Art
Technical staff is it will be clearly understood that any improvement in the present invention, the equivalence replacement to each raw material of product of the present invention and auxiliary element
Interpolation, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosure.
Claims (9)
1. a kind of culture medium for fermentation production of acetone acid, described culture medium includes carbon source, nitrogen source, phosphorus source and inorganic salt, its
It is characterised by, described carbon source includes sodium gluconate, the mass percent that described sodium gluconate accounts for carbon source is 80-100%.
2. culture medium according to claim 1 is it is characterised in that described carbon source is sodium gluconate.
3. culture medium according to claim 1 and 2 it is characterised in that described nitrogen source be yeast extract, peptone, Semen Maydis pulp,
In molasses, ammonia, ammonium salt or carbamide any one or at least two mixing.
4. the culture medium according to any one of claim 1-3 is it is characterised in that the pH of described culture medium is 6-8, preferably
For 6.5-7.5.
5. a kind of method of the culture medium fermentation production of acetone acid with as any one of claim 1-4 it is characterised in that
Comprise the steps：With the culture medium culturing escherichia coli as any one of claim 1-4.
6. method according to claim 5 is it is characterised in that described escherichia coli are E.coliYP01 and/or E.coli
YP02.
7. the method according to claim 5 or 6 is it is characterised in that described colibacillary inoculum concentration is 0.1-10%.
8. the method according to any one of claim 5-7 is it is characterised in that the colibacillary training of described culture medium culturing
Foster temperature is 35-39 DEG C, preferably 37 DEG C.
9. the method according to any one of claim 5-8 is it is characterised in that the colibacillary training of described culture medium culturing
The foster time is 15-60h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107867759A (en) * | 2017-11-07 | 2018-04-03 | 周晓斌 | A kind of Novel sewage handles outer Ensure Liquid cultivation agent and preparation method thereof |
| CN113801900A (en) * | 2021-09-27 | 2021-12-17 | 中国科学院过程工程研究所 | Method for preparing pyruvic acid by using forest trees and application thereof |
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| CN104946576A (en) * | 2015-04-27 | 2015-09-30 | 中国科学院过程工程研究所 | Escherichia coli gene engineering strain and construction method thereof, and application of strain in pyruvic acid production |
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2016
- 2016-09-07 CN CN201610807128.8A patent/CN106434431A/en active Pending
Patent Citations (2)
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| CN101319235A (en) * | 2008-07-01 | 2008-12-10 | 江南大学 | Method for improving production volume of pyruvic acid preparation of zymotechnics with additive gluconic acid sodium salt |
| CN104946576A (en) * | 2015-04-27 | 2015-09-30 | 中国科学院过程工程研究所 | Escherichia coli gene engineering strain and construction method thereof, and application of strain in pyruvic acid production |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107867759A (en) * | 2017-11-07 | 2018-04-03 | 周晓斌 | A kind of Novel sewage handles outer Ensure Liquid cultivation agent and preparation method thereof |
| CN113801900A (en) * | 2021-09-27 | 2021-12-17 | 中国科学院过程工程研究所 | Method for preparing pyruvic acid by using forest trees and application thereof |
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