CN108795791A - A kind of culture medium and its preparation method and application - Google Patents
A kind of culture medium and its preparation method and application Download PDFInfo
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- CN108795791A CN108795791A CN201710295907.9A CN201710295907A CN108795791A CN 108795791 A CN108795791 A CN 108795791A CN 201710295907 A CN201710295907 A CN 201710295907A CN 108795791 A CN108795791 A CN 108795791A
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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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- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01018—Lysine decarboxylase (4.1.1.18)
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Abstract
The present invention relates to a kind of culture mediums, further relate to the preparation method of the culture medium, further relate to the purposes of the culture medium.A kind of culture medium, including pH buffer；The pH buffer includes citric acid and citrate, and the molar ratio of the citric acid and citrate is 0.69:1-0.05:1, preferably 0.47:1-0.075:1；In the culture medium, the total concentration of the citrate of pH buffer is 0.01M-0.3M, preferably 0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium is 4.5-8, preferably 5-7.5, more preferably 5.5-6.6.The present invention solves in existing incubation the not control ph then low technical problem of conversion ratio, and used pH buffer environmental pollution is small, can reduce cost for wastewater treatment.
Description
Technical field
The present invention relates to a kind of culture mediums, further relate to the preparation method of the culture medium, further relate to the purposes of the culture medium.
Background technology
L-lysine decarboxylase, which is one kind, can be catalyzed L-lysine decarboxylic reaction and generate carbon dioxide and 1,5- penta 2
The biological enzyme of amine.The 1,5- pentanediamines of one of its product are a relatively common diamine compounds.Because 1,5- pentanediamines can be with
It polymerize with binary acid and generates novel polyamide and be concerned.Glucose or L-lysine catalysis are generated 1 by bioanalysis,
5- pentanediamines, and then prepare polyamide 5.6 etc., the nylon 6.6 dependent on petroleum resources is substituted, is a green chemistry leather
Life has preferable foreground.L-lysine decarboxylase is primarily present in Corynebacterium glutamicum (Corynebacterium
Glutamicum), Escherichia coli (E.coli), corpse bacillus (Bacterium cadaveris), hafnia alvei
(H.alvei) etc. bacteriums, exist in the higher plants such as cucumber (Sabo, D.L., E.A.Boeker, B.Byers, H.Waron
and E.H.Fischer(1974)."Purification and physical properties of inducible
Escherichia coli lysine decarboxylase."Biochemistry13(4):662-670；Fecker,L.F.,
H.Beier and J.Berlin(1986)."Cloning and Characterization of a Lysine
Decarboxylase Gene from Hafnia-Alvei."Molecular&General Genetics 203(1):177-
184).In order to efficiently express L-lysine decarboxylase, lysine decarboxylase gene is usually passed through into molecular biology skill
Art carry out overexpression, then by express L-lysine decarboxylase recombinant plasmid transformed to Escherichia coli or Escherichia coli in into
Row fermentation expression.
Shake flask fermentation culture is a kind of easy, practical cultural method, is developed into also therefore just microorganism training quickly
Epochmaking technology in supporting can be widely applied to Research for Industrial Microbial Germ screening, the experiment of laboratory large scale fermentation, seed training
It supports.
There are many Shake flask mediums for microculture and to produce L-lysine decarboxylase at present, such as glucose 2%, ferment
Female cream 2%, corn steep liquor 4%, MgSO40.03%, KH2PO40.01%, NaCl 0.5%, L-lysine 0.5%, vitamin
B60.1% (Zhu Jing, Du Lianxiang, Lu Fuping, Zhang Ju (2008) hafnia alveis production L-lysine decarboxylase culture optimization
.2008 the Tianjin year whole nation enzyme preparation research and development application technology conference Papers collection:135-138.), for another example：Sucrose 2%, jade
Rice & peanut milk 2%, yeast extract 0.5%, beef extract 0.3%, peptone 1%, NaC1 0.5%, ammonium sulfate 0.2%, vitamin B6
0.5%, L-lysine 0.1%, 2- (N- morpholinoes) ethanesulfonic acid 2.13%, pH=7.0 (Zhu Jing, Du Lianxiang, Lu Fuping, Fan Xin
It meets, Wu Wei (2009) " research of induction hafnia alvei production L-lysine decarboxylase condition " industrial microorganism (3):1-5),
For another example peptone 1%, beef extract 0.3%, sodium chloride 0.5% (Jiang Lili, Wu Xiaoyan, Liu Yi, Liu Qian, Jiao Qingcai
(2006) " lysine decarboxylase zymotechnique and zymologic property " fine chemistry industry 23 (11):1060-1063,1067.).This three
In a culture medium prescription, first relatively inexpensive, latter two cost is higher than first.It is general to select in order to control fermentation costs
Use first culture medium as the fermentation medium of microbial fermentation production L-lysine decarboxylase production.It is used in fermentation tank
It, can be by strictly controlling the parameter in fermentation process such as oxygen supply, pH, stir speed (S.S.), cultivation temperature when this culture medium ferments
Etc. enabling fermentation process to continue progress, and ensure to obtain higher cell density in final zymotic fluid, higher L- relies ammonia
Acid decarboxylase output, higher unit zymotic fluid are to the conversion ratio of L-lysine and higher carbon source, nitrogen source utilization rate；But
Since it is desired that controlling fermentation pH, the opposite complexity for improving zymotechnique by fed-batch mode；In addition, being trained in shaking flask
The problems such as oxygen supply, pH being unable to control during supporting, when these culture mediums are only capable of maintaining during shake flask fermentation shorter fermentation
Between, cause L-lysine decarboxylation production of enzyme not high, zymotic fluid is weak to the conversion capability of L-lysine, the glucose in zymotic fluid
Residual volume is larger, this is all loss to cost and output.
Therefore it needs to be improved culture medium so that it is optimized in fermentation.
Invention content
The first aspect of the present invention is designed to provide a kind of culture medium, makes to produce L-lysine decarboxylation using the culture medium
The zymotic fluid that enzyme is formed is to L-lysine high conversion rate, to solve in existing incubation the not control ph then low skill of conversion ratio
Art problem.
The present invention solves above-mentioned technical problem by the following technical programs, achieves the object of the present invention.
A kind of culture medium, including following component：
Carbon source；
Nitrogen source；
PH buffer；
The pH buffer includes citric acid and citrate, and the citric acid and citrate molar ratio are 0.69:
1-0.05:1, preferably 0.47:1-0.075:1；In the culture medium, the total concentration of the citrate of pH buffer is
0.01M-0.3M, preferably 0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium is 4.5-
8.0, preferably 5.0-7.5, more preferably 5.5-6.6.
Further, the content of the carbon source is 5g/L-30g/L；Preferably, the carbon source includes glucose, sucrose, molasses
It is one or more in starch, fiber hydrolysate.
On the basis of any of the above-described technical solution further, the content of the nitrogen source is：10.1g/L-49g/L；It is preferred that
Ground, the nitrogen source include corn steep liquor, yeast extract, hair hydrolysis liquid, urea, ammonium sulfate, ammonium chloride, soybean meal hydrolysate and yeast powder
In it is one or more.
On the basis of any of the above-described technical solution further, the citrate includes sodium citrate, potassium citrate, lemon
It is one or more in lemon acid iron and magnesium citrate.
On the basis of any of the above-described technical solution further, the culture medium further includes the inorganic salts containing trace element.
The content of inorganic salts containing trace element is：1.1g/L-5.5g/L；Preferably, the inorganic salts packet containing trace element
It includes one or more in soluble magnesium salt, sylvite and sodium salt；It is highly preferred that the inorganic salts containing trace element include solvable
Property magnesium salts and sodium salt；Again it is highly preferred that the content of the soluble magnesium salt is 0.1g/L-0.5g/L, the content of the sodium salt is
1g/L-5g/L。
On the basis of any of the above-described technical solution further, the present invention provides a kind of low-cost culture medium, institutes
A kind of culture medium stated, including following component：
0.47:1-0.075:1；In the culture medium, the total concentration of the citrate of pH buffer is 0.01M-0.3M, preferably
0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium be 4.5-8, preferably 5.0-7.5, more
Preferably 5.5-6.6.
The second aspect of the present invention purpose is to propose the preparation method of above-mentioned culture medium.
A kind of preparation method of culture medium, the culture medium include the following steps as described in any one technical solution above：
1) in deionized water by the carbon source, the nitrogen source, the inorganic salts dissolving containing trace element；
2) pH buffer is added；
3) it sterilizes.
The third aspect of the present invention purpose is to propose the purposes of above-mentioned culture medium.
A kind of purposes of culture medium, the culture medium is as described in any one technical solution above, the purposes of the culture medium
It is used as the culture medium of the bacterium of fermented and cultured expression L-lysine decarboxylase.
Further, the bacterium of the expression L-lysine decarboxylase is wild or recombinant expression L-lysine decarboxylase
Bacterial strain, including the Escherichia coli (E.coli) of wild type or recombination, bacillus subtilis (B.subtilis), streptomyces coelicolor
(S.coelicolor), hafnia alvei (H.alvei), corynebacterium glutamicum (C.glutamicum) or production acid Cray primary
Salmonella (Klebsiella oxytoca).
The culture medium of the present invention, of low cost, preparation method is simple；The culture medium of the present invention in use, is not necessarily to
PH value is adjusted, zymotechnique can be simplified；And the buffer salt of addition is organic acid buffer salt, environmental pollution is small, reduces wastewater treatment
Cost；The L-lysine decarboxylase production bacterial strain cultivated in the medium of the present invention, cell OD are obviously improved, produced L-
Lysine decarboxylase yield also significantly improves, and zymotic fluid also significantly improves the conversion ratio of the L-lysine of same concentrations.
Specific implementation mode
The present invention propose it is a kind of it is of low cost, be used as fermented and cultured (including shake flask fermentation, ferment tank) and express L-
The culture medium of the bacterium of lysine decarboxylase.The bacterium of the expression L-lysine decarboxylase is wild-type e. coli or recombination
Type Escherichia coli.Culture medium in present embodiment, including following component：Carbon source；Nitrogen source；Containing the inorganic of trace element
Salt；PH buffer；The pH buffer includes citric acid and sodium citrate, and the molar ratio of citric acid and sodium citrate is
0.69:1-0.05:1, preferably 0.47:1-0.075:1；In the culture medium, the total concentration of the citrate of pH buffer
For 0.01M-0.3M, preferably 0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium is
4.5-8.0, preferably 5.0-7.5, more preferably 5.5-6.6.
Preferably, the sal limonis includes one kind or more in sodium citrate, potassium citrate, ironic citrate and magnesium citrate
Kind.It is furthermore preferred that the sal limonis includes sodium citrate.
The content of the carbon source is 5g/L-30g/L；Further, in order to provide a kind of low-cost culture medium, the carbon
Source includes one or more in glucose, sucrose and molasses.
The content of the nitrogen source is：10.1g/L-49g/L.The nitrogen source include corn steep liquor, yeast extract, hair hydrolysis liquid,
It is one or more in urea, ammonium sulfate, ammonium chloride, soybean meal hydrolysate and yeast powder.
The content of inorganic salts containing trace element is：1.15g/L-5.7g/L.The inorganic salts containing trace element
Including one or more in soluble magnesium salt, sylvite and sodium salt；Further, it is described containing trace element inorganic salts include can
Soluble magnesium salt, sylvite and sodium salt；Further, the content of the magnesium salts of the solubility is 0.1g/L-0.5g/L, the sylvite
Content is 0.05g/L-0.2g/L, and the content of the sodium salt is 1g/L-5g/L.Soluble magnesium salt such as magnesium chloride, magnesium sulfate, nitric acid
Magnesium.Sylvite such as potassium chloride, potassium sulfate, potassium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate.Sodium salt such as sodium chloride, sodium sulphate, nitric acid
Sodium, sodium dihydrogen phosphate, disodium hydrogen phosphate.
Further, the present invention provides a kind of low-cost culture medium, a kind of culture medium, including with the following group
Part：
0.47:1-0.075:1；In the culture medium, the total concentration of the citrate of pH buffer is 0.01M-0.3M, preferably
0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium be 4.5-8.0, preferably 5.0-7.5,
More preferably 5.5-6.6.
The preparation method of above-mentioned culture medium, the culture medium include the following steps as described in any one technical solution above：
1) in deionized water by the carbon source, the nitrogen source, the inorganic salts dissolving containing trace element；
2) pH buffer is added；
3) it sterilizes.
The culture medium of the present invention, of low cost, preparation method is simple, and the buffer salt of addition is organic buffer salt, to environment
It pollutes small；The culture medium of the present invention in use, without adjusting pH value, can simplify zymotechnique；In the culture medium of the present invention
Middle culture L-lysine decarboxylase produces bacterial strain, and cell OD is obviously improved, and produced L-lysine decarboxylation production of enzyme is also notable
It improves, zymotic fluid also significantly improves the conversion ratio of the L-lysine of same concentrations.
Comparative example 1
(Zhu Jing, Du Lianxiang, Lu Fuping, Zhang Ju (2008) hafnia alveis produce the training of L-lysine decarboxylase to bibliography
It supports base and optimizesNational enzyme preparation researchs and develops application technology conference Papers collection within 2008Tianjin:135-138.) in match
The Shake flask medium of system, formula are as follows：Glucose 2% (i.e. 20g/L), yeast extract 2%, corn steep liquor 4%, MgSO40.03%,
KH2PO40.01%, NaCl 0.5%, L-lysine 0.5%, vitamin B60.1%, initial pH=6.5.Preparation method is such as
Under：500ml deionized waters are added in beaker, and by the corn steep liquor (name of an article：Corn steep liquor, appearance：Brown liquid, wherein dry matter
Content 41.2wt%, protein content 43.96wt%, ammonia nitrogen nitrogen content 1.41wt% in dry matter, following comparative example, embodiment
Together), the yeast extract (name of an article：Yeast extract, product type：Paste, product hierarchy：Level-one, following comparative example, embodiment are same),
Sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, vitamin B6It is added in beaker according to the content in formula with glucose, L-lysine,
It is settled to 900ml after being sufficiently stirred dissolving.Adjusting pH to 6.6 (mode of adjusting pH value, which uses, is added NaOH, following comparative example,
Embodiment is same).In packing to the triangle shake bottle of 250ml, every bottle of packing 50ml.121 DEG C of sterilizing 15min.
The Escherichia coli L-lysine decarboxylase superior strain used, which is conversion, constitutive expression Escherichia coli CadA bases
The plasmid recombinant bacterium of cause, preparation method is as follows, and will derive from Escherichia coli MG1655 (has purchased from Beijing day bounties biotechnology
Limit company) CadA genes be inserted on plasmid pUC18 after lac promoters, structure obtains pUCadA, then by recombinant plasmid pUCadA
It is transformed into e. coli bl21 (purchased from Beijing day bounties Bioisystech Co., Ltd), it is (following real to obtain recombination bacillus coli
It is same to apply example, comparative example).By recombination bacillus coli in the test tube seed culture medium of LB overnight incubation (16 hours, compare below
Example, embodiment are same) after above-mentioned Shake flask medium is forwarded to the switching amount of 2% (volume ratio, following comparative example, embodiment are same)
In, it is 37 DEG C in temperature, culture in the shaking table (amplitude 40mm, following shaking table are same) that rotating speed is 220rpm.Culture to 18h,
For 24 hours, sampling analysis when 42h and 48h.Analysis project mainly has glucose residual volume, cell OD600, conversion to L-lysine
Rate, the conversion ratio to L-lysine refer to that the culture solution of 700ul reacts 2h with the L-lysine that the mass concentration of 900ul is 40%
When conversion results.As a result as follows：
The specific detection method of important parameter is following (comparative example, embodiment hereafter is same) in this comparative example：
The measurement of L-lysine conversion ratio：Take 700ul shake flask fermentation liquid and 900ul a concentration of 40% (mass concentration,
Following comparative example, embodiment are same) L lysine HCL and 10ul 0.1M phosphopyridoxal pyridoxal phosphate mixing.It is 37 in temperature
DEG C, react 2h in the shaking table that rotating speed is 220rpm.Reaction solution supernatant is collected by centrifugation after reaction.Supernatant passes through nuclear magnetic resonance point
It analyses (Bruker 400MHZ) and measures remaining L-lysine and 1, the 5- pentanediamines of generation in reaction solution respectively, pass through calculating
The mole divided by L-lysine of 1,5- pentanediamines and the integral molar quantity of 1,5- pentanediamines turn L-lysine up to zymotic fluid
Rate.
OD600Measurement method：The culture medium for not cultivating cell of 3ml is added in the cuvette of 1cm wide, in UV-8000
School light (ABS) zero is absorbed in ultraviolet-uisible spectrophotometer (Shanghai Yuan Xi Instrument Ltd.) under 600nm.Cuvette is cleaned
The culture medium that 2.9ml does not cultivate bacterium is added after drying, is added after the zymotic fluid mixing of 0.1ml in UV-8000 UV, visible lights point
The absorption light value under 600nm is tested in light photometer.Instrument show numerical value be multiplied by 30 be zymotic fluid OD600.(other wavelength
Lower absorption value uses similar approach).
Glucose content measures：10 25mL tool plug scale test tube number 1-10 are taken, often 3, the 5- dinitros of 1.5ml are added in pipe
Base salicylic acid (DNS), is separately added into the glucose standard 0 of 1mg/mL in 1-10 test tubes, and 0.2,0.4,0.6,0.8,
1.0,1.2,1.4,1.6,1.8ml, with distilled water polishing to total volume 3.5ml；Each pipe is shaken up, is accurately heated in boiling water bath
5min takes out, and ice bath is cooled to room temperature, and 25mL is settled to distilled water, and mixing is overturned after jumping a queue, is measured on spectrophotometer
Absorption value under 540nm measures the absorption value of 2~No. 10 pipes with No. 1 pipe zeroising；Using absorption value as ordinate, glucose contains
It is abscissa to measure (mg), makes standard curve.It takes 1ml zymotic fluids to take 100ul supernatants after centrifugation, is added to 25mL tool plug scales
In test tube, the DNS of 1.5ml is added, with water polishing to 3.5ml；5min is accurately heated in boiling water bath, is taken out, and ice bath is cooled to
Room temperature is settled to 25mL with distilled water, and mixing is overturned after jumping a queue, measures the absorption value under 540nm；Participation is conversed by mark song
Glucose content.
Comparative example 2
The component of culture medium in this comparative example and the concentration of component are as follows：
Concentration of glucose in the medium is 16g/L；
A concentration of 38g/L of corn steep liquor in the medium；
Dusty yeast concentration in the medium is 2g/L；
Sodium sulfate concentration in the medium is 3g/L；
A concentration of 0.1g/L of potassium dihydrogen phosphate in the medium；
A concentration of 0.3g/L of magnesium chloride in the medium；
The initial pH of culture medium is 6.6.
Preparation method is as follows：500ml deionized waters are added in beaker, and by glucose, corn steep liquor, magnesium chloride, sulfuric acid
Sodium is added according to the content in formula in beaker, and 900ml is settled to after being sufficiently stirred dissolving.Adjust pH to 6.6.Packing is extremely
In the triangle shake bottle of 250ml, every bottle of packing 50ml.121 DEG C of sterilizing 15min.
Recombination bacillus coli is forwarded to after overnight incubation with 2% switching amount in the test tube seed culture medium of LB above-mentioned
In Shake flask medium, it is 37 DEG C in temperature, is cultivated in the shaking table that rotating speed is 220rpm.Culture to 18h, for 24 hours, 42h and 48h when
Sampling analysis.Analysis project mainly has glucose residual volume, cell OD600, to the conversion ratio of L-lysine, to L-lysine
Conversion ratio refers to conversion results when reacting 2h with a concentration of the 40% of 900ul L-lysine with the culture solution of 700ul.Knot
Fruit is as follows：
Comparative example 3
The component of culture medium in this comparative example and the concentration of component are as follows：
Concentration of glucose in the medium is 18g/L；
A concentration of 40g/L of corn steep liquor in the medium；
Dusty yeast concentration in the medium is 1g/L；
Sodium sulfate concentration in the medium is 3g/L；
A concentration of 0.1g/L of potassium dihydrogen phosphate in the medium；
A concentration of 0.3g/L of magnesium chloride in the medium；
The final mass of calcium carbonate is 1,2,3,4,5 and 6g/L in the medium；
The initial pH value of culture medium is 6.6.
Preparation method is as follows：500ml deionized waters are added in beaker, and mentioned component is added according to the content in formula
Enter in beaker, 900ml is settled to after being sufficiently stirred dissolving.Adjust pH to 6.6.In packing to the triangle shake bottle of 250ml, every bottle point
50ml.And the calcium carbonate of 1-6g is added.121 DEG C of sterilizing 15min.
Recombination bacillus coli is forwarded to after overnight incubation with 2% switching amount in the test tube seed culture medium of LB above-mentioned
In Shake flask medium, it is 37 DEG C in temperature, is cultivated in the shaking table that rotating speed is 220rpm.Culture to 18h, for 24 hours, 42h and 48h when
Sampling analysis.For analysis project mainly to the conversion ratio of L-lysine, the conversion ratio to L-lysine refers to the culture solution with 700ul
Conversion results when 2h are reacted with a concentration of the 40% of 900ul L-lysine.As a result as follows：
The pH value variation of fermentation process see the table below
Embodiment 1
The component of culture medium in the present embodiment and the concentration of component are as follows：
Concentration of glucose in the medium is 20g/L；
A concentration of 30g/L of corn steep liquor in the medium；
Dusty yeast concentration in the medium is 1g/L；
Sodium sulfate concentration in the medium is 3g/L；
A concentration of 0.3g/L of magnesium chloride in the medium；
In the medium in pH buffer citric acid and trisodium citrate the total concentration of citrate be 0.02,0.06,
0.08,0.1,0.2M, the citrate molal quantity wherein in citric acid account for citrate total mole number ratio in pH buffer and are
14%, it is 86% (following real that citrate molal quantity, which accounts for citrate total mole number ratio in pH buffer, in trisodium citrate
It is same to apply example)；
The initial pH value of culture medium is 6.6.
Preparation method is as follows：500ml deionized waters are added in beaker, and by glucose, corn steep liquor, yeast powder, sulfuric acid
Sodium and magnesium chloride are added according to the content in formula in beaker, and 800ml is settled to after being sufficiently stirred dissolving.Adjust pH to 6.6.Point
It is filled in the triangle shake bottle of 250ml, every bottle of packing 45ml.In order to make the citrate containing 0.02M in culture medium, add
The trisodium citrate of the citric acid of the 2M of 0.035ml and the 1.88M of 0.493ml；In order to make the citric acid containing 0.06M in culture medium
Salt adds the trisodium citrate of the citric acid of the 2M of 0.105ml and the 1.88M of 1.48ml；In order to make to contain 0.008M in culture medium
Citrate, add the trisodium citrate of the citric acid of the 2M of 0.14ml and the 1.88M of 1.974ml；In order to make in culture medium
Citrate containing 0.1M adds the trisodium citrate of the citric acid of the 2M of 0.175ml and the 1.88M of 2.47ml；In order to make training
The citrate containing 0.2M in base is supported, the trisodium citrate of the citric acid of the 2M of 0.35ml and the 1.88M of 4.93ml is added；121
DEG C sterilizing 15min.
Recombination bacillus coli is forwarded to after overnight incubation with 2% switching amount in the test tube seed culture medium of LB above-mentioned
In Shake flask medium, it is 37 DEG C in temperature, is cultivated in the shaking table that rotating speed is 220rpm.Culture to 18h, for 24 hours, 42h and 48h when
Sampling analysis.Analysis project is mainly the conversion ratio to L-lysine, and the conversion ratio to L-lysine refers to the culture with 700ul
Liquid reacts conversion results when 2h with a concentration of the 40% of 900ul L-lysine.As a result as follows：
Embodiment 2
The component of culture medium in the present embodiment and the concentration of component are as follows：
Concentration of glucose in the medium is 20g/L；
A concentration of 30g/L of corn steep liquor in the medium；
Dusty yeast concentration in the medium is 1g/L；
Sodium sulfate concentration in the medium is 3g/L；
A concentration of 0.3g/L of magnesium chloride in the medium；
The total concentration of citrate is 0.08M in citric acid and trisodium citrate in the medium；The initial pH of culture medium
It is 5.0,5.6,6.0,6.6,7.0.
Preparation method is as follows：
In beaker be added 500ml deionized waters, and by glucose, corn steep liquor, yeast powder, sodium sulphate and magnesium chloride according to
Content in formula is added in beaker, and 800ml is settled to after being sufficiently stirred dissolving.In packing to the triangle shake bottle of 250ml, every bottle
Dispense 45ml.In order to make Medium's PH Value be respectively 5.0,5.6,6.0,6.6, citrate total concentration is 0.08M, is trained in 45ml
Support the buffer salt that following volumes is separately added into base：
Be added 1.25ml 1.88M citric acid three sodium solution and 0.82ml 2M citric acid solution, and adjust pH to
5.0。
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 1.54ml and the 2M of 0.55ml is added, and adjust pH
To 5.6.
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 1.72ml and the 2M of 0.38ml is added, and adjust pH
To 6.0.
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 1.83ml and the 2M of 0.28ml is added, and adjust pH
To 6.2.
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 1.97ml and the 2M of 0.14ml is added, and adjust pH
To 6.6.
121 DEG C of sterilizing 15min.
Recombination bacillus coli is forwarded to after overnight incubation with 2% switching amount in the test tube seed culture medium of LB above-mentioned
In Shake flask medium, it is 37 DEG C in temperature, is cultivated in the shaking table that rotating speed is 220rpm.Culture to 18h, for 24 hours, 42h and 48h when
Sampling analysis.For analysis project mainly to the conversion ratio of L-lysine, the conversion ratio to L-lysine refers to the culture solution with 700ul
Conversion results when 2h are reacted with a concentration of the 40% of 900ul L-lysine.As a result as follows：
The variation of pH value see the table below in fermentation process：
Embodiment 3
The component of culture medium in the present embodiment and the concentration of component are as follows：
Concentration of glucose in the medium is 20g/L；
A concentration of 30g/L of corn steep liquor in the medium；
Dusty yeast concentration in the medium is 1g/L；
Sodium sulfate concentration in the medium is 3g/L；
A concentration of 0.3g/L of magnesium chloride in the medium；
The total concentration of citric acid and trisodium citrate is 0.0.02M in the medium；The initial pH of culture medium be 5.0,
5.6、6.0、6.2、6.6。
Preparation method is as follows：
In beaker be added 500ml deionized waters, and by glucose, corn steep liquor, yeast powder, sodium sulphate and magnesium chloride according to
Content in formula is added in beaker, and 800ml is settled to after being sufficiently stirred dissolving.In packing to the triangle shake bottle of 250ml, every bottle
Dispense 45ml.In order to make Medium's PH Value be respectively 5.0,5.6,6.0,6.6, citrate total concentration is 0.02M, is trained in 45ml
Support the buffer salt that following volumes is separately added into base：
Be added 0.31ml 1.88M citric acid three sodium solution and 0.21ml 2M citric acid solution, and adjust pH to
5.0。
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 0.38ml and the 2M of 0.14ml is added, and adjust pH
To 5.6.
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 0.43ml and the 2M of 0.10ml is added, and adjust pH
To 6.0.
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 0.46ml and the 2M of 0.07ml is added, and adjust pH
To 6.2.
Or the citric acid solution of the citric acid three sodium solution of the 1.88M of 0.49ml and the 2M of 0.01ml is added, and adjust pH
To 6.6.
121 DEG C of sterilizing 15min.
Escherichia coli are forwarded to above-mentioned shaking flask after overnight incubation in the test tube seed culture medium of LB with 2% switching amount
In culture medium, it is 37 DEG C in temperature, is cultivated in the shaking table that rotating speed is 220rpm.Culture to 18h, for 24 hours, 42h and 48h when sample
Analysis.Analysis project mainly to the conversion ratio of L-lysine, the conversion ratio to L-lysine refer to the culture solution of 700ul with
Conversion results when a concentration of 40% L-lysine reaction 2h of 900ul.As a result as follows：
The variation of pH value see the table below in fermentation process：
Comparative example 2, implements OD under 3 different conditions at embodiment 2600The result of value see the table below
From comparative example 3, the result of embodiment 2 as can be seen that in the case that initial pH value is 6.6, after fermenting 48 hours
The zymotic fluid pH value for adding buffer solution of the present invention is significantly higher than addition calcium carbonate, and zymotic fluid pair in embodiment 2
The conversion ratio of L-lysine is significantly larger than comparative example 3.From comparative example 2 and embodiment 2 and embodiment 3 as can be seen that in initial pH
Value be it is identical and fermentation 48 hours after, zymotic fluid is higher than the conversion ratio of L-lysine far away in embodiment 2 and embodiment 3
Comparative example 2.In example 2, in the culture medium that citric acid salt concentration is 0.08M, the initial pH of culture medium is gradually carried from 5.0
For height to 6.6, corresponding zymotic fluid also steps up the conversion ratio of L-lysine, wherein with gained under conditions of initial pH6.6
Changing effect highest of the zymotic fluid to L-lysine；In embodiment 3, in the culture medium that citric acid salt concentration is 0.02M, just
Beginning pH is equally stepped up by 5.0 to 6.6, but corresponding zymotic fluid does not step up the conversion ratio of L-lysine,
But stablize in a relatively high value.Thus it is considered that improving the initial pH of fermentation medium, and maintain fermentation process
PH value may not be able to improve conversion ratio of the zymotic fluid to L-lysine in a high level, and suitable buffer concentration
It is aided with the conversion ratio that suitable fermentation initial pH value can effectively improve zymotic fluid to L-lysine, illustrates to add citrate
That buffer solution plays is not only the pH for improving zymotic fluid, it is also possible to due to certain unknown booster actions, cause zymotic fluid
L-lysine conversion ratio is greatly improved.
Claims (8)
1. a kind of culture medium, including following component：
Carbon source；
Nitrogen source；
PH buffer；
It is characterized in that, the pH buffer includes citric acid and citrate, the molar ratio of the citric acid and citrate
Example is 0.69:1-0.05:1, preferably 0.47:1-0.075:1；In the culture medium, the citrate of pH buffer it is total
A concentration of 0.01M-0.3M, preferably 0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium
For 4.5-8, preferably 5-7.5, more preferably 5.5-6.6.
2. a kind of culture medium as described in claim 1, wherein the content of the carbon source is 5g/L-30g/L；Preferably, described
Carbon source includes one or more in glucose, sucrose, molasses, starch and fiber hydrolysate.
3. a kind of culture medium as claimed in claim 1 or 2, wherein the content of the nitrogen source is：10.1g/L-49g/L；It is preferred that
Ground, the nitrogen source include corn steep liquor, yeast extract, hair hydrolysis liquid, urea, ammonium sulfate, ammonium chloride, soybean meal hydrolysate and yeast powder
In it is one or more.
4. a kind of culture medium as described in any one of claims 1-3, wherein the citrate includes sodium citrate, lemon
It is one or more in sour potassium, ironic citrate and magnesium citrate.
5. a kind of culture medium according to any one of claims 1-4, wherein further include the inorganic salts containing trace element；Institute
Stating the content containing micro- inorganic salts is：1.1g/L-5.5g/L；Preferably, the inorganic salts containing trace element include
It is one or more in soluble magnesium salt, sylvite and sodium salt；It is highly preferred that the inorganic salts containing trace element include solubility
Magnesium salts and sodium salt；Again it is highly preferred that the content of the magnesium salts is 0.1g/L-0.5g/L, the content of the sodium salt is 1g/L-5g/
L。
6. a kind of culture medium as described in any one in claim 1-5, wherein the culture medium includes following component：
0.47:1-0.075:1；In the culture medium, the total concentration of the citrate of pH buffer is 0.01M-0.3M, preferably
0.05M-0.2M, more preferably 0.075M-0.125M；The initial pH value of the culture medium be 4.5-8, preferably 5-7.5, it is more excellent
It is selected as 5.5-6.6.
7. a kind of preparation method of culture medium, which is characterized in that the culture medium is described as described in claim any one of 1-6
Preparation method includes the following steps：
1) in deionized water by the carbon source, the nitrogen source, the inorganic salts dissolving containing trace element；
2) pH buffer is added；
3) it sterilizes.
8. a kind of purposes of culture medium, which is characterized in that the culture medium is as described in claim any one of 1-6, the culture
The purposes of base is used as the culture medium of the bacterium of fermented and cultured expression L-lysine decarboxylase；Preferably, the bacterium includes open country
Raw or recombination bacillus coli (E.coli), bacillus subtilis (B.subtilis), streptomyces coelicolor (S.coelicolor),
Hafnia alvei (H.alvei), corynebacterium glutamicum (C.glutamicum) or acid-producing Klebsiella bacterium (Klebsiella
oxytoca)。
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| KR102590534B1 (en) * | 2023-03-28 | 2023-10-16 | 국민대학교산학협력단 | Method for the production of cadaverine with Hafnia Alvei as biocatalizer |
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