CN101538599B - Method for improving yield of denitrified pseudomonas vitamin B12 - Google Patents
Method for improving yield of denitrified pseudomonas vitamin B12 Download PDFInfo
- Publication number
- CN101538599B CN101538599B CN 200810034948 CN200810034948A CN101538599B CN 101538599 B CN101538599 B CN 101538599B CN 200810034948 CN200810034948 CN 200810034948 CN 200810034948 A CN200810034948 A CN 200810034948A CN 101538599 B CN101538599 B CN 101538599B
- Authority
- CN
- China
- Prior art keywords
- trimethyl
- glycine
- concentration
- vitamins
- substratum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention discloses a method for producing vitamin B12 by using denitrified pseudomonas. The method comprises the following steps that: the initial concentration of lycine in a denitrified pseudomonas culture medium is regulated to be between 10 and 20g/L; under suitable conditions, the denitrified pseudomonas is cultured to produce the vitamin B12; and when the concentration of the lycine in the culture medium is reduced to certain degree, the lycine is replenished and the concentration of the lycine is kept between 3 and 7g/L. The method can effectively improve the yield of the vitamin B12, is low in cost and suitable for large-scale production and thus has good industrial value.
Description
Technical field
The invention belongs to bioengineering field, more specifically, the present invention relates to a kind of utilization and utilize denitrified pseudomonas high yield vitamins B
12Novel method.
Background technology
Vitamins B
12(VB
12) being commonly used to represent the cobalami compounds, it is a kind of important biologically active substance, can be used for treating surra, also is the somatomedin of many microorganisms and animal simultaneously.Occurring in nature, vitamins B
12Biosynthesizing have two kinds of different approach, i.e. aerobic approach and anaerobism approach, but VB
12Biosynthesizing strictly be confined in some microorganisms, at present be used for commercially producing VB
12Microorganism Xie Shi propionibacterium (Propionibacterium shermanii), Fei Shi propionibacterium (P.freudenreichii) and denitrified pseudomonas (Pseudomonas denitrificans) etc. are mainly arranged.The aerobic synthesise vitamins B of denitrified pseudomonas
12Approach illustrate in detail [Warren M J etc., The biosynthesis ofadenosylcobalamin (vitamin B
12) [J] .Nat.Prod.Rep, 2002,19:390-412].Just because of to vitamins B
12The understanding of route of synthesis, thus can transform bacterial strain by means such as mutagenesis and molecular biology, so that the vitamins B of denitrified pseudomonas bacterial strain
12Throughput improves greatly.Because vitamins B
12Productive rate is high, in industrial production nearly all is at present to use denitrified pseudomonas to be used as aerobic production vitamins B
12Industrial strain.
The aerobic synthesise vitamins B of denitrified pseudomonas
12Process need altogether methylate [BattersbyA R, Leeper F J.Biosynthesis of Vitamin B of 8 steps
12[J] .Topics in Current Chemistry, 1998,195:143-193].Each molecule of trimethyl-glycine (Trimethyl glycine) contains three methyl, and it can be used as the biosynthesizing vitamins B
12Methyl donor.Have been reported and point out in denitrified pseudomonas, trimethyl-glycine-homocysteine methylferase is to vitamins B
12A large amount of synthetic effect arranged.Trimethyl-glycine-homocysteine methylferase catalysis trimethyl-glycine shifts a methyl to homocysteine, forms respectively N-methylsarcosine and methionine(Met), so trimethyl-glycine is participating in vitamins B
12Methylation reaction in the biosynthetic process has the effect of outbalance.There are some researches show that also trimethyl-glycine is to VB in the denitrified pseudomonas
12Excessive generation synthetic and porphyrin play a part outbalance.In addition, trimethyl-glycine is a kind of good osmotic pressure regulator, has report trimethyl-glycine and cholinergic to promote cell surface to the secretion of meta-bolites, thereby to the Production by Bacteria vitamins B
12Has effect of stimulation.
Although this area has been found that trimethyl-glycine is at vitamins B
12Has very important effect in the biosynthetic process.Yet, in the real attenuation process, do not have at present systematic research for the caused denitrified pseudomonas metabotic change of trimethyl-glycine situation, systematically do not study the control strategy of the better and less expensive of trimethyl-glycine in denitrified pseudomonas large scale fermentation production process yet.
Summary of the invention
The object of the present invention is to provide a kind of denitrified pseudomonas high yield vitamins B that utilizes
12Method, the method mainly is to realize by regulating the concentration of trimethyl-glycine in fermention medium.
In a first aspect of the present invention, provide a kind of denitrified pseudomonas that utilizes to produce vitamins B
12Method, described method comprises:
(1) regulates that trimethyl-glycine concentration is 10-20g/L in the initial denitrified pseudomonas substratum, cultivate denitrified pseudomonas, thereby produce vitamins B
12With
(2) when the concentration of trimethyl-glycine in the substratum is reduced to 3-7g/L, replenish the adding trimethyl-glycine, and trimethyl-glycine concentration is that 3-7g/L is to fermentation ends in the maintain base.
In another preference, in step (1), regulate that trimethyl-glycine concentration is 12-18g/L in the initial denitrified pseudomonas substratum; That better is 13-17g/L; That best is 14-16g/L, such as 15g/L.
In another preference, in step (2), when the concentration of trimethyl-glycine in the substratum is reduced to 5-7g/L, replenish the adding trimethyl-glycine.
In another preference, in step (2), trimethyl-glycine concentration is 5-7g/L in the maintain base.
In another preference, in step (2), when replenishing trimethyl-glycine, replenish CoCl
2, making its concentration in substratum is 0.05 ± 0.02g/L.
In another preference, in step (2), replenish trimethyl-glycine by the mode that stream adds.
In another preference, described initial denitrified pseudomonas substratum contains:
Sucrose 80 ± 20g/L, corn steep liquor 30 ± 10g/L, trimethyl-glycine 15 ± 5g/L, (NH
4)
2SO
43 ± 1g/L, MgSO
47H
2O 2 ± 1g/L, CoCl
26H
2O 0.15 ± 0.05g/L, 5,6-dimethylbenzimidazole, 0.08 ± 0.03g/L, ZnSO
47H
2O 0.1 ± 0.05g/L, CaCO
31.0 ± 0.5g/L.
In another preference, the temperature of cultivation is 32 ± 2 ℃.Better, the temperature of cultivation is 32 ± 1 ℃; Better, the temperature of cultivation is 32 ± 0.5 ℃.
In another preference, described method also comprises step:
(3) from substratum, isolate vitamins B
12
In another preference, the time of cultivation is 120-200 hour.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
Fig. 1 has shown pH and the thalli growth variation tendency of denitrified pseudomonas fermenting process under the different concns trimethyl-glycine.Wherein ,-■-0g/L trimethyl-glycine;-●-15g/L trimethyl-glycine; The 30g/L of-▲-trimethyl-glycine.
(a) variation of pH in culture medium under the different concns trimethyl-glycine;
(b) variation of cell concentration in the substratum under the different concns trimethyl-glycine.
Fig. 2 has shown that the different concns trimethyl-glycine is to denitrified pseudomonas fermenting process ALA and vitamins B
12Synthetic impact.Wherein ,-■-0g/L trimethyl-glycine;-●-15g/L trimethyl-glycine; The 30g/L of-▲-trimethyl-glycine.
(a) variation of ALA in the different concns trimethyl-glycine bottom fermentation liquid;
(b) vitamins B under the different concns trimethyl-glycine
12Synthetic situation.
Fig. 3 has shown 120-m
3The dynamic changing process of the trimethyl-glycine concentration in the fermentor tank under the different trimethyl-glycine control strategies.Wherein ,-▲-trimethyl-glycine is controlled at 3-5g/L;-●-trimethyl-glycine is controlled at 5-7g/L;-■-trimethyl-glycine is controlled at 8-10g/L.
Embodiment
The inventor is to utilizing denitrified pseudomonas (Pseudomonas denitrificans) fermentation to produce vitamins B
12(Vatamin B
12, VB
12) the each side factor conduct in-depth research and test, found that the trimethyl-glycine (Trimethyl glycine, the C that comprise proper concn in the substratum
5H
11NO
2) for the fermentative production vitamins B
12Highly beneficial.The inventor has also optimized better trimethyl-glycine concentration in interpolation opportunity of trimethyl-glycine and the fermented liquid, thereby has effectively improved vitamins B
12Output, and described method is particularly useful on a large scale (50-1000m for example
3) fermentative production in, with low cost, thereby have fabulous industry using value.
Therefore, the invention provides a kind of denitrified pseudomonas that utilizes and produce vitamins B
12Method, described method comprises: (1) is regulated that trimethyl-glycine concentration is 10-20g/L in the initial denitrified pseudomonas substratum, is cultivated denitrified pseudomonas under suitable condition, thereby produce vitamins B
12(2) when the concentration of trimethyl-glycine in the substratum is reduced to 3-7g/L, replenish the adding trimethyl-glycine, and trimethyl-glycine concentration is that 3-7g/L is to fermentation ends in the maintain base.
Among the present invention, except trimethyl-glycine itself, the salt of trimethyl-glycine or hydrate also can be used to as effective added ingredients, if they in being added into substratum after, also have the methyl donor effect the same with trimethyl-glycine, and can be utilized by denitrified pseudomonas.Described salt is the hydrochloride of trimethyl-glycine for example.The hydrate of described trimethyl-glycine for example can be sloughed water molecules under optimum conditions.
The inventor is surprised to find that, when the trimethyl-glycine concentration of adding in the fermention medium is crossed low or do not added, is unfavorable for vitamins B
12Synthetic, and during excessive concentration, then can produce to the growth of thalline obvious restraining effect.And, be different (cell concentration is low when initial) at different fermentation stage cell concentrations, the amount that is fit to of trimethyl-glycine also may be different, and therefore grasping trimethyl-glycine addition manner suitable in the different steps and addition is the important factor that obtains high yield.Through repeatedly groping, the inventor finds that trimethyl-glycine concentration is that 10-20g/L is suitable in the denitrified pseudomonas substratum, and this concentration hypothallus is with suitable speed growth, and the vitamins B that produces
12Tire highlyer, also be that thalline is in better growth, metabolism and production status.And when trimethyl-glycine concentration is higher than 20g/L, vitamins B then
12The obvious reduction of having tired.Better, regulate that trimethyl-glycine concentration is 12-18g/L in the initial denitrified pseudomonas substratum, that better is 13-17g/L; That best is 14-16g/L, such as 15g/L.
Fermenting to the regular hour (according to appointment 40-60 hour after), cell concentration has had remarkable rising, vitamins B
12Synthetic significantly accelerate, at this time the consumption of trimethyl-glycine significantly rises, and therefore needs to replenish to add trimethyl-glycine to satisfy thalli growth and to produce required.Yet should the stage biomass etc. condition different from the fermentation initial stage, so the suitable concentration of trimethyl-glycine in nutrient solution also is different.Through repeatedly groping, the inventor finds when the concentration of trimethyl-glycine in the substratum is reduced to 3-7g/L, replenish adding trimethyl-glycine is that suitable trimethyl-glycine replenishes opportunity, and afterwards in the maintain base trimethyl-glycine concentration be 3-7g/L, this additional way can obtain higher vitamins B
12Tire.And when trimethyl-glycine in substratum during excessive concentration, not only can not improve vitamins B
12Tire the remarkable reduction of having tired on the contrary.Better, when the concentration of trimethyl-glycine in the substratum is reduced to 5-7g/L, replenish the adding trimethyl-glycine, and trimethyl-glycine concentration is 5-7g/L in the maintain base.
As a kind of mode of the present invention, when replenishing trimethyl-glycine, replenish CoCl
2, making its concentration in substratum is 0.05 ± 0.02g/L.In fact, CoCl
2The interpolation strategy have no particular limits in the present invention, can be according to this area conventional addition manner that adopts in relevant fermentation strain is produced.
As optimal way of the present invention, the mode that stream adds after the first disposable adding of employing is added trimethyl-glycine in substratum, can stably provide trimethyl-glycine to denitrified pseudomonas in the mode of taking stream to add to the certain phase of fermenting, so that thalline is produced vitamins B under suitable working condition
12, keep desirable production or metabolism state.Certainly, those skilled in the art are understandable to be, it is sensu lato that described stream adds, and it had both comprised continual adding mode; Also comprise enough short batch add mode of intermittent time, as long as this mode also can keep trimethyl-glycine concentration basicly stable in the substratum (namely this concentration does not exceed 3-7g/L, the scope of 5-7g/L more preferably), for example the intermittent time is 1-5 minute.
After fermentation culture finishes, also can comprise step: from fermention medium, separate vitamins B
12Isolated or purified vitamins B from cultured products
12Can adopt technology well known to those skilled in the art.For example can adopt ammonium sulfate precipitation, DEAE-Sepharose ion-exchange, gel filtration method purifying; Or employing affinity chromatography purifying.
The raw material (such as carbon source, phosphorus source, organic nitrogen source, inorganic salt, trace element) and the usage quantity thereof that are used for the preparation fermention medium all can utilize denitrified pseudomonas to produce vitamins B according to this area routine
12The raw material of Shi Suoyong and usage quantity and decide.As optimal way of the present invention, described initial denitrified pseudomonas substratum contains: sucrose 80 ± 20g/L, corn steep liquor 30 ± 10g/L, trimethyl-glycine 15 ± 5g/L, (NH
4)
2SO
43 ± 1g/L, MgSO
47H
2O 2 ± 1g/L, CoCl
26H
2O 0.15 ± 0.05g/L, 5,6-dimethylbenzimidazole, 0.08 ± 0.03g/L, ZnSO
47H
2O 0.1 ± 0.05g/L, CaCO
31.0 ± 0.5g/L.During the fermentation, when determining trimethyl-glycine concentration and be lower than 3-7g/L, beginning is added trimethyl-glycine in substratum, and control trimethyl-glycine concentration is at the 3-7g/L substratum.As another optimal way, described initial denitrified pseudomonas substratum contains: glucose 80 ± 20g/L, yeast extract paste 20 ± 10g/L, trimethyl-glycine 15 ± 5g/L, (NH
4)
2 HPO
41 ± 0.3g/L, (NH
4)
2 SO
42 ± 0.5g/L, MgSO
47H
2O 2 ± 0.5g/L, CoCl
26H
2O 0.15 ± 0.05g/L, 5,6-dimethylbenzimidazole, 0.08 ± 0.02g/L, ZnSO
47H
2O0.1 ± 0.03g/L, CaCO
31.0 ± 0.3g/L.
Cultivate other condition of denitrified pseudomonas, such as temperature, humidity, air flow etc., can adopt condition known in the art.For conditions such as air flow, stirring velocitys, those skilled in the art also can be rule of thumb and the scale of fermentation carry out suitable accommodation.
As optimal way of the present invention, the temperature of cultivation is 32 ± 2 ℃.Better, the temperature of cultivation is 32 ± 1 ℃; Better, the temperature of cultivation is 32 ± 0.5 ℃
In addition, those skilled in the art are understandable to be, method of the present invention is applicable to the denitrified pseudomonas bacterial strain of wild-type, also is applicable to some by the improved denitrified pseudomonas bacterial strain of the means such as mutagenesis or molecular biology, as long as they can produce vitamins B with essentially identical mechanism
12Described essentially identical mechanism for example is: all can promote by trimethyl-glycine the formation of δ-amino-laevulic acid (ALA) synthase, and then promote the synthetic of ALA, promote afterwards synthesise vitamins B
12Synthetic.Most preferred, described denitrified pseudomonas is denitrified pseudomonas J741.
In an embodiment of the present invention, at first in shaking flask, investigated trimethyl-glycine to vitamins B
12Synthetic impact, the result shows that trimethyl-glycine can significantly promote vitamins B
12Synthetic.But when the trimethyl-glycine excessive concentration of adding in the fermention medium, can produce to the growth of thalline obvious restraining effect, more be unfavorable on the contrary vitamins B
12Synthetic, so the trimethyl-glycine concentration in the initial fermention medium of empirical tests can be selected 15g/L best.Then, the inventor has further studied the trimethyl-glycine of 0g/L, 15g/L and 30g/L to the impact of denitrified pseudomonas fermenting process by shaking flask, the result shows that certain density trimethyl-glycine can significantly improve the resultant quantity of δ-amino-laevulic acid in the fermented liquid, thereby promotes vitamins B
12Synthetic.Then, according to the result of shake flat experiment, at 120-m
3Successfully set up reasonable and economic trimethyl-glycine stream in the fermentor tank and added strategy.Add trimethyl-glycine by stream, not only can reduce trimethyl-glycine to the inhibition of thalli growth, but also can avoid affecting vitamins B because of the deficiency of trimethyl-glycine
12Synthetic.120-m
3The concrete stream of trimethyl-glycine adds strategy and is in the fermentor tank: by fermenting when being lower than 7g/L to trimethyl-glycine concentration, the beginning Continuous Flow is controlled at 5-7g/L with the beet alkaline solution with the trimethyl-glycine concentration in the fermented liquid.Add under the strategy vitamins B of (the 162nd hour) during fermentation ends at this stream
12Tire and reached 177.49 ± 2.98 μ g/ml.
Major advantage of the present invention is:
(1) found adjusting denitrified pseudomonas fermentative production vitamins B
12The influence factor of key, and optimized better trimethyl-glycine concentration in interpolation opportunity of trimethyl-glycine and the fermented liquid, thereby effectively improved vitamins B
12Output.
(2) method of the present invention also is applicable in the large-scale fermentative production, and is with low cost, simple to operate, thereby has fabulous industry using value.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
I. materials and methods
Bacterial classification and substratum
Bacterial classification: denitrified pseudomonas (Pseudomonas denitrificans) J741[is referring to White RF, Kaplan L, Birnbaum J.Betaine-Homocysteine Transmethylase in Pseudomonasdenitrificans, a Vitamin B12 Overproducer.Journal of Bacteriology, 1973,113:218-223].
Seed culture medium (g/L): sucrose 36, corn steep liquor 10, (NH
4)
2SO
42.0, (NH
4)
2HPO
40.5, MnSO
4H
2O 0.2, MgSO
47H
2O 1.0, ZnSO
47H
2O 0.1, and pH 7.2～7.4.
Fermention medium (g/L): sucrose 80, corn steep liquor 30, trimethyl-glycine 15, (NH
4)
2SO
43.0, MgSO
47H
2O 2.0, CoCl
26H
2O 0.15,5,6-dimethylbenzimidazole (5,6-dimethylbenzimidazole, DMBI) 0.08, ZnSO
47H
2O 0.1, CaCO
31.0, pH7.0～7.2.
Supplemented medium (g/L): trimethyl-glycine 39, CoCl
26H
2O 0.3, and DMBI 0.3.
Instrument and reagent
Instrument: volume is 120m
3Fermentor tank, the agitator main flow is axially to mix; 8453 type ultraviolet-visible spectrophotometers (Agilent company); HP 1100 type chromatographic instruments (Agilent company).
Reagent: Vitral reference substance (content＞99%, Sigma company.Trimethyl-glycine (Xinshi District, Baoding promoting agent factory), DMBI (large chemical industry company limited of Hebei section), CoCl
26H
2O (Huifeng, Lin County, Shandong cobalt factory), δ-amino-laevulic acid (Sigma company), other reagent is domestic analytical pure.
The cultural method of shake flask fermentation
Seed culture
Scrape with sterilized water and to wash the inclined-plane thalline, make bacteria suspension.Get the access of 1mL bacteria suspension and be equipped with in the 300mL triangular flask of 60mL seed culture medium, 30 ℃, the rotary shaking table shaking culture of 260r/min is to thalline light absorption value (OD
700) be about 10.
Shake-flask culture
The substratum loading amount is 36mL in the 300mL triangular flask, 10%, 32 ℃ of inoculum size, and (260r/min) is cultured to 96 hours in rotary shaking table.
120-m
3Cultural method in the fermentor tank
Adopt three grade fermemtation: the thallus suspension liquid on 4 inclined-planes is inoculated in the first order seed substratum that loading amount is the 60L/100-L first class seed pot, at 32 ± 0.5 ℃ of tank temperature, tank pressure 0.05～0.06MPa, air flow quantity 2～2.5m
3/ hour, under the mixing speed 200rpm, be cultured to thalline light absorption value (OD7
00) be about 15～20, first order seed being accessed loading amount is 7m again
3/ 9-m
3In the secondary seed medium of secondary seed tank, at 32 ± 0.5 ℃ of tank temperature, tank pressure 0.05～0.06MPa, air flow quantity 80～100m
3/ hour, under the mixing speed 200rpm, be cultured to thalline light absorption value (OD
700) be about 15～20; At last the seed liquor in the secondary seed tank being moved into loading amount is 70m
3/ 120-m
3In the fermention medium of fermentor tank, at 32 ± 0.5 ℃ of tank temperature, tank pressure 0.05～0.06MPa, air flow quantity 600～800m
3/ hour, cultivated about 160 hours under the mixing speed 60rpm.
Cell concentration is measured
Adopt the dry cell weight method (Dry cell weight, DCW) of measuring.Inhale the 10mL fermented liquid, after centrifugal and washing, thalline is dried to constant weight at 105 ℃, claims dry cell weight.
Vitamins B
12Measure
Adopt high performance liquid chromatography (HPLC) method.VB in the fermented liquid
12Not only be present in the born of the same parents but also be present in outside the born of the same parents, therefore need to be with bacterial cell disruption during the VB12 in measuring fermented liquid, concrete operation steps is as follows:
(1) sample preparation
Get the 10mL fermented liquid, add each 2.5mL of 8% sodium nitrite solution and Glacial acetic acid, shake up, in 95-100 ℃ of water-bath 30min; Be cooled to room temperature after the water-bath, add deionized water and be settled to 50mL, filter; Gained filtrate is filtered 1mL to sample bottle with 0.22 μ m millipore filtration syringe filters, draws sodium cyanide solution 20 μ L with microsyringe and puts into sample bottle, and sample bottle is put into 35-40 ℃ of water-bath reaction 1 hour, makes vitamins B multi-form in the fermented liquid
12Be converted into cyanocobalamin, under specified criteria, carry out liquid-phase chromatographic analysis;
(2) high-efficient liquid phase chromatogram condition
Moving phase be 250mmol/L phosphate aqueous solution-acetonitrile (30: 70, v/v); Chromatographic column is Dalian Yi Lite Hypersil NH
2Post (4.6mm * 250mm, 5 μ m); The detection wavelength is 361nm; Sample size is 20 μ L; Flow velocity is 1.7mL/min.
Trimethyl-glycine is measured
The employing colorimetry [referring to Cui Yanhong, Huang Xianqing; Beet alkali content is measured and industrial production process research [J]. livestock industry, 2002,6:38-40] and, utilize trimethyl-glycine and Reinecke's salt to react.
The mensuration of δ-amino-laevulic acid (δ-aminolevulinic acid, ALA) content
According to document Burnham B F. δ-aminolevulinic acid sythase[J] .Methods in Enzymology, the method for 1970,17A:195-200 is measured.
II. embodiment
Add trimethyl-glycine in fermention medium, concentration is respectively 0,5,10,15,20,30g/L.Shake flask fermentation was put bottle after 96 hours.In initial fermention medium, add the trimethyl-glycine of different concns to thalli growth and vitamins B
12Synthetic impact sees Table 1.
Table 1
As can be seen from Table 1, in the fermenting process of denitrified pseudomonas, it is very necessary adding a certain amount of trimethyl-glycine, and it is to vitamins B
12syntheticly have an obvious promoter action.Fermention medium does not add trimethyl-glycine, although obtain maximum thalline biomass, does not have vitamins B
12Synthetic.The effect of adding the 15g/L trimethyl-glycine is best, vitamins B
12Tire and reach 52.18 ± 0.81 μ g/ml.Along with the increase of trimethyl-glycine concentration, biomass descends gradually, and this explanation trimethyl-glycine has certain restraining effect to the growth of thalline.
Add 0g/L in order to study trimethyl-glycine to the impact of denitrified pseudomonas fermenting process, to be chosen in the initial fermention medium, the trimethyl-glycine of 15g/L and 30g/L is made further experiment in shaking flask, with the shake flat experiment that do not add trimethyl-glycine in contrast.
1. trimethyl-glycine is on the impact of pH and thalli growth
Fig. 1 is under above-mentioned three kinds of different trimethyl-glycine concentration, the pH of fermenting process and thalli growth variation tendency.By Fig. 1 (a) as seen, do not add trimethyl-glycine in the substratum, pH obviously descended after the 24th hour, and added the trimethyl-glycine of 15g/L and 30g/L, and pH is comparatively stable.The biomass variation that Fig. 1 (b) shows shows that do not add trimethyl-glycine in the substratum, thalli growth is the fastest.In addition, the biomass of the biomass under the 15g/L trimethyl-glycine under the 30g/L trimethyl-glycine.
Above presentation of results does not add trimethyl-glycine in the substratum, may be because the thalli growth metabolism be vigorous, and the sugar consumption is accelerated, thereby causes pH to descend rapidly.And trimethyl-glycine has certain restraining effect to the growth of thalline, so pH can obviously not descend because thalli growth is too fast.
2. trimethyl-glycine is to δ-amino-laevulic acid (ALA) and vitamins B
12Synthetic impact
Fig. 2 is that above-mentioned three kinds of concentration trimethyl-glycines are to fermenting process ALA and vitamins B
12The synthetic result that affects.
By Fig. 2 (a) as seen, do not add trimethyl-glycine in the substratum, the ALA content in the fermented liquid is starkly lower than the ALA content under 15g/L and the 30g/L trimethyl-glycine.Before 72 hours, the ALA content under the 15g/L trimethyl-glycine is also a little more than the ALA content under the 30g/L trimethyl-glycine.
By Fig. 2 (b) as seen, do not add trimethyl-glycine in the substratum, the vitamins B of whole fermenting process
12Resultant quantity is 0.Because the thalli growth under the 15g/L trimethyl-glycine is very fast, its vitamins B
12Tire vitamins B under the 30g/L trimethyl-glycine
12Tire.
At the aerobic synthesise vitamins B of denitrified pseudomonas
12Approach in, ALA is the key precursor thing that generates the corrin ring, ALA synthase (ALA synthase) plays very important regulating effect to ALA synthetic, it also is whole vitamins B
12One of key enzyme of route of synthesis.ALA and vitamins B by Fig. 2
12Variation can be inferred, because trimethyl-glycine can promote the raising of ALA synthase activity, thereby has promoted ALA and vitamins B
12Synthetic.But trimethyl-glycine excessive concentration in the fermented liquid can produce to the growth of thalline certain restraining effect, can reduce the ALA resultant quantity on the contrary, thereby reduces vitamins B
12Synthetic.
Each parameter changing conditions of fermenting process under comprehensive these three kinds of trimethyl-glycine concentration, can obviously reach a conclusion: trimethyl-glycine is to vitamins B
12syntheticly have an important promoter action, but should according to the Expenditure Levels of trimethyl-glycine in the fermenting process and trimethyl-glycine on the impact of thalli growth, select suitable trimethyl-glycine to add concentration.Along with trimethyl-glycine concentration in the substratum increases, can affect because trimethyl-glycine is excessive vitamins B on the contrary
12Synthetic, and this control to fermentation costs also is very disadvantageous.
The trimethyl-glycine regulating strategy of denitrified pseudomonas fermentation under embodiment 3 industrially scalables
By above shaking flask result as can be known, in view of denitrified pseudomonas synthesise vitamins B
12The demand of trimethyl-glycine and trimethyl-glycine to the restraining effect of thalli growth, therefore, are produced vitamins B in the denitrified pseudomonas fermentation
12Process in, preferably adopt the trimethyl-glycine supplement art.By adding of trimethyl-glycine, satisfying vitamins B
12On the basis of synthetic necessary trimethyl-glycine consumption, can not affect the growth of thalline because of trimethyl-glycine excessive concentration in the fermented liquid again.In order to obtain 120-m
3The best stream of trimethyl-glycine adds strategy in the tank, the trimethyl-glycine concentration of adding in initial fermention medium is 15g/L, ferment after the trimethyl-glycine spending rate is obviously accelerated to the substratum, respectively the trimethyl-glycine concentration in the fermenting process is controlled at 3-5g/L, 5-7g/L and 8-10g/L by adding continuously trimethyl-glycine.Fig. 3 has shown the trimethyl-glycine change in concentration situation of fermented liquid in these three control strategy bottom fermentation processes.
As seen from Figure 3, the trimethyl-glycine consumption that ferments front 24 hours is very little, and this biomass with this moment is less than normal relevant.Along with the gradually increase of biomass, the spending rate of trimethyl-glycine is obviously accelerated, and this shows, should consider in the fermented liquid that stream adds trimethyl-glycine in addition could satisfy denitrified pseudomonas synthesise vitamins B
12Demand to trimethyl-glycine.Therefore, one of them tank begins Continuous Flow with the beet alkaline solution after being low to moderate about 7g/L recording trimethyl-glycine concentration, and the flow acceleration by the control trimethyl-glycine, so that the trimethyl-glycine concentration in the fermented liquid is 5-7g/L; Another tank begins Continuous Flow with the beet alkaline solution after being low to moderate about 5g/L recording trimethyl-glycine concentration, and the flow acceleration by the control trimethyl-glycine, so that the trimethyl-glycine concentration in the fermented liquid is 3-5g/L; Last tank begins Continuous Flow with the beet alkaline solution after being low to moderate about 7g/L recording trimethyl-glycine concentration, and the flow acceleration by the control trimethyl-glycine, so that the trimethyl-glycine concentration in the fermented liquid is 8-10g/L.
Table 2 has shown under these three kinds of trimethyl-glycine Concentration Controls, the biomass of fermenting process and vitamins B
12The variation tendency of output.
Table 2
Wherein, Run
1, Run
2And Run
3Being illustrated respectively in ferments be reduced to certain numerical value to trimethyl-glycine concentration after, the stream rate of acceleration by adjusting trimethyl-glycine is with 120-m
3Trimethyl-glycine concentration in the fermentor tank is controlled at respectively 3-5g/L, 5-7g/L and 8-10g/L.
As can be seen from Table 2, the biomass under these three tanks are criticized was more or less the same in fermentation in front 54 hours.But along with beginning to control different trimethyl-glycine stream rates of acceleration, the biomass under after this three tanks are criticized presents obvious difference.When the trimethyl-glycine concentration in the fermented liquid was controlled at 8-10g/L, the tank that its biomass is starkly lower than under 3-5g/L and the control of 5-7g/L trimethyl-glycine concentration was criticized.The maximum dry cell weight that the tank of (3-5g/L, 5-7g/L and 8-10g/L) is criticized under these three kinds of trimethyl-glycine Concentration Controls is respectively 33.80 ± 1.13g/L, 33.19 ± 0.75g/L and 30.89 ± 1.05g/L.Can reach a conclusion thus, the trimethyl-glycine concentration in the fermented liquid is controlled highlyer, will produce more serious restraining effect to the thalli growth of denitrified pseudomonas, and this is consistent with the result who obtains in shake flat experiment.
Be it can also be seen that by table 2, change equally with the biomass of earlier fermentation, beginning before stream adds trimethyl-glycine the vitamins B under three tanks are criticized
12Output is almost equal.Yet, along with coming trimethyl-glycine concentration different in the controlled fermentation liquid, the vitamins B under three tanks are criticized with homogeneous turbulence rate of acceleration not
12Obvious variation has occured in output.Tank under 3-5g/L and the control of 5-7g/L trimethyl-glycine concentration is criticized its vitamins B
12Resultant quantity obviously will be higher than the tank that trimethyl-glycine concentration is controlled at 8-10g/L and criticize.During fermentation ends (the 162nd hour), these three tanks are criticized the vitamins B under (trimethyl-glycine controlled concentration: 3-5g/L, 5-7g/L and 8-10g/L)
12Output is respectively 174.62 ± 3.03 μ g/ml, 177.49 ± 2.98 μ g/ml and 160.80 ± 3.56 μ g/ml.When trimethyl-glycine concentration is controlled at 8-10g/L, vitamins B
12Output 160.80 ± 3.56 μ g/ml are only arranged, to be subject to even more serious inhibition relevant with thalli growth under this concentration trimethyl-glycine for this.
In addition, the inventor has also verified at 120-m
3In the fermentor tank, adding trimethyl-glycine concentration in the initial fermention medium is 15g/L, the situation of fermenting and not replenishing trimethyl-glycine after the trimethyl-glycine spending rate is obviously accelerated to the substratum.Found that, ferment the 160th hour the time maximum dry cell weight 36.10 ± 0.67g/L, vitamins B
12Output only is 98.46 ± 1.46 μ g/mL.
In addition, the inventor has also verified at 120-m
3In the fermentor tank, adding trimethyl-glycine concentration in the initial fermention medium is 15g/L, the situation of fermenting and replenishing trimethyl-glycine after the trimethyl-glycine spending rate is obviously accelerated to the substratum and keeping trimethyl-glycine concentration 1-2g/L in the substratum.Found that ferment the 160th hour the time, maximum dry cell weight only is 34.45 ± 0.53g/L, vitamins B
12Output only is 136.41 ± 2.79 μ g/mL.
In addition, the inventor has verified also that in initial fermention medium adding trimethyl-glycine concentration is 15g/L, the again disposable situation of adding trimethyl-glycine 15g/L of fermenting after the trimethyl-glycine spending rate is obviously accelerated to the substratum.Found that ferment the 160th hour the time, maximum dry cell weight only is 28.10 ± 0.82g/L, vitamins B
12Output only is 109.68 ± 2.07 μ g/mL.
Above result shows, is fermenting to certain phase, and controlling suitable trimethyl-glycine concentration is to improve the denitrified pseudomonas fermentation to produce vitamins B
12The available strategy of output.By regulating trimethyl-glycine concentration, not only can reduce trimethyl-glycine to the inhibition of thalli growth, but also can satisfy fermenting process to the demand of trimethyl-glycine, can not affect because of the deficiency of trimethyl-glycine vitamins B
12Synthetic.And, the vitamins B of criticizing according to the tank under above three different trimethyl-glycine concentration control
12Change, better trimethyl-glycine concentration in the fermented liquid is controlled at 5-7g/L after being reduced to 7g/L to trimethyl-glycine concentration fermenting.The inventor has carried out the experimental demonstration that multiple tank is criticized, and the result proves that it is comparatively reasonable and economical using this trimethyl-glycine control strategy in the fermentative production.
The impact of embodiment 4 fermention mediums
The inventor has also designed another group fermention medium following (g/L):
Glucose 80g/L, yeast extract paste 20g/L, trimethyl-glycine 15g/L, (NH
4)
2HPO
41g/L, (NH
4)
2SO
42g/L, MgSO
47H
2O 2g/L, CoCl
26H
2O 0.15g/L, 5,6-dimethylbenzimidazole 0.08g/L, ZnSO
47H
2O 0.1g/L, CaCO
31.0g/L.
Utilize this substratum to repeat above test, found that, in the constant situation of trimethyl-glycine control strategy, this fermention medium also can be used for fermentation well.
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document is quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (6)
1. one kind is utilized denitrified pseudomonas to produce vitamins B
12Method, it is characterized in that described method comprises:
(1) regulates that trimethyl-glycine concentration is 10-20g/L in the initial denitrified pseudomonas substratum, cultivate denitrified pseudomonas, thereby produce vitamins B
12Described initial denitrified pseudomonas substratum contains: sucrose 80 ± 20g/L, corn steep liquor 30 ± 10g/L, trimethyl-glycine 15 ± 5g/L, (NH
4)
2SO
43 ± 1g/L, MgSO
47H
2O 2 ± 1g/L, CoCl
26H
2O 0.15 ± 0.05g/L, 5,6-dimethylbenzimidazole, 0.08 ± 0.03g/L, ZnSO
47H
2O 0.1 ± 0.05g/L, CaCO
31.0 ± 0.5g/L; With
(2) when the concentration of trimethyl-glycine in the substratum is reduced to 3-7g/L, replenish the adding trimethyl-glycine, and trimethyl-glycine concentration is that 3-7g/L is to fermentation ends in the maintain base; When replenishing trimethyl-glycine, replenish CoCl
2, making its concentration in substratum is 0.05 ± 0.02g/L;
Wherein, 32 ± 2 ℃ of the culture temperature of denitrified pseudomonas were cultivated 120-200 hour.
2. the method for claim 1 is characterized in that, in step (1), regulates that trimethyl-glycine concentration is 12-18g/L in the initial denitrified pseudomonas substratum.
3. the method for claim 1 is characterized in that, in step (2), when the concentration of trimethyl-glycine in the substratum is reduced to 5-7g/L, replenishes the adding trimethyl-glycine.
4. the method for claim 1 is characterized in that, in step (2), trimethyl-glycine concentration is 5-7g/L in the maintain base.
5. the method for claim 1 is characterized in that, in step (2), replenishes trimethyl-glycine by the mode that stream adds.
6. the method for claim 1 is characterized in that, described method also comprises step:
(3) from substratum, isolate vitamins B
12
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810034948 CN101538599B (en) | 2008-03-21 | 2008-03-21 | Method for improving yield of denitrified pseudomonas vitamin B12 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810034948 CN101538599B (en) | 2008-03-21 | 2008-03-21 | Method for improving yield of denitrified pseudomonas vitamin B12 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101538599A CN101538599A (en) | 2009-09-23 |
| CN101538599B true CN101538599B (en) | 2013-04-17 |
Family
ID=41121986
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200810034948 Active CN101538599B (en) | 2008-03-21 | 2008-03-21 | Method for improving yield of denitrified pseudomonas vitamin B12 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101538599B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719504B (en) * | 2012-07-04 | 2014-12-10 | 山东祥维斯生物科技有限公司 | Vitamin B12 complex fermenting addition agent and application method thereof to fermenting vitamin B12 |
| CN106636258B (en) * | 2016-12-20 | 2018-06-01 | 安徽省新康饲料有限公司 | Vitamin B is produced using rumen fluid12Rice straw fermented culture medium and its methods and applications |
| CN109825543B (en) * | 2018-11-24 | 2021-04-30 | 浙江华康药业股份有限公司 | Method for fermenting vitamin B12 by xylose mother liquor microorganism based on illumination regulation |
| CN109837320A (en) * | 2019-03-27 | 2019-06-04 | 山东泓达生物科技有限公司 | A method of it improving denitrified pseudomonas and produces vitamin B12 |
| CN110146612A (en) * | 2019-05-24 | 2019-08-20 | 河北华荣制药有限公司 | A kind of method of cyanide ion concentration in measurement solution |
| CN111073922B (en) * | 2019-12-26 | 2023-04-07 | 湖北广济药业股份有限公司 | Vitamin B 12 Fermentation supplementing medium and supplementing method |
| CN111254173A (en) * | 2019-12-26 | 2020-06-09 | 湖北广济药业股份有限公司 | Production of vitamin B by fermentation of pseudomonas denitrificans12Screening method and screening culture medium for high-yield strains |
-
2008
- 2008-03-21 CN CN 200810034948 patent/CN101538599B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| 李昆太，李永亮，储炬，庄英萍，张嗣良.甜菜碱促进脱氮假单胞菌合成维生素B12.《中国医药工业杂志》.2008,第39卷(第3期),172-175. * |
| 郑建仙.《维生素生产关键技术与典型范例》.《维生素生产关键技术与典型范例》.科学技术文献出版社,2006,75页第3段2-4行及最后一段,76页第1段1-2行及表2-4,78页第3段. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101538599A (en) | 2009-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101538599B (en) | Method for improving yield of denitrified pseudomonas vitamin B12 | |
| CN102517239B (en) | A microorganism having enhanced L-valine productivity and process for preparing L-valine using the same | |
| CN101570771A (en) | Method for producing S-adenosylmethionine through fermentation of recombinant pichia pastoris | |
| CN101748177B (en) | Optimized method for producing vitamin B12 through pseuomonas denitrifican fermentation and synthetic medium | |
| CN109593807A (en) | A kind of method of high level fermenting and producing apramycin | |
| CN101967501B (en) | Method for producing lysine by feedback supplement based on pH | |
| CN102517369A (en) | Method for synthesizing benzene-containing polyhydroxyalkanoate by mixed fermentation of high-density microbes under nitrogen restriction | |
| CN106591401B (en) | Fermentation promoter for increasing yield of gentamicin C1a and addition method thereof | |
| JPH10127298A (en) | Fermentation by controlling osmol concentration for preparation of acarbose | |
| JP2005518780A (en) | Metabolically controlled fermentation process for pseudomonic acid production | |
| CN109207536B (en) | Method for increasing content of milbemycin A3 in milbemycin fermentation product | |
| CN102399845B (en) | Based on CO in tail gas 2the vitamin B12 fermentative production Controlling Technology of concentration | |
| EP1613759B1 (en) | Fermentation processes with low concentrations of carbon- and nitrogen-containing nutrients | |
| CN101006184B (en) | Process for production of 5-ene-3-one or 3,6-dione derivatives of sterols, processes for production of lipid metabolism improvers, foods, drinks, and animal feeds, and analytical method | |
| CN110452945A (en) | Utilize the novel method of S. erythraea fermentations production erythromycin | |
| CN117126898B (en) | Process for preparing valine by biotechnology | |
| JP3653766B2 (en) | Production method of ε-poly-L-lysine | |
| Bekers et al. | Sugar beet juice fermentation by Zymomonas mobilis attached to stainless steel wire spheres | |
| RU2148648C1 (en) | Method of heteropolysaccharide producing | |
| SU745942A1 (en) | Method of culturing bacillus thuringiensis var. galleriae | |
| CN115572753A (en) | Abamectin fermentation production process and abamectin | |
| RU2233874C2 (en) | Method for growing nutrient yeast | |
| RU2022020C1 (en) | Method of ubiquinone-10 preparing | |
| CN111718919A (en) | Culture medium for producing esterifying enzyme and preparation method of crude esterifying enzyme preparation | |
| KR100339277B1 (en) | A process for preparing erythritol by optimizing the fermentation conditions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |