CN101538599A - Method for improving yield of denitrified pseudomonas vitamin B12 - Google Patents
Method for improving yield of denitrified pseudomonas vitamin B12 Download PDFInfo
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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 biological material, 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, promptly aerobic approach and anaerobism approach, but VB
12The biosynthesizing strictness be confined to be used to commercially produce VB at present in some microorganisms
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 sets forth 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 so can transform bacterial strain by means such as mutagenesis and molecular biology, makes the vitamins B of denitrified pseudomonas bacterial strain
12Throughput improves greatly.Because vitamins B
12The productive rate height nearly all is to use denitrified pseudomonas to be used as aerobic production vitamins B at present in industrial production
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.Existing report points out that 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 N-methylsarcosine and methionine(Met) respectively, 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 that a kind of good penetration is pressed conditioning agent, has report trimethyl-glycine and cholinergic to promote the secretion of cell surface to 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 important effect in the biosynthetic process.Yet, in the real attenuation process, do not have systematic research at present for the caused denitrified pseudomonas metabotic change of trimethyl-glycine situation, systematically do not study the control strategy of the preferable 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, this 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 adds trimethyl-glycine, and keep that trimethyl-glycine concentration is that 3-7g/L is to fermentation ends in the substratum.
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, 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), keep that trimethyl-glycine concentration is 5-7g/L in the substratum.
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 ℃.Preferable, 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.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the 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
12The synthetic influence.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
3Trimethyl-glycine dynamic changes of concentration process 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 produces vitamins B to utilizing denitrified pseudomonas (Pseudomonas denitrificans) fermentation
12(Vatamin B
12, VB
12) the each side factor carried out deep 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 preferable trimethyl-glycine concentration in interpolation opportunity of trimethyl-glycine and the fermented liquid, thereby has improved vitamins B effectively
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 adds trimethyl-glycine, and keep that trimethyl-glycine concentration is that 3-7g/L is to fermentation ends in the substratum.
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, and can be utilized by denitrified pseudomonas with trimethyl-glycine.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 the obvious suppression effect to the growth of thalline.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 groping repeatedly, 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 higherly, also be that thalline is in preferable growth, metabolism and production status.And when trimethyl-glycine concentration is higher than 20g/L, vitamins B then
12The tangible reduction of having tired.Preferable, 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, 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 with the fermentation initial stage, so the suitable concentration of trimethyl-glycine in nutrient solution also is different.Through groping repeatedly, 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 keeps afterwards that trimethyl-glycine concentration is 3-7g/L in the substratum, and 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.Preferable, when the concentration of trimethyl-glycine in the substratum is reduced to 5-7g/L, replenishes and add trimethyl-glycine, and keep that trimethyl-glycine concentration is 5-7g/L in the substratum.
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 employing disposable adding is earlier added trimethyl-glycine in substratum, can stably provide trimethyl-glycine in the mode of taking stream to add to the certain phase of fermenting, make thalline under suitable working condition, produce vitamins B to denitrified pseudomonas
12, keep ideal 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 batch add mode that the intermittent time is enough short, as long as this mode also can keep trimethyl-glycine concentration basicly stable in the substratum (promptly 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 (as carbon source, phosphorus source, organic nitrogen source, inorganic salt, trace element) and the usage quantity thereof that are used to prepare 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 when being lower than 3-7g/L, trimethyl-glycine is added in beginning 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)
2HPO
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,, can adopt condition known in the art as temperature, humidity, air flow etc.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 ℃.Preferable, 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 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 the formation of δ-An Jiyixianbingsuan (ALA) synthase by trimethyl-glycine, and then promote the synthetic of ALA, promote synthesise vitamins B afterwards
12Synthetic.Most preferred, described denitrified pseudomonas is denitrified pseudomonas J741.
In an embodiment of the present invention, at first in shaking bottle, investigated trimethyl-glycine to vitamins B
12The synthetic influence, 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 the obvious suppression effect, be unfavorable for vitamins B on the contrary more the growth of thalline
12Synthetic, so the trimethyl-glycine concentration in the initial fermention medium of empirical tests can be selected 15g/L for use best.Then, the inventor has further studied the influence of the trimethyl-glycine of 0g/L, 15g/L and 30g/L to the denitrified pseudomonas fermenting process by shaking bottle, the result shows that certain density trimethyl-glycine can significantly improve the resultant quantity of δ-An Jiyixianbingsuan 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 the inhibition of trimethyl-glycine, but also can avoid influencing vitamins B because of the deficiency of trimethyl-glycine to thalli growth
12Synthetic.120-m
3The concrete stream of trimethyl-glycine adds strategy and is in the fermentor tank: by fermenting when trimethyl-glycine concentration is lower than 7g/L, 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 preferable trimethyl-glycine concentration in interpolation opportunity of trimethyl-glycine and the fermented liquid, thereby improved vitamins B effectively
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 to the present invention is described 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)
28O
42.0, (NH
4)
2HPO
40.5, MnSO
4H
2O 0.2, MgSO
47H
2O 1.0, ZnSO
47H
2O 0.1, pH7.2～7.4.
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, and the 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: Dobetin 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-An Jiyixianbingsuan (Sigma company), other reagent is homemade 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 jar 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 (OD
700) be about 15～20, first order seed being inserted loading amount is 7m again
3/ 9-m
3In the secondary seed medium of secondary seed jar, at 32 ± 0.5 ℃ of jar 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 jar being moved into loading amount is 70m
3/ 120-m
3In the fermention medium of fermentor tank, at 32 ± 0.5 ℃ of jar temperature, tank pressure 0.05～0.06MPa, air flow quantity 600～800m
3/ hour, mixing speed 60rpm cultivated about 160 hours down.
Cell concentration is measured
Employing measurement dry cell weight method (Dry cell weight, DCW).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 be during the VB12 in measuring fermented liquid with bacterial cell disruption, concrete operation steps is as follows:
(1) specimen 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; The water-bath postcooling adds deionized water and is settled to 50mL to room temperature, filters; 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.
δ-An Jiyixianbingsuan (δ-aminolevulinic acid, ALA) Determination on content
According to document Burnham B F. δ-aminolevulinic acid sythase[J] .Methods in Enzymology, 1970, the method for 17A:195-200 is measured.
II. embodiment
Embodiment 1. trimethyl-glycine concentration are to vitamins B
12The synthetic influence
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
12The synthetic influence 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 facilitation.Fermention medium does not add trimethyl-glycine, though 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 the influence of trimethyl-glycine to the 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 bottle, with the shake flat experiment that do not add trimethyl-glycine in contrast.
1. trimethyl-glycine is to the influence 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 of thalli growth is too fast.
2. trimethyl-glycine is to δ-An Jiyixianbingsuan (ALA) and vitamins B
12The synthetic influence
Fig. 2 is that above-mentioned three kinds of concentration trimethyl-glycines are to fermenting process ALA and vitamins B
12Synthetic influences the result.
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 fermentation 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 certain restraining effect to the growth of thalline, 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 select suitable trimethyl-glycine to add concentration according to the consumption situation of trimethyl-glycine in the fermenting process and trimethyl-glycine to the influence of thalli growth.Along with trimethyl-glycine concentration in the substratum increases, can influence vitamins B because trimethyl-glycine is excessive on the contrary
12Synthetic, and this control to fermentation costs also is very disadvantageous.
The trimethyl-glycine regulation and control strategy of denitrified pseudomonas fermentation under embodiment 3 industrially scalables
Shake bottle result as can be known by above, 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 trimethyl-glycine to add technology.By adding of trimethyl-glycine, satisfying vitamins B
12On the basis of synthetic necessary trimethyl-glycine consumption, can not influence the growth of thalline again because of trimethyl-glycine excessive concentration in the fermented liquid.In order to obtain 120-m
3The best stream of trimethyl-glycine adds strategy in jar, 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 trimethyl-glycine continuously.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 preceding 24 hours is very little, and this biomass with this moment is less than normal relevant.Along with the increase gradually of biomass, the spending rate of trimethyl-glycine is obviously accelerated, and this shows, should consider in fermented liquid that stream adds trimethyl-glycine in addition could satisfy denitrified pseudomonas synthesise vitamins B
12Demand to trimethyl-glycine.Therefore, one of them jar begins Continuous Flow with the beet alkaline solution after being low to moderate about 7g/L recording trimethyl-glycine concentration, and passes through to control the flow acceleration of trimethyl-glycine, makes that the trimethyl-glycine concentration in the fermented liquid is 5-7g/L; Another jar begins Continuous Flow with the beet alkaline solution after being low to moderate about 5g/L recording trimethyl-glycine concentration, and passes through to control the flow acceleration of trimethyl-glycine, makes that the trimethyl-glycine concentration in the fermented liquid is 3-5g/L; Last jar begins Continuous Flow with the beet alkaline solution after being low to moderate about 7g/L recording trimethyl-glycine concentration, and passes through to control the flow acceleration of trimethyl-glycine, makes 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 control strategies, 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 3-5g/L, 5-7g/L and 8-10g/L respectively.
As can be seen from Table 2, the biomass under these three jars are criticized was more or less the same in fermentation in preceding 54 hours.But along with beginning to control different trimethyl-glycine stream rates of acceleration, the biomass under after this three jars are criticized presents tangible difference.When the trimethyl-glycine concentration in the fermented liquid was controlled at 8-10g/L, the jar 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 jar of (3-5g/L, 5-7g/L and 8-10g/L) is criticized under these three kinds of trimethyl-glycine concentration control strategies 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 highly more, will produce serious more restraining effect to the thalli growth of denitrified pseudomonas, and this is consistent with the result who obtains in shake flat experiment.
It can also be seen that by table 2, change equally, beginning before stream adds trimethyl-glycine the vitamins B under three jars are criticized with the biomass of earlier fermentation
12Output is almost equal.Yet, along with controlling trimethyl-glycine concentration different in the fermented liquid, the vitamins B under three jars are criticized with homogeneous turbulence rate of acceleration not
12Obvious variation has taken place in output.Jar under 3-5g/L and the control of 5-7g/L trimethyl-glycine concentration is criticized its vitamins B
12Resultant quantity obviously to be higher than trimethyl-glycine concentration be controlled at 8-10g/L the jar batch.During fermentation ends (the 162nd hour), these three jars are criticized the vitamins B under (trimethyl-glycine controlling concn: 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 subjected 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, and the trimethyl-glycine spending rate that ferments to the substratum is obviously accelerated the situation that the back replenishes trimethyl-glycine and keeps 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 disposable once more situation of adding trimethyl-glycine 15g/L after the trimethyl-glycine spending rate is obviously accelerated to the substratum of fermenting.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 the inhibition of trimethyl-glycine, but also can satisfy the demand of fermenting process trimethyl-glycine to thalli growth, can not influence vitamins B because of the deficiency of trimethyl-glycine
12Synthetic.And, according to jar vitamins B of criticizing under above three different trimethyl-glycine concentration controls
12Change, preferable 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 this trimethyl-glycine control strategy of utilization is comparatively reasonable and economical in the fermentative production.
The influence 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, under 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 quoted as a reference separately as each piece document.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 institute restricted portion equally.
Claims (10)
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
12With
(2) when the concentration of trimethyl-glycine in the substratum is reduced to 3-7g/L, replenish adds trimethyl-glycine, and keep that trimethyl-glycine concentration is that 3-7g/L is to fermentation ends in the substratum.
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), keeps that trimethyl-glycine concentration is 5-7g/L in the substratum.
5. the method for claim 1 is characterized in that, in step (2), when replenishing trimethyl-glycine, replenishes CoCl
2, making its concentration in substratum is 0.05 ± 0.02g/L.
6. the method for claim 1 is characterized in that, in step (2), replenishes trimethyl-glycine by the mode that stream adds.
7. the method for claim 1 is characterized in that, 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.
8. the method for claim 1 is characterized in that, the temperature of cultivation is 32 ± 2 ℃.
9. the method for claim 1 is characterized in that, described method also comprises step:
(3) from substratum, isolate vitamins B
12
10. the method for claim 1 is characterized in that, the time of cultivation is 120-200 hour.
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| CN102719504A (en) * | 2012-07-04 | 2012-10-10 | 潍坊祥维斯化学品有限公司 | Vitamin B12 complex fermenting addition agent and application method thereof to fermenting vitamin B12 |
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