CN101012470A - Method for zymolytic production of S-adenosine methionine - Google Patents
Method for zymolytic production of S-adenosine methionine Download PDFInfo
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Abstract
The invention discloses a manufacturing method of S-adenosylmethionine (SAM), which is characterized by the following: adopting pulse supplementing method to add carbon source; putting fitful quantity of alcohol into ferment liquid.
Description
Technical field
The invention belongs to biotechnology and microorganism field; More specifically, the present invention relates to a kind of method of producing S-adenosylmethionine.
Background technology
S-adenosylmethionine (S-Adenosyl-Methionine, SAM) be a kind of important intermediary metabolism substance that is present in all living body biologicals, mainly participated in transmethylase, change sulphur, the synthetic metabolism approach that waits of polyamines medically can be used to treat diseases such as hepatopathy and nervous disorders.At present, SAM is as a kind of new health care product, and market outlook are wide.
SAM can adopt chemosynthesis, external enzymatic to synthesize or obtain through the microbial fermentation extraction.The chemosynthesis productive rate is low, and product is racemic mixture, therefore seldom is used; Enzyme process be owing to will add ATP, so cost is too high, and only suitable synthetic isotope mark SAM.Some bacterial strains of certain micro-organisms, especially saccharomyces (Saccharomyces) can accumulate SAM in vivo in a large number.By microbial fermentation, in substratum, add a certain amount of methionine(Met), can obtain a large amount of SAM.The main bacterial strain that uses has yeast saccharomyces cerevisiae and recombination yeast.
Mostly the recombination yeast that adopts in the SAM fermentation is the methyl alcohol nutritional type recombination yeast of alcohol oxidase expression system, it belongs to induction type, need methanol induction to express, yet this kind SAM production method exists fermentation time long, methyl alcohol can be to problems such as operator's toxigenicities.
A kind of improved mode is to adopt the composing type recombination yeast to produce SAM, and this SAM production method only has a spot of report at present.The recombination yeast of composing type has and need not to induce, the advantage that fermentation time is short.Owing to it is believed that higher dissolved oxygen is favourable for the accumulation of SAM, therefore the zymotechnique that utilizes the composing type recombination yeast to produce SAM of the prior art all adopts carbon source restriction flow feeding method, promptly adopts the carbon source restriction flow feeding strategy of the certain dissolved oxygen of control (for example 5-40%) in the bio-transformation phase.Yet this method complicated operation and SAM output in production practice is not high, can't satisfy the needs of scale operation.
Therefore, this area also needs to be optimized the technology of fermentative production SAM, in the hope of the production technique of further simplifying SAM or the output that improves SAM.
Summary of the invention
The object of the present invention is to provide a kind of method of improved production S-adenosylmethionine, this method has SAM output height, the short advantage of fermentation time.
In a first aspect of the present invention, a kind of method of producing S-adenosylmethionine is provided, comprise step: the fermentation culture S-adenosylmethionine is produced bacterium in fermented liquid, thereby produces S-adenosylmethionine,
Wherein, in culturing process, adopt pulse feed supplement mode to add carbon source in fermented liquid, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is that (preferred, add carbon source to final concentration is 20-80g/L to 10-160g/L in fermented liquid; Preferred, adding carbon source to final concentration is 20-60g/L, as 40g/L), treat that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration once more is 10-160g/L.
In a preference of the present invention, contain the L-methionine(Met) in the described fermented liquid.
In another preference of the present invention, the L-methionine(Met) is for add in batches in the fermented liquid, and 5-25 hour pitch time, add-on is 2-20g/L.
In another preference of the present invention, when the cell density of S-adenosylmethionine production bacterium is 100-200gDCW/L in fermented liquid, add the L-methionine(Met).
In another preference of the present invention, it is that the yeast that derives from the subordinate: Hansenula belongs to that described S-adenosylmethionine is produced bacterium, and Candida belongs to, and Torulopsis belongs to, or Pichia belongs to.
In another preference of the present invention, it is the yeast that Pichia belongs to that described S-adenosylmethionine is produced bacterium, as pichia spp.
In another preference of the present invention, it is that preserving number is the pichia spp of CGMCC NO.0746 that described S-adenosylmethionine is produced bacterium.
In another preference of the present invention, described S-adenosylmethionine is produced bacterium and is carried the S-adenosylmethionine synthetase-coding gene, and with Glycerose 3-phosphate dehydrogenase (GAP) as promoter expression S-adenosylmethionine synthetic enzyme.
In another preference of the present invention, described SAM synthetic enzyme is a SAM synthetic enzyme 2.
In another preference of the present invention, described carbon source is selected from: glycerine, glucose, sorbyl alcohol, peptone, yeast extract, soybean cake powder or its combination.
In another preference of the present invention, described method comprises that also stream adds ethanol in fermented liquid, makes the alcohol concn in the fermented liquid be 0.2-6% according to volume ratio (v/v).
In another preference of the present invention, the concentration of ethanol in the fermented liquid is 1-4% according to volume ratio; More preferably, the concentration of ethanol in the fermented liquid is 1.5-3% according to volume ratio; Most preferably, the concentration of ethanol in the fermented liquid is 2-2.5% according to volume ratio.
In a second aspect of the present invention, a kind of method of preferred production S-adenosylmethionine is provided, comprise step:
(1) in fermented liquid, cultivates the S-adenosylmethionine that contains the S-adenosylmethionine synthetase-coding gene and produce bacterium, make it to express the S-adenosylmethionine synthetic enzyme;
(2) in producing the fermented liquid of bacterium, the S-adenosylmethionine of (1) adds the L-methionine(Met), (the L-methionine(Met) is produced bacterium by S-adenosylmethionine and is absorbed), the ATP and the L-methionine(Met) that make S-adenosylmethionine produce bacterium metabolism generation react under the catalysis of S-adenosylmethionine synthetic enzyme, generate S-adenosylmethionine and
(3) separate S-adenosylmethionine;
Wherein,
In step (2), adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, treats that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration again is 10-160g/L.
In another preference of the present invention, repeat this feed supplement mode several times, for example 1-50 time, preferably 1-20 time.
In a third aspect of the present invention, provide a kind of fermentation culture S-adenosylmethionine to produce the method for bacterium, comprise step: the described S-adenosylmethionine of fermentation culture is produced bacterium in fermented liquid,
Wherein, in described fermentation culture process, adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, treat that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration once more is 10-160g/L.
In a fourth aspect of the present invention, a kind of method of producing S-adenosylmethionine is provided, comprise step: the fermentation culture S-adenosylmethionine is produced bacterium in fermented liquid, thereby produces S-adenosylmethionine,
Wherein,
In described culturing process, intermittently fluctuation between 0%-100% of control dissolved oxygen (descend and rise even dissolved oxygen is intermittent, the following of decline is limited to 0%, is limited to 100% in the rising).
In another preference of the present invention, in fermented liquid, add carbon source by pulse feed supplement mode and control dissolved oxygen, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, treat that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration once more is 10-160g/L.
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. the glycerine specific consumption rate of different feed supplement strategies of bio-transformation phase.
Fig. 2. the variation of bio-transformation phase different feed supplement strategy biomass.
Fig. 3. the variation of SAM output in the different feed supplement strategies of bio-transformation phase.
Fig. 4. the ratio output of SAM in the different feed supplement strategies of bio-transformation phase.
Fig. 5. the enzyme of SAM synthetic enzyme is lived and is changed in the different feed supplement strategies of bio-transformation phase.
Fig. 6. the SAM synthetic enzyme variation more alive in the different feed supplement strategies of bio-transformation phase than enzyme.
Fig. 7. the content of ATP in born of the same parents during bio-transformation phase utilization Different Strategies.
Embodiment
At the not high problem of S-adenosylmethionine in the prior art (SAM) output, the inventor is through deep research, find first in utilizing the synthetic enzyme-producing bacteria fermentative production SAM process of S-adenosylmethionine, adopt pulse to add the mode of carbon source, can improve the output of SAM greatly, and fermentation time is short; In addition, the inventor also finds, in producing the SAM process alcohol concn in the fermented liquid is controlled at 0.2-6% (v/v) and will further improves the output of SAM.Finished the present invention on this basis.
Reaction principle
The ultimate principle that the present invention produces SAM is: utilize SAM to produce the characteristics that bacterium self can produce ATP, fermentation culture SAM produces bacterium, and the ATP and the L-methionine(Met) that make SAM produce the bacterium generation react under the catalysis of SAM synthetic enzyme, thereby generates SAM.
The reaction skeleton symbol is as follows:
Preferred, can be by transforming the SAM synthetase-coding gene that suitable bacterial strain makes it to carry external source, utilize this bacterial strain can express the characteristic of SAM synthetic enzyme and self generation ATP, external source gives L-methionine(Met), the L-methionine(Met) is absorbed the back by bacterial strain and reacts with ATP, thereby generates SAM under the catalysis of SAM synthetic enzyme.
S-adenosylmethionine is produced bacterium
Among the present invention, it can be any bacterial strain of producing S-adenosylmethionine that is applicable to that the S-adenosylmethionine of employing is produced bacterium (SAM produces bacterium), as long as it can synthesize S-adenosylmethionine with ATP and L-methionine(Met) under the catalysis of SAM synthetic enzyme.
As optimal way of the present invention, described SAM produces bacterium and carries the encoding gene of SAM synthetic enzyme, thereby can express the SAM synthetic enzyme under suitable culture condition.And described SAM produces bacterium can produce ATP in self metabolic process.
As optimal way of the present invention, it is to derive from (but being not limited to) yeast with the subordinate: Hansenula to belong to that described SAM produces bacterium, and Candida belongs to, and Torulopsis belongs to, or Pichia belongs to.As preferred mode of the present invention, it is to derive from the yeast that Pichia belongs to that described SAM produces bacterium, as pichia spp.
As optimal way of the present invention, described SAM produces in the bacterium and carries expression plasmid, and described expression plasmid contains the encoding gene of SAM synthetic enzyme, and, contain Glycerose 3-phosphate dehydrogenase (GAP) in the described expression plasmid as promotor.GAP is a kind of promotor of constitutive expression.As used herein, described " promotor " is meant a kind of nucleotide sequence, and it is present in the upstream (5 ') of goal gene encoding sequence usually, can be transcribed into mRNA by the guiding nucleus acid sequence, thus the expression of guiding target protein.Described " promotor of constitutive expression " is meant that this promotor guides the expression of target protein sustainably.Persistence express have usually need not to induce, the expression amount height, and low characteristics of cost.
Method well-known to those having ordinary skill in the art can be used to make up recombinant protein (as the SAM synthetic enzyme), contain as described in the recombinant expression plasmid of recombinant protein and/or suitable promotor, and the recombinant bacterial strain that contains described recombinant expression plasmid.Described method for example can be referring to people such as Sambrook, molecular cloning: described in the lab guide (New York:Cold Spring Harbor Laboratory Press, 1989).
Except the feed supplement mode of carbon source and/or alcoholic acid adding etc. by the pointed special conditions of the present invention, the present invention has no particular limits for cultivation (fermentation) condition (for example temperature, pH value, substratum etc.) that SAM produces other each side of bacterium, can adopt this area routine to be used for SAM and produce the various conditions that bacterium is cultivated.
The present invention also provides a kind of fermentation culture S-adenosylmethionine to produce the method for bacterium, and this method is: the described SAM of fermentation culture produces bacterium in fermented liquid; Wherein, in described fermentation culture process, adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, treat that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration once more is 10-160g/L.
Adopt described method fermentation culture SAM to produce bacterium, can improve the biomass of S-adenosylmethionine production bacterium in the fermented liquid, improve the generation of ATP, improve the activity of SAM synthetic enzyme, thereby improve the output of SAM.
The production of S-adenosylmethionine
The invention provides a kind of method of improved producing S-adenosylmethionine through fermentation, described method comprises step: the fermentation culture S-adenosylmethionine is produced bacterium in fermented liquid, thereby produces S-adenosylmethionine; Wherein, in culturing process, adopt pulse feed supplement mode to add carbon source in fermented liquid, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is that (preferred, add carbon source to final concentration is 20-80g/L to 10-160g/L in fermented liquid; Preferred, adding carbon source to final concentration is 20-60g/L, as 40g/L), treat that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration once more is 10-160g/L.
In optimal way of the present invention, contain the L-methionine(Met) in the described fermented liquid.As optimal way of the present invention, in biotransformation, the L-methionine(Met) is for add in batches in the fermented liquid, and 5-25 hour pitch time, add-on is 2-20g/L.Add the L-methionine(Met) in batches and can guarantee that it is utilized by SAM production bacterium better, thereby obtain high SAM output.
In optimal way of the present invention, before in fermented liquid, adding the L-methionine(Met), cultivate SAM in advance and produce bacterium, make it the biomass that reaches certain, to guarantee in biotransformation, having enough SAM to produce bacterial strain as " producer ".Further preferred, SAM being produced after bacterium is inoculated in the fermented liquid, earlier SAM is produced bacterium and carry out batch culture and/or carbon source restriction feeding culture, make it the cell density that in fermented liquid, reaches certain.For example, when SAM production bacterium is a primary yeast, when the zymic cell density reaches 100-200gDCW/L in the fermented liquid, adds the L-methionine(Met) and can obtain comparatively ideal effect.
In optimal way of the present invention, described carbon source is selected from: glycerine, glucose, sorbyl alcohol, peptone, yeast extract, soybean cake powder or its combination.
The carbon source add-on of the multiplicity of carbon source pulse feed supplement and the kind that depends on used carbon source pitch time between twice feed supplement, speed speed that carbon source is consumed and single and deciding.Usually, the multiplicity of carbon source pulse feed supplement is 1-50 time, preferably 1-20 time; Be 0.5-20 hour the pitch time between twice feed supplement, preferably 1-10 hour, be more preferably 1.5-7 hour, and most preferred is 3-5 hour.
As particularly preferred mode of the present invention, also comprise in the described method: stream adds ethanol in fermented liquid, and making the alcohol concn in the fermented liquid is 0.2-6% (v/v); Preferably, the alcohol concn in the fermented liquid is 1-4% (v/v); More preferably, the alcohol concn in the fermented liquid is 1.5-3% (v/v); Most preferably, the alcohol concn in the fermented liquid is 2-2.5% (v/v).The inventor finds, adds in pulse under the prerequisite of carbon source, keeps the ethanol content of appropriateness can make the output of SAM be further improved in the fermented liquid, and the ratio of raising is about 10-30%.
As a kind of optimal way of the present invention, described method comprises step: (1) is cultivated the S-adenosylmethionine that contains the S-adenosylmethionine synthetase-coding gene and is produced bacterium in fermented liquid, make it to express the S-adenosylmethionine synthetic enzyme; (2) in producing the fermented liquid of bacterium, the S-adenosylmethionine of (1) adds the L-methionine(Met), the L-methionine(Met) is produced bacterium by S-adenosylmethionine and is absorbed, the ATP and the L-methionine(Met) that make S-adenosylmethionine produce bacterium metabolism generation react under the catalysis of S-adenosylmethionine synthetic enzyme, generate S-adenosylmethionine, separate S-adenosylmethionine with (3); Described method is characterised in that, in step (2), adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, treat that (preferred, carbon source concentration was lower than 1g/L when carbon source concentration was lower than 2g/L in the fermented liquid; Preferred, carbon source concentration is lower than 0.5g/L), adding carbon source to final concentration again is 10-160g/L.
The restricted stream that the control dissolved oxygen is compared in the pulse feed supplement of carbon source adds, and the specific consumption rate of carbon source is higher, can be SAM production bacterium more material and energy are provided, thereby reached higher cell density, higher ratio enzyme work.Simultaneously, the ATP content of unit thalline also has raising.These all help the accumulation of SAM.
The variation of having compared in an embodiment of the present invention, SAM output when adopting different feed supplement strategy in the bio-transformation phase of SAM.With glycerine is carbon source, and the feed supplement strategy of employing is as follows: the restricted stream of glycerine of 1. controlling 20～25% dissolved oxygens adds; 2. controlling dissolved oxygen and be 5～10% the restricted stream of glycerine adds; 3. the glycerine pulsed is non-limiting adds.Found that, in biotransformation, adopt the extremely excellent effect that has of pulse feed supplement mode.
Carbon source provides basic substance and energy derive for the vital movement of cell.It need a large amount of ATP because SAM is synthetic, so competent carbon source can guarantee for provide energy.The inventor finds, compares with the carbon source restriction fed-batch mode of control dissolved oxygen, and the carbon source specific consumption rate of carbon source pulse feed supplement mode and average wear rate all increase.The carbon source consumption of visible impulse feed supplement and wear rate maximum illustrate that the ATP amount of its conversion is maximum, for synthetic SAM provides competent ATP.
In biomass, SAM output and the comparison of SAM than output, the inventor finds, provides nonrestrictive carbon source for SAM produces bacterium, although dissolved oxygen may be near 0, cell is still grown vigorous, has reached the density than carbon source restriction stream Jia Genggao.Cell is basic SAM production unit, and " producer " is many, helps output and improves.And, using the also very bigger of different tactful SAM than output difference, the SAM of pulse feed supplement is higher than output.
The SAM synthetic enzyme can catalysis ATP and the synthetic SAM of L-methionine(Met), and its enzyme is lived and also is related to SAM synthetic speed.The inventor finds, compares with the carbon source restriction fed-batch mode of control dissolved oxygen, and the enzyme of the SAM synthetic enzyme of carbon source pulse feed supplement mode is lived and lived all higher than enzyme.Its reason may be synthetic needs of enzyme to consume a large amount of materials and energy, and the carbon source specific consumption rate is faster in the pulse feed supplement, for cell provides more competent energy and material source.High enzyme compares work also provides guarantee for the synthesis rate of SAM.
ATP and L-methionine(Met) are two substrates of SAM building-up reactions.The synthesis rate of SAM not only depends on the vigor of SAM synthetic enzyme, and concentration of substrate is also most important.Because the L-methionine(Met) that external source is added can be excessive, so ATP becomes SAM synthetic restrictive factor.According to report of the prior art, ATP is used in the synthetic competition of other power consumption process and SAM in the born of the same parents, causes SAM to synthesize required ATP deficiency probably.The inventor has measured bio-transformation phase content of ATP in the born of the same parents when using different feed supplement strategy respectively, finds to compare with the carbon source restriction fed-batch mode of control dissolved oxygen, and the ATP content of carbon source pulse feed supplement mode is the highest all the time in the bio-transformation phase.The synthetic main of ATP not only consumes a large amount of oxygen by oxidative phosphorylation, also needs a large amount of carbon sources.Though pulse feed supplement dissolved oxygen may be near 0, its carbon source specific consumption rate is more faster than other two kinds of feed supplement strategies, so ATP content is also higher.Because oxidative phosphorylation needs a large amount of oxygen, the accumulation to SAM as single factors of high dissolved oxygen is favourable, and the carbon source restriction flow feeding of control 20～25% dissolved oxygens also just is being based on this point.But high dissolved oxygen is a cost to sacrifice the carbon source feed rate.Fact proved that the pulse feed supplement of suitably having sacrificed dissolved oxygen has obtained more carbon sources, the ATP content of unit thalline and enzyme are lived all much higher, thereby more help the SAM accumulation.
In addition, the present invention has no particular limits for the method for (or fermented liquid) isolated or purified SAM in thalline, can adopt this area routine to be used for the method for isolated or purified.
Major advantage of the present invention is:
(1) finds first in utilizing the synthetic enzyme-producing bacteria producing S-adenosylmethionine through fermentation process of S-adenosylmethionine, adopt pulse to add the mode of carbon source in the bio-transformation phase, can improve the output of SAM greatly, and fermentation time short (time is about 65-120 hour).Adopt method of the present invention, SAM output is compared conventional carbon source restriction feeding method and has been improved more than 1 times, for suitability for industrialized production is laid a good foundation.
(2) by contrast, the pulse feed supplement of carbon source is obviously more easy in operation than flow feeding, and controllability is stronger.
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 (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Experiment material and method
Use following material and method in the specific embodiment of the invention, below the material and the method that are provided be not to be used for limitation of the present invention.
1. bacterial strain
The fermentation strain that uses is the composing type pichia spp, and preserving number is CGMCC, and NO.0746 is provided by Chinese Academy of Sciences's Shanghai school of life and health sciences biological chemistry and RESEARCH ON CELL-BIOLOGY.This composing type pichia spp is to transform Pichi strain GS115 acquisition by having SAM synthetic enzyme 2 expression of gene plasmid pGAP-SAMS2 electricity.
2. bio-reactor
The FUS-15L fermentor tank that the fermentor tank that uses is produced as Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai.The data collection and analysis software of fermenting process is Tophawk (Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai's exploitation).
3. substratum
A. seed culture medium
Yeast extract 0.5g, the basic nitrogenous source 1g of yeast,
Peptone 0.375g, vitamin H is an amount of,
Glycerine 0.5g;
Dissolved in distilled water is settled to 50ml, 121 ℃ of sterilization 30min.
B. fermentation tank culture medium
85% phosphoric acid 200.25ml, anhydrous calciumsulphate 6.975g,
Vitriolate of tartar 136.5g, magnesium sulfate heptahydrate 111.75g,
Potassium hydroxide 30.975g, glycerine 300g,
Defoamer 7.5ml;
Dissolved in distilled water is settled to 7.5L, 121 ℃ of sterilization 30min.
4. analytical procedure
Cell density
Sentence deionized water in 600nm after the fermented liquid dilution suitable multiple and carry out colorimetric estimation OD for contrast
600=OD reading * extension rate.
Dry cell weight is calculated as follows: 1OD
600=0.245g DCW/L.
SAM is extracting fast in a small amount
Get 20% perchloric acid that a certain amount of fermented liquid adds 4 times of volumes in 4 ℃ of extractings 2 hours, centrifugal after, supernatant filters the back and goes up sample HPLC and analyze.
The HPLC standard measure of SAM
(4.6mm * 250mm), moving phase: 0.5mol/L ammonium formiate (pH4.0), flow velocity 2.0ml/min detects wavelength 154nm to strong cation type ion exchange column Hypercil 10 SCX.External standard method quantitative Analysis SAM content.SAM mark product (Sigma) are the SAM tosilate of purity 93%.
SAM synthetic enzyme vigor is measured
(Li Dongyang is in building first-class among Yuan to press document; Utilize reorganization Pichia pastoris to produce adenosylmethionine; Biotechnology journal, May 2002 Vol.18 No.3) method that provides is measured, and define the enzyme unit (U) that lives is that 37 ℃ of reaction 60min transform the required enzyme amounts of generation 1 μ mol SAM.
Carbon source concentration is measured in the fermented liquid
Adopt serum triglyceride test kit (available from the prompt pupil's thing in Shanghai technology company) to measure in the fermented liquid glycerol concentration as carbon source.
Determining concentration of alcohol in the fermented liquid
Fermented liquid centrifuging and taking supernatant is measured with GC-920 gas-chromatography view.Filler is the Chromosorb101 type, chromatogram column length 1m, and internal diameter 2mm, 135 ℃ of column temperatures, 170 ℃ of temperature of vaporization chamber, 170 ℃ of detector temperatures, the CDMC chromatographic working station is analyzed.
The mensuration of ATP in the born of the same parents
The mensuration of ATP is according to document Pierro DD etc., An ion-pairing high-Performanceliquid chromatographic method for the direct simultaneousdetermination of nucleotides, deoxynucleotides, nicotiniccoenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts.Anal.Biochem.1995,231,407-412; Or Xiao-qing Hu etc., the method for A novel feedingstrategy during the production phase for enhancing the enzymaticsynthesis of S-adenosyl-L-methionine by methylotrophic Pichiapastoris.Enzyme and Microbial Technology report is carried out.
Inclined-plane inoculation group moulding Pichi strain 500ml shakes bottle, cultivates 24 hours for 220rpm30 ℃.Insert fermentor tank, inoculum size 300ml.The canned liquid 8L that ferments, 30 ℃ of temperature, 28% ammoniacal liquor is transferred pH6.0.
Whole fermentation process is divided into 3 stages:
(1) batch culture;
(2) the restricted stream of glycerine adds and makes thalline reach high-density (about 140gDCW/L);
(3) add methionine(Met) and begin bio-transformation SAM.
In the batch culture process, air flow 1.0VVM improves mixing speed dissolved oxygen is maintained more than 20%.After initial glycerine exhausted, dissolved oxygen rose to, and hungry 0.5 hour begins stream and adds 50% (v/v) glycerine (containing PTM1 12ml/L), regulating feed rate is controlled at dissolved oxygen between 20% to 30%, and improve rotating speed and be up to 700r/min, air flow improved 0.2VVM in per 10 hours, until 1.6VVM.When cell density reached the 140g/L left and right sides in 52 hours, add L-methionine(Met) (totally three times, 52 hours, 72 hours, 84 hours add respectively 10g/L, 5g/L, 5g/L), enter the bio-transformation phase.The regulation and control strategy in preceding two stages is identical, and adopts following three kinds of different feed supplement strategies in the bio-transformation phase:
(a) the restricted stream of glycerine of control 20～25% dissolved oxygens adds;
(b) the control dissolved oxygen is that 5～10% the restricted stream of glycerine adds;
(c) the glycerine pulsed is non-limiting adds (52hr begins), it is the pure glycerin of disposable adding 40g/L (pressing fermentating liquid volume calculates), after treating that glycerine has consumed (being lower than 0.5g/L) dissolved oxygen bounce-back (being that dissolved oxygen is increased near 100%) substantially, add 40g/L (being that dissolved oxygen is reduced near 0%) again, repeatable operation.According to surveying and determination, in this example, add the feed supplement of 40g/L glycerine at every turn after, approximately at interval glycerine has consumed substantially and need add once more after 3-5 hour.
After cultivation finished, SAM was present in the cell, needed the yeast cell fragmentation when separating.
The comparison of the specific consumption rate of embodiment 2 bio-transformation phase glycerine and average wear rate
Glycerine is as carbon source unique in the substratum, for the vital movement of cell provides basic substance and energy derive.Need a large amount of ATP because SAM is synthetic, so competent carbon source can guarantee that the specific consumption rate of glycerine just seems and is even more important for it provide energy.Therefore, the glycerine specific consumption rate of different feed supplement strategies and average wear rate in present embodiment comparing embodiment 1 method.
Different time points, glycerol concentration serum triglyceride kit measurement in the fermented liquid.The results are shown in Figure 1.In three kinds of feed supplement strategies, the glycerine specific consumption rate maximum of pulse feed supplement, the control dissolved oxygen is to take second place restricted stream added-time of glycerine of 5～10%, controls the restricted stream added-time minimum of glycerine of 20～25% dissolved oxygens.The three is along with the prolongation of fermentation time is on a declining curve, being to cause the glycerine specific consumption rate to descend because the thalline specific growth rate descends on the one hand, on the other hand, may be because of the carrying out along with fermentation, the vigor of cell descends to some extent, and the specific consumption rate of glycerine is constantly descended.In the fermentation later stage, the specific consumption rate of glycerine is low excessively, and the output of SAM also no longer rises.Three kinds of feed supplement strategies consume amounts of glycerol in the bio-transformation phase and are respectively 327g/L, 278g/L, 237g/L.Mean consumption speed was respectively 7.4g/L hour, and 5.8g/L hour, 4.9g/L hour.
The glycerine consumption of visible impulse feed supplement and wear rate maximum illustrate that the ATP amount of its conversion is maximum, for synthetic SAM furnishes ample material.
Embodiment 3 biomasss, SAM output and SAM are than the comparison of output
The biomass of different feed supplement strategies, SAM output and SAM compare output in present embodiment comparing embodiment 1 method.
In the method for embodiment 1, fermentation culture to 52 hour, i.e. the bio-transformation phase, cell density was about 140g/l when beginning.Yet the bio-transformation phase is used different feed supplement strategies, has caused the difference of final cell density, sees Fig. 2.
During the restricted stream of glycerine of control 20～25% dissolved oxygens added, it is maximum that 64 hour cell density reach, and is 158g/l, after this is fluctuation status, kept stable; The control dissolved oxygen is that cell density is up to 168g/l, and was higher slightly than the former during 5～10% the restricted stream of glycerine added; The pulse feed supplement can reach the highest cell density, is 190g/L, has increased 50g/L when beginning than the bio-transformation phase.
The above results shows that for cell provides nonrestrictive glycerine, although dissolved oxygen can be near 0, cell is still grown vigorous, has reached the density than the restricted stream of glycerine Jia Genggao.Cell is basic SAM production unit, and " producer " is many, helps output and improves.In the pulse feed supplement, the maximum concentration of SAM has reached 4.8g/L (Fig. 3), in addition two kinds of feed supplement strategies only are 2.04g/L (the control dissolved oxygen is that 20～25% the restricted stream of glycerine adds) and 3.52g/L (the control dissolved oxygen is that 5～10% the restricted stream of glycerine adds), and the visible impulse feed supplement is compared other the SAM output of two kinds of methods and improved 135% and 36% respectively.Yet the difference of SAM output is not main because the difference of cell density.This from SAM than output difference (Fig. 4) as can be seen, use the also very bigger of different tactful SAM than output difference, the SAM of pulse feed supplement is higher than output.
Table 1 has provided the fermentation result who uses three kinds of different feed supplement strategies respectively.The utilization of pulse feed supplement makes SAM output reach 4.8g/L, has improved 36% and 135% respectively than other two kinds of strategies; Reached 0.025g/gDCW than output, improved 19% and 79% respectively than other two kinds of strategies; Productive rate reaches 0.052g/L hour, improves 41% and 148% respectively.The transformation efficiency of methionine(Met) also improves a lot, and reaches 9.0% in the pulse feed supplement, and two kinds of strategies only are 6.6% and 3.8% in addition.SAM is to the yield Y of glycerine in the pulse feed supplement
SAM/Gly=0.0080, improved 29% and 100% respectively.
The fermentation result of the different feed supplement strategies of table 1 utilization relatively
| The feed supplement strategy | SAM | The transformation efficiency of L-methionine(Met) (%) | Glycerine wastage in bulk or weight (g) | SAM is to the yield Y of glycerine SAM/Gly | ||
| Output (g/L) | Than output (g/gDCW) | Productive rate (g/L hour) | ||||
| #1 | 2.04 | 0.014 | 0.021 | 3.8 | 505 | 0.0040 |
| #2 | 3.52 | 0.021 | 0.037 | 6.6 | 570 | 0.0062 |
| #3 | 4.80 | 0.025 | 0.052 | 9.0 | 603 | 0.0080 |
#1 represents: the restricted stream of glycerine of control 20～25% dissolved oxygens adds;
#2 represents: the control dissolved oxygen is that 5～10% the restricted stream of glycerine adds;
#3 represents: pulsed glycerine is non-limiting to be added.
Embodiment 4 SAM synthetic enzyme vigor and the comparison of living than enzyme
The SAM synthetic enzyme can catalysis ATP and the synthetic SAM of L-methionine(Met), and its enzyme work is directly connected to SAM synthetic speed.The SAM synthetic enzyme vigor of different feed supplement strategies and more alive in present embodiment comparing embodiment 1 method than enzyme.
Measure the enzyme of three kinds of feed supplement mode SAM synthetic enzyme respectively and lived, the results are shown in Figure 5.The live common trend that changes of enzyme is to raise gradually along with the carrying out of fermentation earlier in three kinds of feed supplement strategies, is fluctuation status after reaching certain value, and the fermentation later stage slightly descends.But enzyme size differences alive is very big in the different feed supplement strategies.In the pulse feed supplement, enzyme work is up to 8.4U/ml, and the control dissolved oxygen is that enzyme work was up to 6.9U/ml during 5～10% the restricted stream of glycerine added, and the restricted stream of glycerine of control dissolved oxygen 20～25% add in enzyme work only up to 4.9U/ml.
Similar than the variation tendency that enzyme is alive and enzyme is alive, the results are shown in Figure 6.In the pulse feed supplement, be up to 48U/gDCW, and in addition two kinds of tactful best results Wei 42U/g and 33U/g than enzyme work.SAM synthetic enzyme in the pulse feed supplement is alive higher than enzyme, and pointing out its reason is that the synthetic of enzyme needs a large amount of materials of consumption and energy, and the glycerine specific consumption rate is faster in the pulse feed supplement, for cell provides more competent energy and material source.High enzyme compares work also provides guarantee for the synthesis rate of SAM.
The Changing Pattern of ATP content in the embodiment 5 bio-transformation phase born of the same parents
ATP and L-methionine(Met) are two substrates of SAM building-up reactions.The synthesis rate of SAM not only depends on the vigor of SAM synthetic enzyme, and concentration of substrate is also most important.Because the L-methionine(Met) that external source is added is excessive, so ATP is a SAM synthetic restrictive factor.The Changing Pattern of ATP content in the born of the same parents of different feed supplement strategies in present embodiment comparing embodiment 1 method.
For this reason, the inventor has measured bio-transformation phase content of ATP in the born of the same parents when using different feed supplement strategy respectively, the results are shown in Figure 7.When the bio-transformation phase began in 52 hours, ATP content was suitable, is about 8.5mmol/gDCW.But owing to adopted different feed supplement strategies subsequently, ATP content shows very big difference.In the pulse feed supplement, ATP content is up to 12.8mmol/gDCW, though obviously descend after 80 hours, content is all the time on other two kinds of strategies.The synthetic main of ATP not only consumes a large amount of oxygen by oxidative phosphorylation, also needs a large amount of carbon sources.Though pulse feed supplement dissolved oxygen is 0, its glycerine specific consumption rate is more faster than other two kinds of feed supplement strategies, so ATP content is also higher.
It is pointed out that because oxidative phosphorylation needs a large amount of oxygen, and the accumulation to SAM as single factors of high dissolved oxygen is favourable, the restricted flow feeding of glycerine of control 20～25% dissolved oxygens also just is being based on this point.But high dissolved oxygen is a cost to sacrifice the carbon source feed rate.Fact proved that the pulse feed supplement of suitably having sacrificed dissolved oxygen has obtained more carbon sources, the ATP content of unit thalline and enzyme are lived all much higher, thereby more help the SAM accumulation.
Add ethanol in the embodiment 6 pulse feed supplement processes
In the present embodiment, strategy (c) (the glycerine pulsed is non-limiting to be added) is identical among basic fermentation process and the embodiment 1, just bio-transformation interim simultaneously also stream add ethanol, make the alcohol concn in the fermented liquid maintain 2～2.5%, until fermentation ends.Measure the output of SAM.
Found that keeping alcohol concn in fermented liquid is 2～2.5%, can further improve the output of SAM, output reaches 5.2g/L, and the amplitude of raising is 8.3%.
In addition, the inventor has also adopted the recombination yeast that derives from Hansenula genus or Candida genus to ferment.The method of fermentation is substantially with described in the embodiment 1.Found that, in these bacterial strains, adopt the non-limiting strategy of adding of glycerine pulsed to improve a lot than the SAM output that adopts the restricted stream of glycerine of controlling 20～30% dissolved oxygens to add strategy.And, add ethanol and can make output higher.
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. method of producing S-adenosylmethionine comprises step: the fermentation culture S-adenosylmethionine is produced bacterium in fermented liquid, thereby produces S-adenosylmethionine,
It is characterized in that, in culturing process, adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, when treating that carbon source concentration is lower than 2g/L in the fermented liquid, adding carbon source to final concentration once more is 10-160g/L.
2. the method for claim 1 is characterized in that, in the described fermented liquid, contains the L-methionine(Met).
3. the method for claim 1 is characterized in that, it is that the yeast that derives from the subordinate: Hansenula belongs to that described S-adenosylmethionine is produced bacterium, and Candida belongs to, and Torulopsis belongs to, or Pichia belongs to.
4. the method for claim 1 is characterized in that, it is to derive from the yeast that Pichia belongs to that described S-adenosylmethionine is produced bacterium.
5. the method for claim 1 is characterized in that, described S-adenosylmethionine is produced bacterium and carried the S-adenosylmethionine synthetase-coding gene, and with Glycerose 3-phosphate dehydrogenase as promoter expression S-adenosylmethionine synthetic enzyme.
6. the method for claim 1 is characterized in that, described carbon source is selected from: glycerine, glucose, sorbyl alcohol, peptone, yeast extract, soybean cake powder or its combination.
7. the method for claim 1 is characterized in that, comprises that also stream adds ethanol in fermented liquid, and making the alcohol concn in the fermented liquid is 0.2-6% according to volume ratio.
8. method of producing S-adenosylmethionine comprises step:
(1) in fermented liquid, cultivates the S-adenosylmethionine that contains the S-adenosylmethionine synthetase-coding gene and produce bacterium, make it to express the S-adenosylmethionine synthetic enzyme;
(2) in the S-adenosylmethionine of (1) is produced the fermented liquid of bacterium, add the L-methionine(Met), make S-adenosylmethionine produce ATP and the L-methionine(Met) that the bacterium metabolism produces and under the catalysis of S-adenosylmethionine synthetic enzyme, react, the generation S-adenosylmethionine and
(3) separate S-adenosylmethionine;
It is characterized in that,
In step (2), adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, and when treating that carbon source concentration is lower than 2g/L in the fermented liquid, adding carbon source to final concentration again is 10-160g/L.
9. the method for a fermentation culture S-adenosylmethionine production bacterium is characterized in that, the described S-adenosylmethionine of fermentation culture is produced bacterium in fermented liquid,
Wherein,
In described fermentation culture process, adopt pulse feed supplement mode in fermented liquid, to add carbon source, described pulse feed supplement mode is: disposable interpolation carbon source to final concentration is 10-160g/L in fermented liquid, when treating that carbon source concentration is lower than 2g/L in the fermented liquid, adding carbon source to final concentration once more is 10-160g/L.
10. method of producing S-adenosylmethionine, comprise step: the fermentation culture S-adenosylmethionine is produced bacterium in fermented liquid, thereby produces S-adenosylmethionine, it is characterized in that, in described culturing process, the control dissolved oxygen intermittently fluctuates between 0%-100%.
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| CN102559726A (en) * | 2010-12-31 | 2012-07-11 | 华东理工大学 | Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine |
| CN114717282A (en) * | 2021-12-31 | 2022-07-08 | 北京盛拓达生物技术有限公司 | Composition and application thereof |
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| CN104928342A (en) * | 2015-03-31 | 2015-09-23 | 浙江大学宁波理工学院 | Method for preparing S-ademetionine and D-methionine |
| CN104878059B (en) * | 2015-03-31 | 2018-06-26 | 浙江大学宁波理工学院 | A kind of method for preparing s-adenosylmethionine |
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| CN102559726A (en) * | 2010-12-31 | 2012-07-11 | 华东理工大学 | Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine |
| CN102559726B (en) * | 2010-12-31 | 2013-10-09 | 华东理工大学 | Application of GAP (GTPase-Activating Protein) promoter library in regulation of metabolic pathway of S-adenosylmethionine |
| CN114717282A (en) * | 2021-12-31 | 2022-07-08 | 北京盛拓达生物技术有限公司 | Composition and application thereof |
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