CN100365129C - Improvement of preparation of sulfuradenosin methionine - Google Patents
Improvement of preparation of sulfuradenosin methionine Download PDFInfo
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- CN100365129C CN100365129C CNB2005100251833A CN200510025183A CN100365129C CN 100365129 C CN100365129 C CN 100365129C CN B2005100251833 A CNB2005100251833 A CN B2005100251833A CN 200510025183 A CN200510025183 A CN 200510025183A CN 100365129 C CN100365129 C CN 100365129C
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- methyl alcohol
- sam
- methanol
- recombination yeast
- methionine
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Abstract
The present invention relates to a method for preparing sulfur adenosine methionine by adopting a biological transformation method. SAM synzyme is mainly obtained by the fermentation of methanol nutritional type recombination yeast (or is named addiction methanol recombination yeast), and adenosine triphosphate and L-methionine react to generate SAM under the existence of the SAM synzyme. The present invention is characterized in that An alternate flow addition mode is adopted by methanol and a non-methanol carbon source in the inducing expression stage of the methanol nutritional type recombination yeast. The flow addition speed of the methanol is from 0.033 to 0.67 g/L. min, the duration time of each time is from 8 to 15h, and duration times are totally 3 to 7 times. The flow addition speed of the non-methanol carbon source is from 0.013 to 0.47 g/L. min, the duration inducing time of each time is from 4 to 8h, and duration inducing times are totally 3 to 7 times. The present invention enhances the yield (which is approximately enhanced by 30%) of SAM by changing the supply and addition mode of a mixed carbon source in the inducing expression stage of the methanol nutritional type recombination yeast. The present invention settles a base for the industrial product of SAM.
Description
Technical field
The present invention relates to a kind of preparation method of sulfuradenosin methionine, particularly a kind of method that adopts biotransformation method to prepare sulfuradenosin methionine.
Background technology
(S-adenosylmethionine is that (methionine, activity form Met) participate in multiple biochemical reaction in the organism to methionine(Met), are a kind of important intermediate metabolitess, also are important many curative effects medicines SAM) to sulfuradenosin methionine.Biologically, SAM is the donor of 1 carbon, 3 carbon and 5 carbon-based groups, have transmethylase, change multiple physiological actions such as aminopropyl and commentaries on classics sulphur, methylating of SAM wide participation body, and in close relations with gene regulating, hormone activity, nerve conduction growth and detoxifcation at the physical function of interior numerous keys.In addition, SAM also can be used as antioxidant, and as raw material synthesizing biotinylated, spermidine and spermine etc.
The production method of SAM mainly contains chemical synthesis, enzymatic reaction method and biotransformation method.Chemical synthesis is because the substrate homocysteine is somewhat expensive, and productive rate is also relatively low, so that this method is used is less; The enzymatic reaction method is to utilize the enzymatic reaction of SAM synthetic enzyme, under this enzyme catalysis, generates SAM by Triphosaden (ATP) and the reaction of L-methionine(Met), owing to be external enzymatic reaction, therefore need to add external source ATP, production cost height, product is easily degraded, and the extraction purification ratio of enzyme is difficult; And biotransformation method is directly to transform in cell paste, synthetic enzyme need not purified, the L-methionine(Met) adds by external source, ATP can be provided by cell self metabolism, and the metabolic regulation of industrial microorganism and amplifying technique comparative maturity, therefore being a kind of high efficiency method that biotransformation method is produced SAM, also is at present the main method of producing SAM.
In the existing method of utilizing methyl alcohol nutritional type recombination yeast (or claim have a liking for methyl alcohol recombination yeast) fermentative preparation sulfuradenosin methionine (SAM), adopt fed-batch mode simultaneously when adding methyl alcohol and non-methyl alcohol carbon source in the abduction delivering stage.Practice shows: this additional way has influenced the productive rate of SAM.Therefore remain room for improvement in the prior art.
Summary of the invention
The preparation method of the said sulfuradenosin methionine of the present invention (SAM), the SAM synthetic enzyme that it is mainly obtained by methyl alcohol nutritional type recombination yeast (or claiming to have a liking for the methyl alcohol recombination yeast) fermentation, and in the presence of this synthetic enzyme, generate SAM by Triphosaden (ATP) and the reaction of L-methionine(Met), it is characterized in that, in the methyl alcohol nutritional type recombination yeast abduction delivering stage, methyl alcohol and non-methyl alcohol carbon source adopt the alternating current add mode, methyl alcohol stream rate of acceleration is 0.033～0.67g/L.min, each time length is 8～15h, amount to 3～7 times, non-methyl alcohol carbon source stream rate of acceleration is 0.013～0.47g/L.min, and each time length is 4～8h, amounts to 3～7 times.
Wherein: said non-methyl alcohol carbon source is the existing carbon source except that methyl alcohol that is used to have a liking for methyl alcohol recombination yeast fermentation inducement expression phase, as (but being not limited to): glycerine, glucose, peptone, each peptide species, amino acid, soybean cake powder and/or aliphatics organic acid, the stream rate of acceleration is meant: the weight that adds carbon source in the unit time to the fermented liquid institute stream of unit volume.
The bacterial strain that the present invention is suitable for is for adopting alcohol oxidase promotor P
AOX1, P
AOX2, transform the gene engineering yeast of SAM by expressing SAM synthetic enzyme 2, as pichia spp (Pichia), debaryomyces hansenii (Hansenula), candiyeast (Candida) or torulopsis (Torulopsis) etc., this recombination yeast can adopt different expression vector (as pPIC9K etc.) when making up, and carrier can have different existing waies (as additive type, integrated etc.) in yeast, and this bacterium can possess different phenotype (Mut
+, Mut
sDeng).
In addition, for the better productive rate that improves SAM, add Sodium Glutamate in the methyl alcohol nutritional type recombination yeast abduction delivering stage, its add-on is 3～10g in every liter of fermented liquid.
The present invention has improved the productive rate (improving about 30%) of SAM by changing the mode that methyl alcohol nutritional type recombination yeast abduction delivering stage mixed carbon source is added, and SAM lays a good foundation for suitability for industrialized production.
Description of drawings
Fig. 1 is the comparison curves of SAM productive rate under different mixed carbon source feed supplement modes.
Among the figure: ■---abduction delivering stage employing alternating current adds the SAM productive rate curve of carbon source mode,
▲---the abduction delivering stage adopts stream simultaneously to add the SAM productive rate curve of carbon source mode.
Embodiment
Below by embodiment the present invention is further illustrated, its purpose only is better to understand content of the present invention, and unrestricted protection scope of the present invention:
Test materials and method brief description
1 bacterial strain: the culture presevation that this experiment is used number is CGMCC, and NO.0648 is provided by Chinese Academy of Sciences's Shanghai school of life and health sciences biological chemistry and RESEARCH ON CELL-BIOLOGY, and its building process is referring to CN1357630A.
2 bio-reactors: the FUS-15L fermentor tank that the fermentor tank that this experiment is used is produced as Guoqiang Biochemical Engineering Equipment Co., Ltd., Shanghai.
3 substratum:
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, and 121 ℃, sterilization 30min.
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, and 121 ℃, sterilization 30min.
Embodiment 1
Picking list bacterium colony on the seed culture flat board inserts seed culture medium, cultivates OD 1 day
600Reach at 24～30 o'clock, insert fermentation tank culture medium, inoculum size 5% (v/v).The parameter of fermentor tank is set to: pH maintains 6.0, and temperature is 30 ℃, and air flow is 7L/min (VVM:0.9), and tank pressure is 0.03MP, and the ammoniacal liquor additional amount is according to the pH feedback regulation, initial mixing speed 300rpm, and initial dissolved oxygen is 100%.Batch fermentation and glycerine stream add the regulation and control strategy of fermentation stage with Pichia protocols or other existing control method.
Glycerine stream stops to add glycerine 1h after adding the fermentation stage end, and carbon source has been consumed, dissolved oxygen rises to 70-80%, enter inductive phase this moment, and this stage oxygen dissolving is controlled at 25%, and methyl alcohol stream rate of acceleration is by the dissolved oxygen feedback control, mean rate is 0.25g/min, after inducing 8h, stop to add methyl alcohol, treat dissolved oxygen bounce-back after, with given pace stream glycerol adding, the stream rate of acceleration is by the dissolved oxygen feedback control, and the average flow rate of acceleration is 1.5g/min, behind the cultivation 4h, stop to add glycerine, after treating dissolved oxygen bounce-back half an hour, add methyl alcohol again, cultivate 8h after, change again and make glycerine cultivation 4h, so alternately to fermentation ends.Wherein methyl alcohol is successively added 6 times, and each 8h amounts to 48h, and glycerine is successively added 6 times, and each 4h amounts to 24h.
In addition, be mixed carbon source still with methyl alcohol and glycerine, guaranteeing under the excessive prerequisite of no glycerine, adopt stream simultaneously to add the strategy of two kinds of carbon sources at induction period.
Repeat above two kinds of strategies each three times, and dual mode is compared, the result shows: when induction period is added mixed carbon source, repeatedly alternately additional way is better, can significantly improve the productive rate of SAM, than while feed supplement mode, the SAM productive rate improves 22%, reaches 18.1g/l, the results are shown in Figure 1.
The HPLC measuring method of SAM productive rate:
In the pichia spp fermenting process, synthetic SAM major part is stayed in the born of the same parents in the born of the same parents, but still has a small amount of SAM can be secreted in the cell free fermentation liquid, particularly along with the cracking of cell, SAM can progressively be discharged into outside the born of the same parents, therefore must carry out extracting simultaneously to the SAM inside and outside the born of the same parents.
Get the 2ml fermented liquid, the trichoroacetic acid(TCA) with 25% is settled to 10ml, and 4 ℃, extracting 3h, 12000rpm, centrifugal 3min, supernatant liquor cross 20 μ m millipore filtrations, and the filtrate cryopreservation is standby.
The present invention has carried out HPLC mensuration to the SAM in the fermented liquid, has obtained SAM productive rate (the SAM productive rate behind the non-separation purifying).Ion pair liquid-phase chromatography method: ODS C18 chromatographic column, moving phase: a sodium laurylsulfonate, b methyl alcohol gradient elution.Flow velocity 1ml/min, gradient elution: 0min, a 100%; 2min, a 100%; 4min, a 30%, and b 70%; 10min, a 20%, and b 80%; 15min, b 100%. detects wavelength 254nm, 25 ℃ of column temperatures, linearity range is 1～5g/l, retention time 10.5～11min.
Embodiment 2
Induction period methyl alcohol peptone replaces feed supplement and transforms production SAM:
Inoculation, batch fermentation and glycerine stream add the fermentation stage operation with embodiment 1, after entering inductive phase, dissolved oxygen is controlled at 5%, methyl alcohol stream rate of acceleration is by the dissolved oxygen feedback control, and mean rate is 0.5g/min, induce 15h after, stop to add methyl alcohol, after treating the dissolved oxygen bounce-back, add peptone with given pace stream, the stream rate of acceleration is by the dissolved oxygen feedback control, the average flow rate of acceleration is 0.1g/min, after cultivating 8h, stop to add peptone, treat dissolved oxygen bounce-back half an hour after, add methyl alcohol again, after cultivating 15h, change again and make peptone cultivation 8h, so alternately to fermentation ends.Wherein methyl alcohol is successively added 3 times, and each 15h amounts to 45h, and peptone is successively added 3 times, and each 8h amounts to 24h.Than while feed supplement mode, the SAM productive rate improves 14%, reaches 16.9g/l.
Embodiment 3
Adopt above-mentioned induction period mixed carbon source to replace the feed supplement mode, the productive rate of SAM is improved, transform the effective transformation efficiency (being converted into the ratio of methionine(Met) in the methionine(Met) that consumes of SAM) that generates SAM for further improving substrate L-methionine(Met), under the condition of embodiment 1 or embodiment 2, add Sodium Glutamate 24g at induction period, effective transformation efficiency of substrate (L-methionine(Met)) brings up to 42% by 30%.
Claims (4)
1. method that adopts biotransformation method to prepare sulfuradenosin methionine, the SAM synthetic enzyme that it is mainly obtained by the fermentation of methyl alcohol nutritional type recombination yeast, and in the presence of this synthetic enzyme, generate sulfuradenosin methionine by Triphosaden and the reaction of L-methionine(Met), it is characterized in that, in the methyl alcohol nutritional type recombination yeast abduction delivering stage, methyl alcohol and glycerine, glucose, peptone, each peptide species, amino acid, soybean cake powder and/or aliphatics organic acid carbon source adopt the alternating current add mode, methyl alcohol stream rate of acceleration is 0.033～0.67g/L.min, each time length is 8～15h, amounts to 3～7 times; Glycerine, glucose, peptone, each peptide species, amino acid, soybean cake powder and/or aliphatics organic acid carbon source stream rate of acceleration are 0.013～0.47g/L.min, and each time length is 4～8h, amounts to 3～7 times.
2. preparation method as claimed in claim 1, it is characterized in that wherein said methyl alcohol nutritional type recombination yeast is recombinant yeast pichia pastoris (Pichia), reorganization debaryomyces hansenii (Hansenula), reorganization candiyeast (Candida) or reorganization torulopsis (Torulopsis).
3. preparation method as claimed in claim 2 is characterized in that, wherein said methyl alcohol nutritional type recombination yeast is recombinant yeast pichia pastoris (Pichia).
4. as described any one preparation method of claim 1～3, it is characterized in that add Sodium Glutamate in the methyl alcohol nutritional type recombination yeast abduction delivering stage, its add-on is 3～10g in every liter of fermented liquid.
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| CN101285085B (en) * | 2008-01-22 | 2011-04-27 | 西北工业大学 | Process for synthesizing adenosine methilanin by intact cell catalysis |
| CN101230373B (en) * | 2008-02-27 | 2011-11-30 | 南京工业大学 | Method for preparing S-adenomethionine |
| CN109402039B (en) * | 2018-10-16 | 2020-12-29 | 江南大学 | Reinforced MutSMethod for expressing heterologous protein by pichia pastoris |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1357630A (en) * | 2001-11-30 | 2002-07-10 | 中国科学院上海生物化学研究所 | Method of producing adenosylmethionine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1357630A (en) * | 2001-11-30 | 2002-07-10 | 中国科学院上海生物化学研究所 | Method of producing adenosylmethionine |
Non-Patent Citations (1)
| Title |
|---|
| S-腺苷甲硫氨酸研究进展. 余志良，杨晟，蔡谨，袁中一.中国医药工业杂志，第34卷第1期. 2003 * |
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