CN101353682A - Method for promoting yield of adenosylmethionine by microbe whole-cell catalytic synthesis - Google Patents
Method for promoting yield of adenosylmethionine by microbe whole-cell catalytic synthesis Download PDFInfo
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Abstract
A method for synthesizing adenosylmethionine by promoting microorganism whole cell catalysis relates to the technical field of synthesizing high-value compounds by utilizing the catalysis of microorganism whole cell. The technical characteristics of the invention lie in that: recombinant coli cells which can express adenosylmethionine synthetases are taken as an enzyme source to increase the permeability of epicyte by adding organic solvent or surfactant with low concentration into a reaction system for synthesizing an SAM by catalysis; and the SAM is synthesized by catalysis with the existence of reaction substrates, namely, triphosadenine and L-methionine, the synthetic amount of the SAM is up to 13.4g/L in reaction of 8 hours, the substrate conversion rate exceeds 95 percent. The method of the invention simplifies the complicated procedures of carrying out permeability treatment to microorganism cells when the synthesis of SAM by catalysis is carried out by utilizing whole cells, thus avoiding the damage of high-concentration penetrating agent to epicyte which results in the enzyme source loss in cells and cell lysis, and finally improving the yield of adenosylmethionine.
Description
Technical field
The present invention relates to a kind of method that promotes yield of adenosylmethionine by microbe whole-cell catalytic synthesis, by in reaction system, directly adding the organic solvent or the tensio-active agent of lower concentration, to increase the permeability of cytolemma, promote yield of adenosylmethionine by microbe whole-cell catalytic synthesis, thereby improve the output of adenosylmethionine, relate to the technical field of utilizing the synthetic high value compound of microbe whole-cell catalysis.
Background technology
S-adenosine-L-methionine(Met) (S-adenosyl-L-methionine.SAM, SAMe or AdoMet), be extensively to be present in the important physiologically active substance that participates in many crucial biochemical reactions in the cell, closely related with many metabolic processes in the human body, main participation hormone in vivo, neurotransmitter, nucleic acid, protein and the biosynthesizing of phosphatide and the formation of metabolism and antioxidant gsh, in close relations with various detoxification processes in the body, be to safeguard cytolemma normal function, human homergy and healthy indispensable important living matter.Evident in efficacy to numerous diseases such as dysthymia disorders, sacroiliitis, fiber headaches, can obviously eliminate liver injury, help liver problem sufferer's rehabilitation.1999, drugs approved by FDA SAM went on the market as healthcare products, had become one of best-selling nutritious prod in the U.S..In recent years, abroad turned to and utilized SAM that hepatic diseases and patients with depression are treated, become the ideal medicament of treatment hepatopathy and dysthymia disorders, its market demand constantly increases.China began import in 2000, had every year 100000000 polybasic to sell, and market outlook are good.The SAM of present domestic use mainly is the product of German Jino (Knol1) and two corporations with foreign capital of Italian RADIUM FARMAS.R.L. (IT), and it costs an arm and a leg, and can not popularize use.China is populous, and a considerable amount of hepatopathys and arthritic are arranged, and along with urban life rhythm is accelerated and the operating pressure increasing, the patients with depression showed increased.Characteristics such as it is little that SAM has side effect simultaneously, evident in efficacy will have great application prospect in China.
SAM is the both hands materials, and two kinds of isomer are arranged: (+) SAM and (-) SAM, have only (-) SAM biologically active in vivo.Present preparation method mainly contains three kinds of chemical synthesis, fermentation method and Enzymatic transformation methods.Chemical method now substantially need not owing to have that productive rate is low, substrate is expensive and problem such as environmental pollution; Fermentation method is the domestic and international main path of suitability for industrialized production SAM in recent years, but the shortcoming of fermentation method is that the end product accumulation volume is low, feed stock conversion is low, the production cycle is long and purifying process complexity etc., thereby caused it to hold at high price, limited widespread production and the application of SAM; The Enzymatic transformation method is compared with fermentation method with chemical synthesis, have substrate conversion efficiency height, product accumulation volume height, separate purify easily, reaction time is short and advantages of environment protection, thereby is comparatively effective industrialized preparing process.Yet SAM synthetic enzyme content in animal, plant and microorganism is few, enzyme is lived low and separation and purification difficulty, therefore produces the restriction that SAM is subjected to SAM synthetic enzyme vigor by the Enzymatic transformation method.Engineered development makes a large amount of highly active SAM synthetic enzyme of acquisition become possibility, increased enterprise's production cost undoubtedly greatly but on industrially scalable, extract a large amount of SAM synthetic enzyme and enzyme is carried out immobilization, and the activity of SAM synthetic enzyme enzyme in purifying and immobilization process can be suffered certain loss.
In application number was 200810017357.5 patent documentation, the method for the synthetic SAM of a kind of recombinant microorganism whole-cell catalytic was disclosed.Utilize the synthetic SAM of whole-cell catalytic can reduce enterprise's production cost, improve the stability and the utilization ratio of enzyme, obtain highly purified product.Along with improving constantly of reorganization SAM synthetase expression level, the method that adopts technology such as genetically engineered and immobilized cell to combine realizes that the synthetic SAM of extensive, low-cost whole-cell catalytic can have very big industrial prospect.Carry out with using resolvase or immobilized enzyme that SAM is synthetic to be compared, in whole-cell biological catalysis, intracellular enzyme source is more stable.But because the permeability barrier of microorganism cells film, make reaction substrate and product be difficult to freely enter cell, the efficient of biocatalysis is starkly lower than pure enzymic catalytic reaction system, thereby how to improve the permeability of cytolemma as much as possible and don't influence the integrity of cytolemma and the activity of desmo enzyme just seems most important.The treatment process that improves the cell permeability at present mainly comprises physical method and chemical processes such as organic solvent and tensio-active agent such as ultrasonic wave, freeze thawing, these methods mainly are microorganism cells to be carried out the permeability pre-treatment of cytolemma, centrifugal-permeability processing-centrifugal-operations such as washing that treatment process is generally, treating processes is comparatively loaded down with trivial details.When microorganism cells being carried out the permeability processing, penetrating dose consumption is difficult to hold simultaneously, if penetrating dose consumption is less, the permeability of cytolemma is lower; If penetrating dose large usage quantity the excessive damage of cytolemma and the phenomenons such as cracking of cell occur through regular meeting, cause losing of desmo enzyme, reduced the efficient of microorganism cells biocatalysis, the enzymic activity transformation period of cell catalysis reaction reduces.
Summary of the invention
In the microbe whole-cell catalytic reaction process, the damage that when penetrating dose of pair cell of high density carries out penetrating the processing, causes, the present invention proposes a kind of method that promotes yield of adenosylmethionine by microbe whole-cell catalytic synthesis, by in the reaction system of the synthetic SAM of microbe whole-cell catalysis, directly adding penetrating dose of lower concentration, damage bigger problem to solve the penetrating dose of pair cell of high density that exists in the synthetic SAM process of microbe whole-cell catalysis.
Technical scheme
A kind of method that promotes yield of adenosylmethionine by microbe whole-cell catalytic synthesis is characterized in that step is as follows:
Step 1, fermentation culture can be expressed the microorganism cells of adenosine methilanin synthase, and the temperature maintenance of nutrient solution is at 15～38 ℃ in the fermenting process, and fermented liquid is collected microorganism cells 1 time with distilled water wash behind the centrifugal 10min of 6000rpm;
Step 2, with the microorganism cells of centrifugal collection as the enzyme source, in the reaction solution of catalysis synthesizing adenosine methilanin, add penetrating dose simultaneously, with Triphosaden and L-methionine(Met) is the catalytic material synthesizing adenosine methilanin, the temperature maintenance of reaction solution is at 35 ℃, 120rpm reacts 8h, and cell concentration is 150g wet thallus/L in the reaction system; The described penetrating dose of volume percentage final concentration in the synthesizing adenosine methilanin reaction solution is 0.25%～2.5%; Described reaction solution is: the Tris-HCl damping fluid 100mmol.L of pH 7.0
-1, Triphosaden 10～35mmol.L
-1, L-methionine(Met) 10～35mmol.L
-1, Repone K 100mmol.L
-1, sal epsom 20～70mmol.L
-1With p-methyl benzenesulfonic acid sodium 400-800mmol.L
-1, all transfer to pH 7.0 after the dissolving.
Described penetrating dose is organic solvent or tensio-active agent; Described organic solvent is toluene or dimethylbenzene, and described tensio-active agent is a cetyl trimethylammonium bromide.
The volume percentage final concentration of described toluene in the adenosylmethionine synthesis reaction solution is 0.5%.
The volume percentage final concentration of described dimethylbenzene in the adenosylmethionine synthesis reaction solution is 1%.
The volume percentage final concentration of described cetyl trimethylammonium bromide in the adenosylmethionine synthesis reaction solution is 0.5%.
Described microorganism is to include the recombinant Bacillus coli cells that derives from intestinal bacteria Escherichia coli adenosine methionine synthetase gene.
Beneficial effect
The present invention proposes a kind of method that promotes yield of adenosylmethionine by microbe whole-cell catalytic synthesis, beneficial effect can be summarized as follows: in using the synthetic SAM process of microbe whole-cell catalysis, the method that the microorganism cells film is carried out permeability mainly comprises cell with the organic solvent of higher concentration or the tensio-active agent place is existing, centrifugal and complicated procedures such as washing.The present invention has simplified the existing program in penetratingization of microorganism cells film place in the preparation SAM process, and just directly adds the organic solvent or the tensio-active agent of lower concentration in the reaction system of synthetic SAM, to improve the permeability of cell.No longer carry out loaded down with trivial details centrifugal, washing supervisor, and avoided the damage of penetrating dose of pair cell of high density even the cracking of cell, cause the loss of SAM synthetic enzyme in the cell, the output of final SAM reaches 13.4g/L.
Embodiment
Now in conjunction with the embodiments the present invention is further described:
Test materials and method:
1. cell and reagent
E. coli k12, e. coli jm109, colibacillus expression plasmid pBR322 are this chamber and preserve, and also can buy to obtain.
Colibacillus engineering carries the wild e. coli k12 adenosine methionine synthetase gene of coding.Use PCR method to obtain adenosine methionine synthetase gene for touching the plate amplification with the e. coli k12 genomic dna, this gene is connected among the expression vector pBR322 and is transformed in the e. coli jm109 express, the expression of adenosine methionine synthetase gene is subjected to the regulation and control of himself promotor.
Used T
4Dna ligase, Taq archaeal dna polymerase and restriction enzyme are all available from Takara company.Plasmid DNA extraction agent box, genome DNA extracting reagent kit, DNA glue reclaim test kit available from the vast Imtech in Beijing.Other all reagent are homemade or the import analytical reagent.
2. substratum
Liquid LB/Tet substratum: (10g/L peptone; The 5g/L yeast powder; 10g/L NaCl; 15 μ g/ml tetracycline hydrochlorides).Peptone, yeast powder and NaCl are regulated pH=7.0 after with deionized water dissolving, 121 ℃ of sterilizations 20 minutes, add tetracycline hydrochloride then when inoculation, making its final concentration is 15 μ g/ml.
Solid LB/Tet substratum: the agar of adding 1.5% in liquid LB/Tet substratum, promptly make the solid Agar Plating.
3. conventional molecular biology operation
Technology such as the extraction of the extraction of bacillus coli gene group DNA, genes of brewing yeast group DNA, the pcr amplification of SAM synthase gene, genetic expression are carried out with reference to fine works molecular biology experiment guide (.1995 such as Ao Sibai).
The present invention illustrates by following embodiment step:
The fermentation of step 1, expression adenosine methilanin synthase recombination bacillus coli:
Picking mono-clonal on solid LB/Tet flat board inserts in the 10ml liquid LB/Tet substratum then, cultivates 16 hours (220 rev/mins) for 37 ℃.Above-mentioned bacterium liquid is transferred in the 2L liquid LB/Tet substratum, cultivate 16 hours (280 rev/mins) for 30 ℃, make thalli growth reach stationary phase, the activity of expression of recombinant e. coli SAM synthetic enzyme is active more than 150 times of an e. coli k12 adenosine methilanin synthase after measured.Bacterium liquid after cultivating is carried out centrifugal, rotating speed is 6000rpm, centrifugal 10 minutes, and abandoning supernatant, the thalline of collecting precipitation is used for follow-up test.
This recombination bacillus coli also can carry out the culture expression adenosine methilanin synthase under 15-38 ℃ condition.When cultivating under 15 ℃ of conditions, cell grows into and need surpass 30 hours stationary phase, and the expression amount of adenosine methilanin synthase is lower, and enzymic activity is wild colibacillary about 8 times; When cultivating under 38 ℃ of conditions, cell grows into only to be needed about 8 hours stationary phase, but the inclusion body that the recombinant adenosine methionine synthetase of expressing forms is more, and enzymic activity is wild colibacillary about 58 times; When under 30 ℃ of conditions, cultivating, reach stationary phase through about 16 hours cells with regard to growing, and the adenosine methilanin synthase activity of expressing is the highest, is active more than 150 times of e. coli k12 adenosine methilanin synthase after measured.So the expression of recombinant e. coli adenosine methilanin synthase is best under 30 ℃ of conditions.
Step 2, the synthetic SAM of penetrating dose of pretreated recombination bacillus coli whole-cell catalytic of process:
Adopt toluene, dimethylbenzene or the CTAB of different concns to carry out penetratingization processing to recombination bacillus coli, treatment process is as follows: the Escherichia coli bacteria liquid of cultivating in the step 1 is carried out centrifugal, rotating speed is 6000rpm, centrifugal 10 minutes, abandoning supernatant is collected bacterial sediment.The thalline of collecting is suspended in the deionized water, and then carries out penetratingization processing.
In another preference, penetratingization reagent treatment adopts toluene: the thalline of collecting is suspended in the deionized water, the adding final concentration is that the toluene of 1-5% is handled 1h at 35 ℃, with centrifugal 10 minutes of reaction solution 6000rpm, the thalline of collecting through penetratingization processing was used for the synthetic SAM of follow-up whole-cell catalytic then.Also the CATB pair cell of the dimethylbenzene of available 1-5% or 1-3% carries out penetratingization processing.
Synthesize being formulated as follows of SAM reaction solution through penetrating dose of pretreated intestinal bacteria whole-cell catalytic: 100mMTris, 35mM ATP, 35mM L-Met, 70mM MgSO4,100mM KCl, 0.8M p-methyl benzenesulfonic acid sodium, penetrating dose of pretreated recombinant Bacillus coli cells 150g of process, add water to 1L, reacting liquid pH value is transferred to 7.0.Above-mentioned reaction solution is added in the reactor, and 35 ℃ of 120rpm reacted 8 hours.After the HPLC monitoring reaction finishes, centrifugal 15 minutes of 10000rpm, collect supernatant liquor be stored in 4 ℃ standby.Simultaneously, also can use concentration of substrate to be the synthetic SAM of the reaction system catalysis of 10mM and 20mM, the remaining reaction condition is same as above.
Experimental result shows that the output through the synthetic SAM of penetrating dose of pretreated recombinant Bacillus coli cells catalysis is 10-12g/L in the reaction system of 35mM concentration of substrate.
In another preference, adopt the CTAB that in the reaction system of the synthetic SAM of whole-cell catalytic, directly adds lower concentration to carry out penetratingization processing: the CTAB that in the reaction system of the synthetic SAM of recombination bacillus coli whole-cell catalytic, directly adds 0.25%～2.5% different concns.The reaction system of the synthetic SAM of whole-cell catalytic is as follows: 100mMTris, 35mM ATP, 35mM L-Met, 70mM MgSO4,100mM KCl, 0.8M p-methyl benzenesulfonic acid sodium, recombinant Bacillus coli cells 150g, add water to 1L, reacting liquid pH value is transferred to 7.0.Above-mentioned reaction solution is added in the reactor, and 35 ℃ of 120rpm reacted 8 hours.After the HPLC monitoring reaction finishes, centrifugal 15 minutes of 10000rpm, collect supernatant liquor be stored in 4 ℃ standby.Simultaneously, also can use concentration of substrate to be the synthetic SAM of the reaction system catalysis of 10mM and 20mM, the remaining reaction condition is same as above.
Experimental result shows that when the final concentration of CTAB in reaction solution reached 0.5%, the optimization output of SAM was 11.5g/L in the reaction system of 35mM concentration of substrate.
In another preference, adopt the dimethylbenzene that in the reaction system of the synthetic SAM of whole-cell catalytic, directly adds lower concentration to carry out penetratingization processing: the dimethylbenzene that in the reaction system of the synthetic SAM of recombination bacillus coli whole-cell catalytic, directly adds 0.25%～2.5% different concns.The reaction system of the synthetic SAM of full cell is as follows: 100mMTris, 35mM ATP, 35mM L-Met, 70mM MgSO4,100mM KCl, 0.8M p-methyl benzenesulfonic acid sodium, recombinant Bacillus coli cells 150g, add water to 1L, and reacting liquid pH value is transferred to 7.0.Above-mentioned reaction solution is added in the reactor, and 35 ℃ of 120rpm reacted 8 hours.After the HPLC monitoring reaction finishes, centrifugal 15 minutes of 10000rpm, collect supernatant liquor be stored in 4 ℃ standby.Simultaneously, also can use concentration of substrate to be the synthetic SAM of the reaction system catalysis of 10mM and 20mM, the remaining reaction condition is same as above.
Experimental result shows that when the final concentration of dimethylbenzene in reaction solution reached 1%, the optimization output of SAM was 12.7g/L in the reaction system of 35mM concentration of substrate.
In another preference, adopt the toluene that in the reaction system of the synthetic SAM of whole-cell catalytic, directly adds lower concentration to carry out penetratingization processing: the toluene that in the reaction system of the synthetic SAM of recombination bacillus coli whole-cell catalytic, directly adds 0.25%～2.5% different concns.The reaction system of the synthetic SAM of full cell is as follows: 100mM Tris, 35mMATP, 35mM L-Met, 70mM MgSO4,100mM KCl, 0.8M p-methyl benzenesulfonic acid sodium, recombinant Bacillus coli cells 150g, add water to 1L, and reacting liquid pH value is transferred to 7.0.Above-mentioned reaction solution is added in the reactor, and 35 ℃ of 120rpm reacted 8 hours.After the HPLC monitoring reaction finishes, centrifugal 15 minutes of 10000rpm, collect supernatant liquor be stored in 4 ℃ standby.Simultaneously, also can use concentration of substrate to be the synthetic SAM of the reaction system catalysis of 10mM and 20mM, the remaining reaction condition is same as above.
Experimental result shows that when the final concentration of toluene in reaction solution reached 0.5%, the optimization output of SAM was 13.4g/L in the reaction system of 35mM concentration of substrate.
As can be seen from the above embodiments, compare with the technical scheme of synthesizing SAM through penetrating dose of pretreated recombination bacillus coli catalysis, the synthetic SAM of penetrating dose of catalysis that directly adds lower concentration in reaction solution has more superiority.
In the above-described embodiments, the preference of the used reaction solution of yield of adenosylmethionine by microbe whole-cell catalytic synthesis is: Triphosaden (ATP) 10～35mmol.L
-1, L-methionine(Met) (L-Met) 10～35mmol.L
-1, Repone K (KCl) 100mmol.L
-1, sal epsom (MgSO4) 20～70mmol.L
-1, p-methyl benzenesulfonic acid sodium 400-800mmol.L
-1, the Tris-HCl damping fluid 100mmol.L of pH7.0
-1, reacting liquid pH value transfers to 7.0.
In the preference of another reaction solution, the used reaction solution of yield of adenosylmethionine by microbe whole-cell catalytic synthesis is: Triphosaden 10mmol.L
-1, L-methionine(Met) 10mmol.L
-1, Repone K 100mmol.L
-1, sal epsom 20mmol.L
-1, p-methyl benzenesulfonic acid sodium 400mmol.L
-1, the Tris-HCl damping fluid 100mmol.L of pH7.0
-1, reacting liquid pH value transfers to 7.0.
In the preference of another reaction solution, the used reaction solution of yield of adenosylmethionine by microbe whole-cell catalytic synthesis is: Triphosaden 20mmol.L
-1, L-methionine(Met) 20mmol.L
-1, Repone K 100mmol.L
-1, sal epsom 40mmol.L
-1, p-methyl benzenesulfonic acid sodium 600mmol.L
-1, the Tris-HCl damping fluid 100mmol.L of pH7.0
-1, reacting liquid pH value transfers to 7.0.
In the preference of another reaction solution, the used reaction solution of yield of adenosylmethionine by microbe whole-cell catalytic synthesis is: Triphosaden 35mmol.L
-1, L-methionine(Met) 35mmol.L
-1, Repone K 100mmol.L
-1, sal epsom 70mmol.L
-1, p-methyl benzenesulfonic acid sodium 800mmol.L
-1, the Tris-HCl damping fluid 100mmol.L of pH7.0
-1, reacting liquid pH value transfers to 7.0.
In addition, and compare without the synthetic SAM of the recombination bacillus coli catalysis of penetratingization processing, the synthetic SAM of penetrating dose of catalysis that directly adds lower concentration in reaction solution equally also has superiority.
Recombinant Bacillus coli cells to the synthetic SAM of catalysis is not done penetratingization pre-treatment, does not add penetrating dose of lower concentration simultaneously in the reaction solution of the synthetic SAM of catalysis yet.Being formulated as follows of the synthetic SAM reaction solution of intestinal bacteria whole-cell catalytic: 100mM Tris, 35mMATP, 35mM L-Met, 70mM MgSO4,100mM KCl, 0.8M p-methyl benzenesulfonic acid sodium, without the penetrating dose of full cell 150g of pretreated recombination bacillus coli, add water to 1L, reacting liquid pH value is transferred to 7.0.Above-mentioned reaction solution is added in the reactor, and 35 ℃ of 120rpm reacted 8 hours.After the HPLC monitoring reaction finishes, centrifugal 15 minutes of 10000rpm, collect supernatant liquor be stored in 4 ℃ standby.Simultaneously, also can use concentration of substrate to be the synthetic SAM of the reaction system catalysis of 10mM and 20mM, the remaining reaction condition is same as above,
Experimental result shows that the optimization output without the synthetic SAM of recombinant Bacillus coli cells catalysis of penetrating dose of processing only is 2.5g/L.
The mensuration of SAM synthase activity can adopt following method in embodiments of the present invention:
Use 0.1mol/L Tris-HCl damping fluid (pH=8.0) to suspend respectively the Bacillus coli cells that fermentation obtains, and be contrast not change the wild bacterium that carries SAM synthase gene plasmid over to.Every gram wet cell adds the 3ml damping fluid and suspends, and the N,O-Diacetylmuramidase that adds 100ug/ml is 35 ℃ of reactions 30 minutes down, then at 0 ℃ of following ultrasonic disruption cell.4 ℃ of centrifugal collection SAM synthetic enzyme crude extracts of 12000rpm are standby.
Set reaction system 1ml, wherein contain 100mM Tris, 100mM KCl, 26mM MgSO4,10mML-Met, 13mM ATP, 0.4M p-methyl benzenesulfonic acid sodium, reacting liquid pH value is transferred to 7.Add an amount of SAM synthetic enzyme crude extract catalyzed reaction, and do contrast with the crude extract that adds wild bacterium simultaneously, do blank with the reaction that does not add L-Met.Behind 37 ℃ of following incubation 30min, add 10% trichoroacetic acid(TCA) termination reaction.The centrifugal precipitation of removing is by the content of SAM in the quantitative supernatant liquor of HPLC.
Adopt high pressure lipuid chromatography (HPLC) (HPLC) to measure SAM content in embodiments of the present invention:
The content of SAM can carry out analyzing and testing by the anti-phase C18 post of HPLC, can monitor the peak of substrate A TP and the peak of product S AM by sampling under the differential responses time, and definite reaction process and reaction end, concrete condition determination is as follows: chromatographic column Kromasil C18 (5 μ m, 4.6 * 250mm); Detect wavelength 260nm; Moving phase be (40mM acetate-sodium acetate buffer+5% methyl alcohol, pH=4.6); Flow velocity 0.6ml/min.The retention time of SAM is about 8min, and the retention time of ATP is about 5.5min.
Adopt external standard method, the typical curve of drawing according to the SAM standard substance peak area of different concns just can carry out quantitatively the SAM in the sample.
Claims (6)
1. method that promotes yield of adenosylmethionine by microbe whole-cell catalytic synthesis is characterized in that step is as follows:
Step 1, fermentation culture can be expressed the microorganism cells of adenosine methilanin synthase, and the temperature maintenance of nutrient solution is at 15～38 ℃ in the fermenting process, and fermented liquid is collected microorganism cells 1 time with distilled water wash behind the centrifugal 10min of 6000rpm;
Step 2, with the microorganism cells of centrifugal collection as the enzyme source, in the reaction solution of catalysis synthesizing adenosine methilanin, add penetrating dose simultaneously, with Triphosaden and L-methionine(Met) is substrate catalysis synthesizing adenosine methilanin, the temperature maintenance of reaction solution is at 35 ℃, 120rpm reacts 8h, and cell concentration is 150g wet thallus/L in the reaction system; The described penetrating dose of volume percentage final concentration in the synthesizing adenosine methilanin reaction solution is 0.25%～2.5%; Described reaction solution consists of: the Tris-HCl damping fluid 100mmol.L of pH 7.0
-1, Triphosaden 10～35mmol.L
-1, L-methionine(Met) 10～35mmol.L
-1, Repone K 100mmol.L
-1, sal epsom 20～70mmol.L
-1With p-methyl benzenesulfonic acid sodium 400-800mmol.L
-1, dissolving back adjust pH to 7.0.
2. the method for promotion yield of adenosylmethionine by microbe whole-cell catalytic synthesis according to claim 1 is characterized in that: described penetrating dose is organic solvent or tensio-active agent; Described organic solvent is toluene or dimethylbenzene, and described tensio-active agent is a cetyl trimethylammonium bromide.
3. the method for promotion yield of adenosylmethionine by microbe whole-cell catalytic synthesis according to claim 2 is characterized in that: the volume percentage final concentration of described toluene in the adenosylmethionine synthesis reaction solution is 0.5%.
4. the method for promotion yield of adenosylmethionine by microbe whole-cell catalytic synthesis according to claim 2 is characterized in that: the volume percentage final concentration of described dimethylbenzene in the adenosylmethionine synthesis reaction solution is 1%.
5. the method for promotion yield of adenosylmethionine by microbe whole-cell catalytic synthesis according to claim 2 is characterized in that: the volume percentage final concentration of described cetyl trimethylammonium bromide in the adenosylmethionine synthesis reaction solution is 0.5%.
6. the method for promotion yield of adenosylmethionine by microbe whole-cell catalytic synthesis according to claim 1 is characterized in that: described microorganism is to include the recombinant Bacillus coli cells that derives from intestinal bacteria Escherichia coli adenosine methionine synthetase gene.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757086A (en) * | 2014-01-09 | 2014-04-30 | 集美大学 | Method for synthesizing S-adenosylmethionine through coupling of escherichia coli and saccharomyces cerevisiae |
| CN110283859A (en) * | 2019-07-15 | 2019-09-27 | 苏州富士莱医药股份有限公司 | The method of microbial enzyme method synthesis N-BETA-Alanyl-L-histidine |
-
2008
- 2008-09-09 CN CNA2008101508699A patent/CN101353682A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103757086A (en) * | 2014-01-09 | 2014-04-30 | 集美大学 | Method for synthesizing S-adenosylmethionine through coupling of escherichia coli and saccharomyces cerevisiae |
| CN110283859A (en) * | 2019-07-15 | 2019-09-27 | 苏州富士莱医药股份有限公司 | The method of microbial enzyme method synthesis N-BETA-Alanyl-L-histidine |
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