CN103757086A - Method for synthesizing S-adenosylmethionine through coupling of escherichia coli and saccharomyces cerevisiae - Google Patents
Method for synthesizing S-adenosylmethionine through coupling of escherichia coli and saccharomyces cerevisiae Download PDFInfo
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Abstract
The invention discloses a method for synthesizing S-adenosylmethionine (SAM) through coupling of escherichia coli and saccharomyces cerevisiae. The method comprises basic processes of firstly, respectively cultivating an escherichia coli engineered strain BL21/pET-28a-SAMS containing S-adenosylmethionine synthetase gene (SAMS) and saccharomyces cerevisiae at proper conditions, then mixing according to a certain proportion, coupling so as to generate SAM; secondly, allowing the synthesized SAM to secrete to outer side of the cell by taking an organic solvent as a permeability agent; and finally, analyzing and detecting the SAM by a high performance liquid chromatography technology. The invention provides a novel method for producing SAM at low cost, and the method is simple and convenient to operate, and can catalyze ATP and L-Met by the whole cell so as to synthesize SAM.
Description
Technical field
The invention belongs to bioengineering field, be specifically related to the preparation method of the synthetic S-adenosylmethionine of intestinal bacteria and yeast saccharomyces cerevisiae coupling.
Background technology
S-adenosylmethionine (S-Adenosyl-methionine, SAM) is to be extensively present in a kind of important mesostate in animal, plant and microbe.In human body, SAM is a kind of important physiologically active substance, participates in multiple biochemical reaction.SAM causes people's extensive concern owing to having important physiological action.Through clinical application research for many years, show, SAM is that current effect is best, function maximum, the widest one of applicable surface both can be used as disease therapeuticing medicine, can be used as again the material of food enrichment.It can be used for treating the diseases such as hepatopathy, dysthymia disorders and sacroiliitis, is also antidotal high-class healthy medicine.Due to its have rapid-action, side effect is little, the feature such as safe and reliable, at present U.S. FDA official approval the enteric agents of SAM and capsule as nonprescription drugs; While U.S. FDA is also ratified SAM and is gone on the market as healthcare products.In China, SAM clinical application mainly concentrates on hepatopathy aspect.China is the popular big country of hepatitis, and according to statistics, China does not also have a kind of bio-pharmaceutical that can really improve liver cell metabolism at present.Therefore, develop at home SAM medicine significant.
The preparation method of SAM mainly contains chemical synthesis, enzymatic synthesis and microbe fermentation method.Wherein, chemical synthesis is to adopt AdoHcy and methyl donor to synthesize, but its productive rate is low, reaction substrate is expensive, therefore fails to be applied to suitability for industrialized production.Enzymatic synthesis is to utilize SAM synthetic enzyme (SAMS) to come catalysis ATP and METHIONINE (L-Met) to synthesize SAM.But one side SAM synthetic enzyme need to carry out separation and purification rear can be used as biological catalyst use, thereby has increased production cost; The synthetic of SAM also needs expensive ATP as substrate on the other hand, and therefore, the SAM of general synthetic isotope mark of enzymatic synthesis is for scientific research, if need further to improve and explore for suitability for industrialized production at present.Microbe fermentation method is now most widely used general, simultaneously also cheapest method of cost.Certain micro-organisms can accumulate SAM by cellular metabolism, especially some bacterial strains of saccharomyces (Saccharomyces), in substratum, add a certain amount of external source METHIONINE (L-Met) and just can in cell, react with L-Met by catalysis ATP, the SAM of synthetic higher concentration.Although microbe fermentation method has overcome the expensive ATP of enzymatic synthesis utilization and need to carry out to SAM synthetic enzyme the shortcomings such as separation and purification, but synthetic SAM is mainly accumulated in yeast cell, in separation and purification SAM process, first need broken thicker yeast cells wall, and then through complicated purification procedures, finally could obtain the SAM that purity is higher.And the broken wall problem of yeast cell is still a Main Bottleneck at present in suitability for industrialized production, complicated purification procedures also makes the ultimate yield of SAM lower simultaneously.With L-Met, calculate, the productive rate of fermentative Production SAM only has 15% ~ 30%, thereby causes the production cost of SAM higher.Therefore, how further the separation purifying technique of efficient synthetic a large amount of SAM and simplification SAM, to increasing substantially the output of SAM, will be conducive to advance the process of China SAM large-scale industrial production.
SAM is that under the condition being existed at ATP by S-adenosylmethionine synthetic enzyme (EC2.5.1.6, S-Adenosyl-methionine-synthetase, SAMS), catalysis METHIONINE (L-Met) is synthetic.The important factor that affects building-up process comprises: the activity of S-adenosylmethionine synthetic enzyme (SAMS) and the supply of ATP.The biosynthetic efficiency of the former major effect, the latter is closely related with the economy of process.Owing to directly adding ATP in reaction process, be that institute is inappropriate economically, therefore, for this class, need the biosynthetic process of ATP, can introduce an effective ATP synthesis system, and coordinate mutually to form a coupled system with SAM synthesis system, be ATP regeneration system rapidly, directly affect economy and the efficiency of reaction process.
Summary of the invention
It is a kind of for above-mentioned the deficiencies in the prior art that technical problem to be solved by this invention is to provide, a kind of preparation method who utilizes intestinal bacteria and the synthetic S-adenosylmethionine of yeast saccharomyces cerevisiae coupling is provided, to solve present stage, produces the problems such as the cost existing in SAM method is high, separation and purification is difficult.
For solving the problems of the technologies described above, technical solution of the present invention is:
A preparation method for the synthetic S-adenosylmethionine of intestinal bacteria and yeast saccharomyces cerevisiae coupling, comprises the following steps:
(1) colibacillus engineering strain BL21/pET-28a-SAMS and the yeast saccharomyces cerevisiae that will contain S-adenosylmethionine synthase gene (SAMS) are collected respectively thalline after suitable condition bottom fermentation is cultivated;
(2) by a certain percentage by above-mentioned intestinal bacteria and yeast saccharomyces cerevisiae thalline mixing coupling, and add coupled system reaction solution; Wherein, intestinal bacteria provide the place of SAMS and synthetic SAM, and yeast saccharomyces cerevisiae provides enough ATP;
(3) with an organic solvent as penetrating dose, coupling cell is carried out to permeabilized processing, make SAM synthetic in intestinal bacteria, and be secreted into extracellular;
(4) reaction finishes rear centrifugal collection supernatant, obtains S-adenosylmethionine.
In described step (1), the culturing process of colibacillus engineering strain BL21/pET-28a-SAMS is: the colibacillus engineering strain BL21/pET-28a-SAMS that contains S-adenosylmethionine synthase gene (SAMS) is cultivated to OD
600it is 0.5 ~ 1.5 o'clock, add 2 ~ 8 mg/mL lactose or 0.1 ~ 1.0 mmol/L isopropyl-β-D-thiogalactoside(IPTG) (IPTG) as inductor, inducing temperature is 25 ~ 37 ℃, and induction time is 5 ~ 16 h, with a certain amount of S-adenosylmethionine synthetic enzyme of abduction delivering SAMS.
In described step (1), the culturing process of yeast saccharomyces cerevisiae is: by yeast saccharomyces cerevisiae under shaking speed 170 ~ 200 r/min, 28 ~ 30 ℃ of conditions of temperature, incubation time 17 ~ 22 h.
In described step (2), described e. coli bl21/pET-28a-SAMS and yeast saccharomyces cerevisiae wet thallus mass mixing ratio are 0.5 ~ 5:1.
In described step (2), coupled system reaction solution comprises: 200 mmol/L glucose, 10 mmol/L adenosines (Ado), 1 mmol/L AMP (AMP), 0.1 mmol/L Reduced nicotinamide-adenine dinucleotide (NAD), 30 mmol/L MgCl
26H
2o, 30mmol/L L-Methionine (L-Met), 200 mmol/L potassium phosphate buffers, with 0.5 mol/L potassium hydroxide tune pH=7.0.
In described step (3), using 0.5 ~ 6% toluene or 0.5% methyl alcohol or 0.5% acetone as described penetrating dose.
In described step (3), its reaction conditions is: shaking speed is that 150 ~ 200r/min, temperature are 25 ~ 37 ℃, linked reaction 2 ~ 8 h.
Adopt after such scheme, the recombination bacillus coli and the stronger yeast saccharomyces cerevisiae of glycolysis path enzyme activity that contain S-adenosylmethionine synthase gene due to utilization of the present invention, through cell is carried out after permeability processing, being fixed do not operate and directly two kinds of thalline mixed, and by its complete enzyme system, builds an ATP regeneration system rapidly and SAM synthesis system.Take e. coli bl21 (DE3) as host cell, conversion is with the recombinant plasmid pET-28a-SAMS of coding S-adenosylmethionine synthase gene (SAMS), having obtained a strain, to have SAM composite reactive, plasmid stability and mitotic stability all good, and can reusable recombinant escherichia coli strain BL21/pET-28a-SAMS.On this basis, utilize the ATP biosynthetic enzyme system in S-adenosylmethionine synthetase series and the yeast saccharomyces cerevisiae in recombination bacillus coli BL21/pET-28a-SAMS, built between a kind take glucose as the energy coupling ATP regenerative system to synthesize SAM, and synthetic product SAM has been secreted into extracellular effectively.One aspect of the present invention has been avoided the ATP of extra interpolation costliness; Because SAM is secreted into extracellular, can greatly simplify its separation purifying technique on the other hand.Therefore, the invention provides a kind of cheap novel method of producing S-adenosylmethionine, is a kind of easy and simple to handle, the method for the synthetic SAM of the simple intact cell catalysis ATP of technique and L-Met.The at present domestic report that utilizes intestinal bacteria and yeast saccharomyces cerevisiae coupling to generate S-adenosylmethionine of not seeing.
More specifically, the present invention has following features:
1. the present invention uses the recombinant bacterium of energy great expression S-adenosylmethionine synthetic enzyme to catalyze and synthesize SAM as enzyme source, and during for coupling, catalyzing and synthesizing SAM provides the enzyme source of a large amount of cheapnesss.
2. the present invention uses the yeast saccharomyces cerevisiae that glycolysis path enzyme activity is stronger to produce ATP, and the ATP of a large amount of cheapnesss is provided for catalyzing and synthesizing SAM.
The present invention with an organic solvent toluene as penetrating dose to carrying out permeability processing for the cell of the synthetic SAM of catalytic coupling, to guarantee substrate and the diffusion transmission of product inside and outside cell, especially guarantee that ATP that yeast generates can be transferred to smoothly engineering bacteria and carry out the synthetic of SAM.The productive rate synthetic through the cell catalysis SAM of penetrating processing is greatly enhanced, and greatly improved the utilization ratio of SAM synthetic enzyme.
4. the present invention utilizes intestinal bacteria and the synthetic S-adenosylmethionine of yeast saccharomyces cerevisiae coupling, because product is to be secreted into outside born of the same parents, can greatly simplify the follow-up separation purifying technique of SAM.
Accompanying drawing explanation
Figure 1A is that HPLC detects SAM standard substance color atlas;
Figure 1B is that HPLC detects intestinal bacteria and the synthetic SAM sample chromatogram figure of yeast saccharomyces cerevisiae coupling;
Fig. 2 is SAM typical curve.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
One, this laboratory of colibacillus engineering strain BL21/pET-28a-SAMS(builds) cultivation:
1. substratum: LB substratum (yeast powder 5 g/L, Tryptones 10 g/L, NaCl 10 g/L, pH 7.0), contains 2% agar in solid LB substratum.Add as required 50 μ g/mL kantlex.
2. cultural method: the mono-colony inoculation of picking colibacillus engineering strain BL21/pET-28a-SAMS is to the 50 μ g/mL kantlex LB(LK that contain that 30 mL are housed from the solid LB substratum that contains 50 μ g/mL kantlex) cultivate in 250 mL triangular flasks of substratum, in shaking speed, be that 180 r/min, temperature are under 37 ℃ of conditions, cultivate 12 h left and right.Nutrient solution is seeded to by the inoculum size of 1% (v/v) in 250 mL triangular flasks of the LK substratum that 100 mL are housed and cultivates, 37 ℃ of temperature, shaking speed 180 r/min, when yeast culture is to logarithmic growth (OD in mid-term
600=1.5 left and right) time, add 4 mg/mL lactose to carry out abduction delivering restructuring SAMS.Wherein, inducing temperature is 30 ℃, and induction time is 8 h.Nutrient solution after abduction delivering finishes at 4 ℃, centrifugal 20 min of 12000 × g, collect thalline, then wash thalline 2 times with ice-cold 20 mmol/L potassium phosphate buffers, for follow-up coupling system.
Two, the cultivation of yeast saccharomyces cerevisiae (this laboratory storage):
1. substratum: slant medium consist of 8
op wheat juice, agar 2%, pH 5.5; Liquid nutrient medium (g/L) consist of glucose 20, peptone 20, yeast extract paste 10, pH 5.5.
2. cultural method
(1) slant culture: postvaccinal inclined-plane is placed in 30 ℃ of constant incubators and cultivates 2 days, is preserved in cold compartment of refrigerator, monthly at least transfers once.
(2) liquid culture: by 30 ℃, inclined-plane seed activation 3-4 h, get in the 250 mL triangular flasks that a ring thalline is seeded to the liquid nutrient medium that 50mL is housed and cultivate, under shaking speed 180 r/min, 30 ℃ of conditions of temperature, incubation time 19 h.Then by cultured nutrient solution according to the inoculum size of 10% (v/v), be seeded in the 250 mL triangular flasks that 50 mL liquid nutrient mediums are housed and cultivate, fermentation, under shaking speed 180 r/min, 30 ℃ of conditions of temperature, cultivate 18 h.Nutrient solution at 4 ℃, centrifugal 20 min of 6000 × g, collect thalline, then wash thalline 2 times with ice-cold 20 mmol/L potassium phosphate buffers, for follow-up coupling system.
Three, the synthetic S-adenosylmethionine of intestinal bacteria and yeast saccharomyces cerevisiae coupling:
In the ratio of 4:1, taking e. coli bl21 (pET-28a-SAMS) mixes with yeast saccharomyces cerevisiae wet thallus, add coupled system reaction solution (to comprise: 200 mmol/L glucose, 10 mmol/L adenosines (Ado), 1 mmol/L AMP (AMP), 0.1 mmol/L Reduced nicotinamide-adenine dinucleotide (NAD), 30 mmol/L MgCl
26H
2o, 30 mmol/L L-Methionines (L-Met), 200 mmol/L potassium phosphate buffers, with 0.5 mol/L potassium hydroxide tune pH=7.0), add 5%(v/v) toluene, under shaking speed 180 r/min, 37 ℃ of conditions of temperature, centrifugal collection supernatant after oscillatory reaction 5 h, measures SAM content in supernatant.
Four, the mensuration of S-adenosylmethionine content
In sample, the content of SAM can carry out analyzing and testing by the anti-phase C18 post of HPLC, concrete condition determination is: moving phase is 0.01mol/L ammonium formiate (formic acid is adjusted pH3.5,0.22 μ m water film filtering): methyl alcohol=97:3, flow velocity 1 mL/min, 30 ℃ of column temperatures, detect wavelength 254 nm.Detected result is shown in Figure 1A and Figure 1B.
Adopt external standard method, the typical curve (see figure 2) of drawing according to the SAM standard substance peak area of different concns is carried out quantitatively the SAM in sample.When adding 50 mg(thalline) during/mL damping fluid, SAM concentration can reach 55 mg/l, and to produce SAM amount be 1.2 to every milligram of thalline.
The above, be only preferred embodiment of the present invention, is not used for limiting scope of the invention process.Therefore variation or the modification in every case done according to claim of the present invention and specification sheets, within all should belonging to the scope that patent of the present invention contains.
Claims (7)
1. a preparation method for the synthetic S-adenosylmethionine of intestinal bacteria and yeast saccharomyces cerevisiae coupling, is characterized in that comprising the following steps:
(1) the colibacillus engineering strain BL21/pET-28a-SAMS that contains S-adenosylmethionine synthase gene and yeast saccharomyces cerevisiae after cultivating, are collected suitable condition bottom fermentation respectively to thalline;
(2) by a certain percentage by above-mentioned intestinal bacteria and yeast saccharomyces cerevisiae thalline mixing coupling, and add coupled system reaction solution; Wherein, intestinal bacteria provide the place of SAMS and synthetic SAM, and yeast saccharomyces cerevisiae provides enough ATP;
(3) with an organic solvent as penetrating dose, coupling cell is carried out to permeabilized processing, make SAM synthetic in intestinal bacteria, and be secreted into extracellular;
(4) reaction finishes rear centrifugal collection supernatant, obtains S-adenosylmethionine.
2. the preparation method of the synthetic S-adenosylmethionine of intestinal bacteria according to claim 1 and yeast saccharomyces cerevisiae coupling, it is characterized in that, in described step (1), the culturing process of colibacillus engineering strain BL21/pET-28a-SAMS is: the colibacillus engineering strain BL21/pET-28a-SAMS that contains S-adenosylmethionine synthase gene is cultivated to OD
600it is 0.5 ~ 1.5 o'clock, add 2 ~ 8 mg/mL lactose or 0.1 ~ 1.0 mmol/L isopropyl-β-D-thiogalactoside(IPTG) as inductor, inducing temperature is 25 ~ 37 ℃, and induction time is 5 ~ 16 h, with a certain amount of S-adenosylmethionine synthetic enzyme of abduction delivering SAMS.
3. the preparation method of the synthetic S-adenosylmethionine of intestinal bacteria according to claim 1 and yeast saccharomyces cerevisiae coupling, it is characterized in that in described step (1), the culturing process of yeast saccharomyces cerevisiae is: by yeast saccharomyces cerevisiae under shaking speed 170 ~ 200 r/min, 28 ~ 30 ℃ of conditions of temperature, incubation time 17 ~ 22 h.
4. the preparation method of the synthetic S-adenosylmethionine of intestinal bacteria according to claim 1 and yeast saccharomyces cerevisiae coupling, is characterized in that: in described step (2), described intestinal bacteria and yeast saccharomyces cerevisiae wet thallus mass mixing ratio are 0.5 ~ 5:1.
5. the preparation method of the synthetic S-adenosylmethionine of intestinal bacteria according to claim 1 and yeast saccharomyces cerevisiae coupling, it is characterized in that: in described step (2), coupled system mixed reaction solution comprises: 200 mmol/L glucose, 10 mmol/L adenosines, 1 mmol/L AMP, 0.1 mmol/L Reduced nicotinamide-adenine dinucleotide, 30 mmol/L MgCl
26H
2o, 30mmol/L L-Methionine, 200 mmol/L potassium phosphate buffers, with 0.5 mol/L potassium hydroxide tune pH=7.0.
6. the preparation method of the synthetic S-adenosylmethionine of intestinal bacteria according to claim 1 and yeast saccharomyces cerevisiae coupling, is characterized in that: in described step (3), using 0.5 ~ 6% toluene or 0.5% methyl alcohol or 0.5% acetone as described penetrating dose.
7. according to the preparation method of the intestinal bacteria described in claim 1 or 6 and the synthetic S-adenosylmethionine of yeast saccharomyces cerevisiae coupling, it is characterized in that: in described step (3), its reaction conditions is: shaking speed is that 150 ~ 200 r/min, temperature are 25 ~ 37 ℃, linked reaction 2 ~ 8 h.
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| CN111117940A (en) * | 2019-12-04 | 2020-05-08 | 天津大学 | Escherichia coli engineering bacterium and method for high yield of pentamethylene diamine |
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Application publication date: 20140430 |