CN104178540A

CN104178540A - Method for synthesizing ademetionine by biological catalytic process

Info

Publication number: CN104178540A
Application number: CN201410351739.7A
Authority: CN
Inventors: 傅小峰; 沈剑锋; 姚黎栋; 王刚; 周军贤; 胡纯琦
Original assignee: ZHEJIANG ZHENYUAN PHARMACEUTICAL CO Ltd
Current assignee: ZHEJIANG ZHENYUAN PHARMACEUTICAL CO Ltd
Priority date: 2014-07-23
Filing date: 2014-07-23
Publication date: 2014-12-03
Anticipated expiration: 2034-07-23
Also published as: CN104178540B

Abstract

The invention relates to a method for synthesizing ademetionine by a biological catalytic process, belonging to the technical field of biological pharmacy. The method comprises the following steps: inoculating Escherichia coli into a seed culture medium, carrying out shake culture over night, adding the over-night culture material into an LB (Langmuir-Blodgett) liquid culture medium, carrying out shake culture to mid-log phase, adding IPTG (isopropyl-beta-D-thiogalactopyranoside) into the bacterium liquid to fix the concentration, continuing carrying out shake culture and SAM synthetase expression, centrifuging to obtain wet bacteria, stirring for extraction treatment, loading to an ion exchange resin column, concentrating by nanofiltration, crystallizing, and carrying out vacuum drying to obtain the crystal. The technical scheme has the advantages of high conversion rate, short period, high purity and the like.

Description

A kind of method of biological catalysis synthesizing adenosine methionine

Technical field

A kind of method that the present invention relates to biological catalysis synthesizing adenosine methionine, belongs to biological pharmacy technical field.

Background technology

SAM (S. adenosyl-L.methionine, SAM) be the important metabolic intermediate matter being extensively present in animal, plant and microbe, also be a kind of important physiologically active substance in human body, mainly play a part in vivo transmethylase, turn sulphur and turn aminopropyl, sacroiliitis, dysthymia disorders, liver dysfunction, pancreatitis etc. are all had to good curative effect.SAM or preventing cancer, cardiovascular disorder and antidotal high-class healthy medicine.1999, U.S. FDA approval SAM went on the market as healthcare products, becomes one of best-selling nutritious prod rapidly in the U.S..Along with the raising of people's quality of life, the renewal of health concept, also will get more and more to the demand of SAM.

The national annual requirement of bulk drug (API) fourth disulfonic acid ademetionine is at least more than 10 tons, and the demand in the whole world is 50 tons every year, its selling price per unit 5000 yuan/more than Kg; And if count in for other as feedstuff industry, demand is just more than 200 tons.The producer of domestic production bulk drug (API) fourth disulfonic acid ademetionine is just only having sea, Zhejiang, and another Zhejiang shake unit of producer has also obtained production certification, but forms and sell not yet.Domestic existing development and research, because the expression level of fermentation method is low, the enzyme of enzymatic synthesis and the cost of ATP are higher.

At present ademetionine industrialized producing technology mainly contains two kinds of yeast fermentation method (intracellular expression method) and enzymatic synthesis (extracellular enzyme is urged method), and these two kinds of methods all exist different technological difficulties:

Yeast fermentation method (intracellular expression method): the Wine brewing yeast strain of the external expression SAM that adopt special cultivation is prepared SAM by intracellular expression more.The domestic people of having works out genetically engineered and changes the yeast saccharomyces cerevisiae of structure, fermentation expression SAM, but what expression was the highest at present also only reaches 8g/L fermented liquid, and most exploration that only completes zymotechnique, producing by this method is at intracellular expression, because the precursor L-Methionine of SAM need see through cell wall, so and the restriction that is subject to space in born of the same parents is expressed, and output is very difficult to be improved, yield is low, and cost is high, is not suitable for extensive preparation.

Enzymatic method (extracellular enzyme is urged method): enzymatic method is mainly with extract purifying adenomethionine synthase from cereuisiae fermentum or Other Engineering bacterium, then transform preparation SAM by optimizing enzymatic reaction condition, this method feed stock conversion is high, SAM is easy to extract purifying, but in this method, the extraction purification procedures complexity of enzyme is in saccharomycetic fragmentation, centrifugal, filter, on concentrated supernatant and supernatant liquor, in column purification, have the loss of enzyme, the uncertain factor that simultaneously affects enzymic activity is more, the cycle of purifying enzyme is longer, cause the activity of enzyme to be subject to impact, the purifying enzyme amount that this method obtains is simultaneously less, cause the cost of enzyme very high, also be not suitable for preparing on a large scale SAM.

Summary of the invention

The invention provides a kind of method of biological catalysis synthesizing adenosine methionine method of carrying out ademetionine extraction utilized, first utilize intestinal bacteria to replace yeast, take peculiar operation to express required SAM synthetic enzyme, produce and obtain SAM by enzymatic method again, to solve, to produce by yeast fermentation process the feed stock conversion that ademetionine exists low, life cycle of the product length, finished product separation and purification program complexity, expensive problem.

The technical scheme that the present invention takes is as follows:

A kind of method of biological catalysis synthesizing adenosine methionine, the intestinal bacteria that preserve at-70 DEG C are inoculated in seed liquid nutrient medium, under 37 DEG C, 200rpm rotating speed, shake overnight incubation, getting overnight culture next day adds in LB liquid nutrient medium, under 37 DEG C, 200rpm rotating speed, concussion is cultured to mid-log phase, in bacterium liquid, add IPTG to regulate after concentration, continue concussion at 18 DEG C to cultivate after 20 hours, carry out enzymatic reaction and obtain SAM; Wherein, the action component of described seed culture medium is: bacto-tryptone 12g, and yeast extract 24g, glycerine 4 mL, are dissolved in 900mL water, add KH ₂pO ₄2.31g, K ₂hPO ₄16.43 in the 100 mL aqueous solution; The action component of LB liquid nutrient medium is the paraxin of 50mg/ml carbenicillin and 34mg/ml.

Further arranging of technique scheme is as follows:

Described enzymatic reaction comprises the steps: that (1) extract: by the intestinal bacteria of cultivating under 4 DEG C, 5000rpm rotating speed after centrifugal treating, add ethyl acetate aqueous solution extracting 15-60 minute, the addition of the ethyl acetate aqueous solution is 150-300mL/kg intestinal bacteria, add again 0.15-0.2M sulphuric acid soln to carry out after acidification 1-2 hour, the addition of sulphuric acid soln is 400-600 mL/kg intestinal bacteria, centrifugal, remove after cell debris, obtain the extraction liquid that contains SAMe; (2) purifying: adjust extraction liquid pH to 3-5, adopt ion exchange resin column to carry out loading wash-out, obtain SAM elutriant, by this SAM elutriant carry out nanofiltration concentrated after, splash into anhydrous methanol, after stirring standing sedimentation and filtering, then use vacuum-drying at twice, 50 DEG C of 90% methanol solution washing crystal can obtain finished product.

In the described ethyl acetate aqueous solution, the volume ratio of ethyl acetate and water is 1:4-3:2.

In technique scheme, utilize biological catalysis that intestinal bacteria are carried out to multiplication culture and form SAM synthetic enzyme, then the SAM synthetic enzyme of acquisition is carried out to enzymatic reaction, thereby obtained SAM, compared with conventional SAM preparation method, technique scheme tool of the present invention has the following advantages:

1. the advantage that the enzymatic method of routine is prepared SAM is that transformation efficiency is high, therefore natural synthetic enzyme is expressed in yeast, the main cereuisiae fermentum that adopts carries out the expression of SAM synthetic enzyme, but because its separation and purification operation is more complicated, the activity of the SAM synthetic enzyme that yeast obtains can be along with the carrying out inactivation gradually of processing, be subject to the impact of enzyme motility rate, finally cannot realize scale operation; In technique scheme of the present invention, substitute yeast with intestinal bacteria, utilize genetic engineering technique, be transformed in intestinal bacteria carrying the plasmid of expressing SAM synthetic enzyme, and by abduction delivering, separation and Extraction and obtain high expression level, highly purified SAM synthetic enzyme.Carry out in SAM synthetic enzyme expression process in the above-mentioned technique of employing, overcome and yielded poorly, the poor problem of separation purity, not only make final active SAM yield greatly improve, and, in the treatment process adopting, adopt suitable substratum, improve the expression of SAM synthetic enzyme, simultaneously, the method that adopts ion exchange column to separate in treatment process, follow-up separation and purification difficulty is reduced, more be conducive to the extraction of SAM, and guarantee that intestinal bacteria have the obviously advantage due to yeast obtaining aspect the productive rate of product and purity, therefore in the time adopting technique scheme, fundamentally solve common enzymatic method and must carry out complex separations purification procedures, effectively avoid the impact of purification procedures on SAM activity, guarantee that the SAM synthetic enzyme that intestinal bacteria adopt biological catalysis to express has very high activity, finally ensure that SAM yield is up to more than 96%, carrying out enzymatic method synthesis yield with employing yeast is only, therefore, adopt such scheme of the present invention to make the synthetic SAM of enzymatic method be applied to suitability for industrialized production, can carry out on a large scale preparation and the purification of SAM.

2. in technique scheme of the present invention, when SAM synthetic enzyme expression process is optimized, follow-up enzymatic reaction technique is also optimized and is improved, the method of utilizing ion exchange column to separate, make the operation of extraction and purifying more easy, extract in purge process, processing intensity reduces greatly, avoid crossing the extraneous impact of interfering activity of enzyme reaction of high strength, simultaneously, carry out recrystallization in the later stage, ensured that again it extracts purification effect, make the yield of SAM and activity all remain on higher level.

Adopt technique scheme of the present invention, not only effectively bring into play intestinal bacteria and obtained products collection efficiency and purity, and the advantage of the aspects such as optimized Separation efficiency, the activity keeping that makes to express the SAM synthetic enzyme obtaining is defined under 30 DEG C of 5mM ATP and 5mML methionine(Met) starting point concentration in the work of 1.25u(enzyme product that per minute in 20 minutes generates 1 umol gland acid anhydride methionine(Met) and is defined as in the level of l more than u), biological catalysis is being expressed to SAM synthetic enzyme in the time that enzymatic method extraction preparation SAM is combined, pass through genetic engineering technique, and change medium component, overcome SAM synthetic enzyme at the low technical barrier of expression in escherichia coli rate, make this be combined into possibility, and active and all good SAM of yield are finally obtained.

Brief description of the drawings

Fig. 1 is the performance test results figure of embodiment 1 prepared product;

Fig. 2 is the performance test results figure of embodiment 2 prepared products.

Embodiment

Experiment material and method:

1. engineering bacteria and reagent: intestinal bacteria BL-21;

2. substratum:

Plate culture medium: LB medium (50 mg/ml Pyocianils and 34 mg/ml paraxin);

Seed culture medium (taking 1L as example): bacto-tryptone 12g, yeast extract 24g, glycerine 4 mL are dissolved in 900mL water, and add KH in this aqueous solution of every 100mL ₂pO ₄2.31g, K ₂hPO ₄16.43g form seed liquid nutrient medium.

3. the expression of SAM synthetic enzyme:

To be kept at-70 DEG C, intestinal bacteria are inoculated in 5 mL seed liquid nutrient mediums, and 37 DEG C, 200 rpm shaking culture are spent the night; Get next day in the LB liquid nutrient medium that 3 mL overnight culture add 2L, 37 DEG C of concuss are cultivated 1 h, to mid-log phase (A ₅₅₀=0.4 ~ 0.6), add IPTG(isopropyl-β-D-thiogalactoside(IPTG) in bacterium liquid, molecular formula is C ₉h ₁₈o ₅s), to bacterium liquid final concentration 0.4 mMol/L, 18 DEG C are continued concussion cultivation 20 h, complete Escherichia coli fermentation and cultivate.Get 1 mL bacterium liquid for detection of enzyme protein expression situation (A ₅₅₀=1.0 ~ 1.2).

4. catalyzer preparation

To detect remaining bacterium liquid at 4 DEG C, centrifugal 10 min of 4000 rpm, collect the bacterial sediment after centrifugal, by the PBS washed twice of 15 mL ice precoolings, add Loading Buffer(50 mM Tris, 500 mM NaCl, 10 % glycerine by 3 ~ 5 mL/g, pH=7.5) and be placed on-70 DEG C of refrigerators and 37 DEG C of water-baths and intersect quick multigelation 5 times, then carry out ultrasonication, electric current 600 HZ, 10s × 5 time; 4 DEG C, centrifugal 10 min of 4000 rpm, remove precipitation (thalline residue), and supernatant is filtered with 0.22 mm or 0.45 mm strainer.

Embodiment 1:

1) get 1 mL and complete the bacterium liquid (A that Escherichia coli fermentation is cultivated ₅₅₀=1.0 ~ 1.2) express after SAM synthetic enzyme, in 4 DEG C, centrifugal under 5000 r/min conditions, gather in the crops 40 kilograms of wet thallus, in wet thallus, add the ethyl acetate aqueous solution that 9.6 liters of volumetric concentrations are 50%, under 500 r/min rotating speeds, vigorous stirring is processed 30 minutes, then uses 20 liter of 0.175 mol/L H ₂s0 ₄continue to process after 1.5 hours, then under 5000 r/min conditions centrifugal 20 minutes, remove cell debris, 40 liters of the extraction liquids that obtains containing SAM, wherein in extraction liquid, the content of SAM is 8.5 g/L.

2) 40 liters of extraction liquids that contain SAM are adjusted to pH5.0 with 5mol/l sodium hydroxide solution, flow velocity with 2 times of resin bed volumes per hour carries out loading processing by 6.7 liters of JK110 ion exchange resin columns, after end of the sample, first use the deionized water rinsing pillar of 30 liters, then carry out wash-out with PBS solution, obtain SAM elutriant 25L, in elutriant, SAM concentration is 13.2g/l, and the SAM rate of recovery is 96%.

3) due to the susceptibility of SAM to temperature, use the crosslinked aromatic polyamide membrane of molecular weight cut-off 200, the nanofiltration of 25 liters of SAM elutriants is concentrated into 120g/L, obtain concentrated solution 2.70L, the SAM rate of recovery is 98%.

4) under whipped state, concentrated solution is slowly splashed in 20 liters of anhydrous methanols with the speed of 8ml/min, mixing speed is 45r/min, be added dropwise to complete rear continuation and stir 1.0 hours, then leave standstill sedimentation, filter and obtain crystal, twice, 50 DEG C of vacuum-drying of methanol solution washing crystal with 90% obtains 300 grams of products, purity 98%.

Embodiment 2:

1) after Escherichia coli fermentation is cultivated and finished, 4 DEG C, 5000r/min is centrifugal, gather in the crops 40 kilograms of wet thallus, is the 20% acetic acid second unstrained spirits aqueous solution to adding 12 liters of volumetric concentrations in this wet thallus, and 500r/min processing 60 minutes, then uses 16 liters of 0.15mmol/LH ₂sO ₄continue to process 2 hours.Centrifugal 20 minutes of 5000r/min, removes cell debris, 38 liters of the extraction liquids that obtains containing SAM, and wherein the content of SAM is 8.7g/l.

2) 38 liters of extraction liquids that contain SAM are adjusted to pH4.5 with 5mol/L sodium hydroxide solution, pass through 9.5 liters of D113 ion exchange resin column loadings with the flow velocity of l per hour times of resin bed volume.After end of the sample, first use the deionized water rinsing pillar of 30 liters, then carry out wash-out with PBS solution, obtain SAM elutriant 25L, its SAM concentration is 13.2g/l, and the SAM rate of recovery is 96%.

All the other subsequent disposal are identical with embodiment 1.

Embodiment 3: catalyst recovery

After to be expressed, get brokenly slightly acidic macropore 1AJ olefin(e) acid Zeo-karb (D11:3) separator column on filtered solution after filtered solution and enzymatic after bacterium.Every 30 minutes detect elutriant, measure its OD value, wavelength 204nm place solution absorb be greater than within 0.4 o'clock, use instead pH2.0 aqueous sulfuric acid wash 4 hours, stop wash.The rate of recovery reaches 80%.

Embodiment 4: the concentrated and crystallization of product adenosylmethionine

Ademetionine sterling solution is put into freeze drier and carry out lyophilize, obtain ademetionine (SAM) the dry powder finished product of white powdery, after this product is detected, data gather as follows:

Purity 98%, ¹h-NMR-500M δ (ppm): 8.49 (d, j=4.1,2H); 6,20 (d, J=3.8,4H); (4.88 q, J=4.1,1H); (4.66 t, J=5.8,1H); 4.61 (t, j=2.5,1H); 4.06-3.97 (m, 3H); 4.06-3.97 (m, 2H); 3.04 (s, 2H); 3.01 (S, 1H); 2.94 (td, 6H); 2.49-2.38 (m, 2H); (1.89-1.81 m, 6H). ¹³c-NMR-500M δ (ppm): 173.40,152.74,150.77,147.27,147.24,122.08,92.75,81.43,75.83,75.51,54.25,53.22,47.04,46.67,27.53,26.28,25.87, MS (ESI), m/z=399.1442 [M+H]+. Anal. Calcd for C ₁₅h ₂₄n ₆o ₅s:C, 44.99; H, 6.04; N, 20.99; Found:C, 44.97; H, 6.08; N, 20.96.

Comparative example: conventional enzymatic method is prepared the concrete case of SAM (taking the reaction system of a liter as example)

1, add reagent

Add respectively the following reagent preparing: borate buffer 60mL; 1 mol/L K ₂s0 ₄90mL; L mol/l MgS0 ₄60mL; 0.4 mol/L adenosine triphosphate atp 20mL; 0.2 mol/L methionine(Met) 60mL; To Isosorbide-5-Nitrae-butane sodium disulfonate 70g; DTT 0.1 mmol; 0.5 mol/L EDTA2mL; Deionized water is settled to 1L.

2, temperature of reaction and the time

Above-mentioned mixed solution is added in reactor, and it is 30 DEG C that temperature of reaction is set, reaction times 10h.

3, stopped reaction

After reaction finishes, adding sulfuric acid to final concentration is 0.05 mol/L stopped reaction.

The embodiment of the present invention is carried out to purity, productive rate and preparation cycle aspect with the conventional enzymatic method in the comparative example who provides and compare, refer to table 1.

Table 1 embodiment of the present invention and comparative example's synthetic contrast table

Comparing result by table 1 can be found out, adopt technique scheme of the present invention, the purity of product, productive rates etc. are all better than conventional enzymatic method, its preparation cycle is short more a lot of than comparative example's cycle, therefore, adopt the present invention not only effectively to bring into play intestinal bacteria and obtaining products collection efficiency, the advantage of the aspect such as purity and separation efficiency, make the activity keeping of expressing the SAM synthetic enzyme obtaining more than 1.25u, simultaneously, in such scheme, pass through genetic engineering technique, and change medium component, overcome SAM synthetic enzyme at the low technical barrier of expression in escherichia coli rate, make biological catalysis express the possibility that is combined into of SAM synthetic enzyme and enzymatic method extraction preparation SAM, in 3 catalyst recovery and embodiment 4, product concentrates and these aftertreatments of crystallization in conjunction with the embodiments, active and all good SAM of yield are finally obtained.

Claims

1. a method for biological catalysis synthesizing adenosine methionine, is characterized in that, comprises the steps:

(1) intestinal bacteria are cultivated and the expression of SAM synthetic enzyme: intestinal bacteria are inoculated in seed culture medium, after concussion overnight incubation, get overnight culture and add LB liquid nutrient medium, its concussion is cultured to after mid-log phase, in its bacterium liquid, add IPTG to determine concentration, continue to have shaken and cultivate and carry out the expression of SAM synthetic enzyme

Wherein, the action component of seed culture medium is: bacto-tryptone 12g, yeast extract 24g, glycerine 4 mL, KH ₂pO ₄, K ₂hPO ₄, first bactopeptone, yeast extract and glycerine are dissolved in 900 mL water and form the aqueous solution, then add 2.31g KH in every this aqueous solution of 100 mL ₂pO ₄, 16.43g K ₂hPO ₄; The action component of LB liquid nutrient medium is: the paraxin of 50mg/ml carbenicillin and 34mg/ml,

(2) Centrifugical extraction: the Escherichia coli bacteria liquid centrifugal treating that step (1) is cultivated obtains wet thallus, drips the ethyl acetate aqueous solution in this wet thallus, stirs after extracting processing, adds H ₂sO ₄after solution acidification, centrifugal removal cell debris, obtains SAM extraction liquid;

(3) loading, wash-out: adjust after the pH value of SAM extraction liquid, carry out loading by ion exchange resin column, use deionized water rinsing ion exchange resin column after end of the sample, then carry out wash-out and obtain SAM elutriant;

(4) nanofiltration, concentrated: SAM elutriant is carried out to nanofiltration concentrated, obtain concentrated solution;

(5) crystallization, vacuum-drying: under whipped state, concentrated solution is splashed in dehydrated alcohol, after continuing to stir, standing, sedimentation, filter to obtain crystal, wash the dry product that to obtain of final vacuum by this crystal.

2. the method for a kind of biological catalysis synthesizing adenosine methionine as claimed in claim 1, it is characterized in that: in step (1), the intestinal bacteria that preserve at-70 DEG C are inoculated in seed culture medium, under 37 DEG C, 200rpm rotating speed, shake overnight incubation, getting overnight culture next day adds in LB liquid nutrient medium, under 37 DEG C, 200rpm rotating speed, concussion is cultured to mid-log phase, in the bacterium liquid that is cultured to mid-log phase, add IPTG to regulate concentration to 0.4mmol/L, at 18 DEG C, continue concussion cultivation and within 20 hours, complete intestinal bacteria cultivation, and carry out the expression of SAM synthetic enzyme.

3. the method for a kind of biological catalysis synthesizing adenosine methionine as claimed in claim 1, it is characterized in that: in step (2), by the Escherichia coli bacteria liquid of cultivating under 4 DEG C, 5000 rpm rotating speeds after centrifugal treating, add ethyl acetate aqueous solution extracting 15-60 minute, in the ethyl acetate aqueous solution, the volume ratio of ethyl acetate and water is 1:4-3:2, and addition is 150-300mL/kg wet thallus, adds the H of 0.15-0.2 mol/L after extracting again ₂sO ₄solution carries out acidification 1-2 hour, and the addition of sulphuric acid soln is 400-600 mL/kg wet thallus, centrifugal, removes after cell debris, obtains SAM extraction liquid.

4. the method for a kind of biological catalysis synthesizing adenosine methionine as claimed in claim 1, it is characterized in that: in step (3), extraction liquid pH is adjusted to 3-5, extraction liquid is that 1-2 times of resin bed volume/h carries out loading by the speed of ion exchange resin column, adopts PBS solution to carry out wash-out after loading.

5. the method for a kind of biological catalysis synthesizing adenosine methionine as claimed in claim 1, is characterized in that: in step (4), and the concentrated crosslinked aromatic polyamide membrane that adopts molecular weight cut-off 200 of nanofiltration.

6. the method for a kind of biological catalysis synthesizing adenosine methionine as claimed in claim 1, it is characterized in that: in step (5), the speed that concentrated solution is added drop-wise in anhydrous methanol is 8mL/min, and mixing speed is 45r/min, the methanol solution washed twice of crystal by adopting 90%, 50 DEG C of vacuum-dryings.

7. the method for a kind of biological catalysis synthesizing adenosine methionine as described in claim 1-6 any one, it is characterized in that, bacterium liquid after described intestinal bacteria cultivation and SAM synthetic enzyme are expressed also can be used for the preparation of catalyzer: bacterium liquid is carried out centrifugal, collect bacterial sediment, and it is carried out after PBS washing, add Loading Buffer to intersect multigelation, ultrasonication, after centrifugal removal precipitation, supernatant liquid filtering gained solution is catalyzer, and the composition of this catalyzer is adenomethionine synthase.