CN100465282C - Method for synthesizing adenosine methionine utilizing biological catalysis - Google Patents

Abstract

This invention relates to a method for synthesizing adenosine methionine by the biological catalysis method characterizing that the catalysis synthesizing reaction and salifiable reaction of the product of the adenosine methionine are combined in one step and the adsorption callback and purification of the product of the catalyst are combined as one to greatly simplify the production technology, reduce the production period by 40% and reduce the cost of production.

Description

Utilize the method for biological catalysis synthesizing adenosine methionine

Technical field

The present invention relates to the production method of ademetionine.

Background technology

Ademetionine SAM is present in all organisms from the unicellular lower eukaryote bacterium to the high multicellular organism mankind, is playing the part of important biological function in cell and body metabolism process.Ademetionine exists with two kinds of isomers forms:

(S，S)S-adenosyl-L-methionine?and(R，S)S-adenosyl-L-methionine。Its main biological function comprises: (it is thymus nucleic acid, protein, hormone, catechol (Catechol) in the body as methyl donor in the transmethylase reaction in vivo, indoles amine (Indoleamine), the biosynthesizing of phosphatidylethanolamine (Phosphatidylethanolamine) and phosphatidylcholine (Phosphatidylcholine) provides methyl); It participates in biochemical reaction generation Methylthioadenosine and homocysteine as the substrate of lyase; Take off behind the shuttle in vivo for the biosynthesizing of the many ammonia of neural mediator agent (polyamines spermidine and spermine) provides aminopropyl, for the biosynthesizing of tRNA provides ammonia butyl precursor; It also participates in the synthetic enzymatic reaction of vitamin H (Biotin) as coenzyme, for the biosynthesizing of vitamin H provides precursor; Participate in the biosynthesizing and the metabolism of hormone in vivo, neurotransmitter, nucleic acid, protein and phosphatide.As seen ademetionine is to safeguard cytolemma normal function and human homergy and healthy indispensable important living matter.SAM also participates in main antioxidant gsh synthetic in the cell, and is closely related with anti-oxidant in the body and various detoxification processes.

Since nineteen fifties was found, people had carried out a large amount of research to the chemical property and the biological function of ademetionine.Up to the present existing adenomethionine synthase gene from tens species is by molecular cloning, comprising intestinal bacteria, Bacillus subtilus, yeast, neurospora, rat and paddy rice etc.

Two a large amount of animals and clinical experiments European during the last ten years and North America the world of medicine show that ademetionine can promote and safeguard the normal function of liver, and multiple hepatitis and liver failure are had the obvious treatment effect; Ademetionine can promote the formation and the wound healing of cartilaginous tissue, safeguards the health in bone joint, and various sacroiliitis and joint injury are had obvious curative effects; Ademetionine participates in the biosynthesizing of phosphatide and polyamines, and is most important to the normal function of safeguarding normal permeability of cytolemma and axoneure.Ademetionine can be regulated the cental system function, and dysthymia disorders is had notable therapeutic effect.Ademetionine content obviously reduces in senile dementia patient's cerebrospinal fluid, and ademetionine content also significantly reduces in the Parkinsonism blood samples of patients.Clinical trial confirms that also ademetionine can obviously alleviate senile dementia.

The eighties people began one's study and utilized the recombinant adenosine methionine synthase at external catalysis synthesizing adenosine methionine last century, for the enzymatic scale operation of ademetionine is done a lot of work.

Although the clinical value of the critical function of ademetionine in body metabolism and it, ademetionine under normal temperature condition water absorbability and the complicacy of unstable and production technique seriously limited the widespread use of ademetionine at medical health field.Therefore, multinomial patent is being declared aspect the stable salify of ademetionine and the production technique by American-European scientific and technological circle.Below be some representative on medicine material market patents: as United States Patent (USP) 6,635,615,5,102,791,4,028,183,4369177,4764603 etc.

Traditional ademetionine production realizes that by yeast fermentation the shortcoming of yeast fermentation process is that feed stock conversion is low, and life cycle of the product is long, and production cycle of fermentation method needs 10 day time, finished product separation and purification program complexity at least.The complexity of production technique has caused costing an arm and a leg of ademetionine product.

Summary of the invention

The invention provides a kind of method of utilizing the biological catalysis synthesizing adenosine methionine.Low to solve the feed stock conversion that exists by yeast fermentation process production ademetionine, life cycle of the product is long, finished product separation and purification program complexity, expensive problem.

The technical scheme that the present invention takes is:

By L-methionine(Met) (L-methionine) and Triphosaden (the product ademetionine that the reaction of adenosine 5 '-triphosphate) forms in catalystic converter system directly and organic sulfonic acid react salify, wherein the concentration of organic sulfonic acid in reaction system is 0.1-0.5mol/L.

The PH scope that building-up reactions, salt-forming reaction, catalyst recovery, crystallization reaction adopt among the present invention is 1-7, the concentration of potassium, magnesium ion be 20-200 the milli rub/liter, organic sulfonic acid concentration be 50-500 the milli rub/liter, L-methionine(Met) concentration be 1-500 millis rub/liter, the mass ratio of weakly base resin such as carboxymethyl cellulose and catalyzer is 1:1-10:1.

The gene of encoding human catalyzer adenomethionine synthase (EC2.5.1.6) is to be obtained by chemical synthesis process among the present invention, and the expression of synthetic enzyme is regulated and control by promotor TAC.

Recycling and reusing of catalyzer is to realize by carboxymethyl cellulose resinoid absorption solidification among the present invention.

The non-covalent absorption solidification of biological catalyst has obviously increased the catalytic activity and the stability of adenomethionine synthase among the present invention.

The separation of catalysis end product adenosylmethionine combines with the absorption solidification process of synthetic enzyme among the present invention, reaches the purifying of product when making catalyst recovery.

The present invention combines with the product salify owing to catalysis is synthetic, and catalyst recovery combines with product purification, product salt crystal stability height, and this processing method has obviously reduced the synthetic production cost of producing ademetionine of enzymatic.

Application of cold temperature crystallization method of the present invention obtains high stable adenosylmethionine crystal formulations, and the crystalline condition is: 1-500 millis adenosylmethionine that rubs/rise adds 1-10 times of acetone to the stoste volume, potential of hydrogen PH1-7,0-20 degrees centigrade of temperature.

The feedback inhibition that the present invention lives to enzyme for product is overcome by the reorganization inorganic pyrophosphatase.

The transformation efficiency of reaction raw materials L-methionine(Met) in system surpasses 95%, reacts the back synthetic enzyme rate of recovery greater than 90% at every turn, productive rate 60%-80% after the salify crystallization, and purity is greater than 95%.

The present invention utilizes genetic engineering technique and protein biochemistry technology to prepare high-activity biological catalyzer adenomethionine synthase earlier; Utilize the biocatalysis engineering that catalytic condition, product separating technique, the product of ademetionine building-up reactions are optimized the stable recycling technology that overcomes technology and catalyzer of catalyst activity feedback inhibition again; Utilize pharmaceutical chemistry and drug preparation technique when increasing product stability, to simplify product salify, purifying and catalyst recovery program at last.

The present invention utilizes genetic engineering technique and enzyme engineering technology that the adenomethionine synthase gene that chemical synthesis obtains is utilized efficient prokaryotic expression systems produce recombinant adenosine methionine synthase, the pure specific enzyme activity of recombinant adenosine methionine synthase is up to 2-5IU/ milligram, can be with greater than 95% reaction raw materials: L-methionine(Met) and ATP are converted into ademetionine under the condition that PH5-9,15-35 degree centigrade and small amounts of inorganic Pyrophosphate phosphohydrolase exist; And the transformation efficiency that yeast fermentation is sent out synthesizing adenosine methionine only is 15-30%, direct and the organic many sulfonic acid salify of the product that forms, after catalyzer was reclaimed by anionite-exchange resin such as carboxymethyl cellulose absorption, product ademetionine purity was nearly 90%, and purity surpasses 95% behind the low temperature crystallization.The present invention is with short production cycle, and production cycle is 3 day time only, and technology is simple, amplifies simple and feasible.Catalyst recovery and purifying products technology in the ademetionine scale production that the present invention is further perfect simplified production technique greatly on original catalysis technique basis, reduced production cost, improved the practicality of biocatalysis technology in scale operation ademetionine product.

Embodiment

Experiment material and method:

1, engineering bacteria and reagent:

The bacillus coli gene recombinant bacterial strain, this bacterial classification has been submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation to, address: Zhongguangcun, Haidian District, Beijing City, preservation date on October 17th, 2005, deposit number: CGMCC No.1492, the classification name: colon bacillus Escherichia coli, carry chemosynthesis adenomethionine synthase (EC2.5.1.6) gene.

Reagent all is homemade analytical pure level.It is standby that each reagent is made into following concentration.1mol/l borate or N-2-hydroxyethyl piperazine-N '-2 ethane sulfonic aicd HEPES damping fluid, the pH value is 7.5 ± 1; 1mol/l K ₂SO ₄1mol/l MgSO ₄0.4mol/l adenosine triphosphate atp; 0.2mol/l L-methionine(Met); Paratoluenesulfonic acid sodium salt; To 1,4-butane sodium disulfonate; 99% beta-mercaptoethanol; 0.5mol/l edta edta; Deionized water.The equal hole of carboxymethyl cellulose CM cellulose weak base anion-exchange resin (SIGMA).

2, substratum

A substratum: peptone 12g/l; Yeast powder 24g/l, K ₂HPO ₄9.4g/L, KH ₂PO ₄2.2g/l Glycerol 0.2%.Mentioned reagent with deionized water dissolving after, sterilization is 20 minutes under 120 ℃, 16PSI pressure.Add amine Bian penicillin before the inoculation to final concentration 25 μ g/ml.

The B substratum: sterilize behind the agar of adding 1.5% in the A substratum, condition adds amine Bian penicillin with the A substratum before not solidifying after the sterilization, and final concentration is 25 μ g/ml, promptly makes Agar Plating after solidifying.

3, fermentation

With above-mentioned Agar Plating, just the adenomethionine synthase genetic engineering bacterium of cultivating on the B substratum is provoked single bacterium colony, inserts in the 40ml A substratum (containing 25 μ g/ml amine Bian penicillin), and 37 ℃ of shaking tables were cultivated 16 hours.Above bacterium liquid is inoculated in the A substratum of 6L (containing 25 μ g/ml amine Bian penicillin), 37 ℃ of shaking tables were cultivated about 20 hours, surveyed the OD of bacterium liquid ₆₀₀More than=1.8.Secondary is cultivated the bacterium liquid of collecting the back carry out centrifugally, rotating speed is 4000rpm, centrifugation time 10 minute hands, and abandoning supernatant, the thalline of collecting precipitation ,-70 ℃ are frozen.

4, Preparation of Catalyst

The engineering bacteria thalline (check back target protein should account for more than 20% of total protein) that fermentation is obtained suspends with the HEPES damping fluid, every gram cell mud is with 4 milliliters of damping fluids, bacterium liquid after the suspension utilizes clarifixator to carry out fragmentation, the pressure that behaviour does is 60MPa, temperature is controlled at below 15 ℃, circulates twice.Collection suspension carries out centrifugal, and rotating speed is 18000rpm, and temperature is 4 ℃.It is standby that the supernatant liquor that contains catalyzer is collected in centrifugal back, uses in 24 hours to be stored in 4 ℃, and standing storage should be at-20 ℃.

Embodiment one enzyme catalysis ademetionine building-up reactions

With one liter reaction system is example:

1, adds reagent

Add the above-mentioned reagent for preparing respectively: 50 milliliters of N-2-hydroxyethyl piperazine-N '-2 ethane sulfonic aicd HEPES damping fluid; 1mol/l K ₂SO ₄100 milliliters; 50 milliliters of 1mol/l MgSO4; 0.4mol/l 20 milliliters of adenosine triphosphate atps; 0.2mol/l 60 milliliters of L-methionine(Met); Paratoluenesulfonic acid sodium salt 77 grams; 1 milliliter of 99% beta-mercaptoethanol; 0.5mol/l 2 milliliters of edta edtas; 0.001-0.01 unit (IU) inorganic pyrophosphatase contains the catalyzer supernatant liquor of 20-30 unit (IU), deionized water is settled to 1 liter.

2, temperature of reaction and time

Above-mentioned mixed solution is added in the reactor, and it is 35 ℃ that temperature of reaction is set, 10 hours reaction times.

3, stopped reaction

It is the 0.05mol/L stopped reaction that reaction finishes back adding sulfuric acid to final concentration.

Embodiment two enzyme catalysis ademetionine building-up reactionss

With one liter reaction system is example:

1, adds reagent

Add the above-mentioned reagent for preparing respectively: 60 milliliters of borate buffers; 1mol/l K ₂SO ₄90 milliliters; 60 milliliters of 1mol/l MgSO4; 0.4mol/l 20 milliliters of adenosine triphosphate atps; 0.2mol/lL-60 milliliters of methionine(Met); To 1,4-butane sodium disulfonate, 70 grams; 1 milliliter of 99% beta-mercaptoethanol; 0.5mol/ 2 milliliters of state EDTA; Deionized water is settled to 1 liter.

2, temperature of reaction and time

Above-mentioned mixed solution is added in the reactor, and it is 30 ℃ that temperature of reaction is set, 10 hours reaction times.

3, stopped reaction

It is the 0.05mol/L stopped reaction that reaction finishes back adding sulfuric acid to final concentration.

Embodiment three catalyst recovery

1, adds the weakly alkaline ion and return resin

Add the equal hole of CM cellulose weak base anion-exchange resin in the above-mentioned mixed solution after catalyzed reaction stops, final concentration is 50g/l, transfers PH to 5.5,20 ℃ of temperature, the slight stirring～30 minutes.

2, centrifugal

It is centrifugal to get the 1.0mL sample, can determine behind supernatant uv-absorbing A280/A260＜1.0 that synthetic enzyme absorption finishes.Sample is through centrifugal 15 minutes of 4 ℃ of following 3000rpm, and precipitation both can be carried out catalyzed reaction again through the 50 millis HEPES damping fluid (pH7.5) that rubs after resuspended.Supernatant detects the ademetionine purity salt through HPLC and reaches 90%.

Concentrating and crystallization (1) of embodiment four product adenosylmethionines

Synthetic enzyme through absorption reclaim the back supernatant liquor through the sodium filter be concentrated into the ademetionine salt concn for～90 rub in the least/can drop into acetone behind the L carries out crystallization.Acetone is 5 to the volumetric ratio of ademetionine salts solution, and potential of hydrogen is a PH basis 5.0, and temperature is 10 degrees centigrade.Crystal by alcohol flushing after the subcooling drying.Crystal prototype detects purity through HPLC, electrospray ionization mass spectrum, carbon-13 nmr spectra and reaches 95%.

Concentrating and crystallization (2) of embodiment five product adenosylmethionines

Synthetic enzyme supernatant liquor after absorption is reclaimed is concentrated into the ademetionine salt concn through the sodium filter and rubs/L for～100 millis.The amount that drops into acetone is: acetone is 10 to the volumetric ratio of ademetionine salts solution, and potential of hydrogen is a PH basis 3.0, and temperature is 10 degrees centigrade.Crystal by washed with methanol after the subcooling drying.Crystal prototype detects purity through HPLC, electrospray ionization mass spectrum, carbon-13 nmr spectra and reaches 95%.

Claims (4)

1, a kind of method of utilizing the biological catalysis synthesizing adenosine methionine, react product the ademetionine direct and organic sulfonic acid reaction salify in catalystic converter system that forms by L-methionine(Met) and Triphosaden, wherein the concentration of organic sulfonic acid in reaction system is 0.1-0.5mol/L, it is characterized in that: the gene of encoding human catalyzer adenomethionine synthase (EC2.5.1.6) is to be obtained by chemical synthesis process, and the expression of synthetic enzyme is regulated and control by promotor TAC.

2, the method for utilizing biological catalysis catalysis synthesizing adenosine methionine according to claim 1, it is characterized in that: recycling and reusing of catalyzer is to realize by carboxymethyl cellulose resinoid absorption solidification, the non-covalent absorption solidification of biological catalyst has obviously increased the catalytic activity and the stability of adenomethionine synthase, the separation of catalysis end product adenosylmethionine combines with the absorption solidification process of synthetic enzyme, reaches the purifying of product when making catalyst recovery.

3, the method for utilizing biological catalysis catalysis synthesizing adenosine methionine according to claim 1, it is characterized in that: the application of cold temperature crystallization method obtains high stable adenosylmethionine crystal formulations, the crystalline condition is: 1-500 milli adenosylmethionines that rub/rise, add 1-10 times of acetone to the stoste volume, acidity-basicity ph value 1-7,0-20 degrees centigrade of temperature.

4, the method for utilizing biological catalysis catalysis synthesizing adenosine methionine according to claim 1 is characterized in that: the feedback inhibition that product is lived to enzyme is overcome by the reorganization inorganic pyrophosphatase.