Utilize fermentation of bacillus to produce the method for guanosine
Technical field
The present invention relates to a kind of method of producing guanosine.
Background technology
Guanosine claims guanosine-again, and chemical name is 9-β-D-ribofuranosyl guanine, and English name is Guanosine, molecular formula C
10H
13N
5O
5, molecular weight 283.24, guanosine be white crystalline powder, do not have smell, tasteless; The uv-absorbing peak of the aqueous solution is at 256 μ m, molar extinction coefficient 12.2 * 10
3Dissolving 1 gram in 18 ℃ of following 1320ml water; In the boiling water bath, dissolving 1 gram in the 33ml water; Dissolve in soda acid, be insoluble to organic solvent; Fusing point 237-240 ℃.
Guanosine belongs to the ucleosides micromolecular compound, is mainly used in antiviral, antitumor drug synthetic key intermediate industrial, and the raw material that is used for extensive synthesised food additive 5 '-guanylic acid (GMP).
5 '-guanylic acid is to be the flavor agent strongly, and its delicate flavour is 160 times of monosodium glutamate (Sodium Glutamate).A spot of GMP mixes with monosodium glutamate can improve the monosodium glutamate delicate flavour more than 10 times.GMP by the first-selected additive of varieties of food items producer as lifting product quality and enhancement product special flavour, has been one of main taste composition of senior seasonings such as special delicious sauce, chickens' extract, chicken powder, specially fresh monosodium glutamate, quality improver, flavour agent at present.
The big purposes of another of 5 '-guanylic acid is the health additive as heath food and infant formula.The elderly and patient's nucleic acid manufacturing capacity descend, and suitably add Nucleotide in meals, can delaying senility, build up health.The homemade milk powder and the Imported Milk of Xiao Shouing in the market, nearly all added a certain amount of various Nucleotide (GMP is one of them important component), mainly be at premature infant and LBWI de novo synthesis and the deficiency of remedying synthesizing ribonucleotide, promote growing of baby.The European Community in 1991 stipulates to be limited to CMP2.5mg, UMP1.75mg, AMP1.5mg, GMP0.5mg, IMP1.0mg in the interpolation of Nucleotide in every 420KJ infant or baby food.Ministry of Health of China in 2005 announces that the Nucleotide total addition level in the baby milk powder of recommending is 0.2~0.58g/kg for No. 15.
Guanosine itself also can be used for producing functional foodstuffs such as cold-resistant food, diet food in addition, so guanosine is very big in the demand of food and field of health care products.
Utilize guanosine can synthesize ribavirin (ribavirin) and acyclovir antiviral such as (acycloguanosine).Ribavirin has the obvious suppression effect to dna virus and RNA viruses, is a kind of wide spectrum ucleosides antiviral.Acycloguanosine also is a kind of broad-spectrum antiviral medicament, comprises that for external and intravital simplexvirus hsv HSV-1 and HSV-2, varicella zoster virus (VIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) etc. have restraining effect.Therefore guanosine has wide development prospect at field of medicaments as medicinal intermediates.
The fermentative Production guanosine has advantages such as output is big, the cycle short, control is easy, thereby is widely used at home and abroad.The sixties in 20th century, Japan at first adopted the nucleic acid metabolism mutant strain large scale fermentation of subtilis to produce guanosine.China starts from the eighties in 20th century to the fermentation research of guanosine, the research report is a lot, but makes little progress always, compares with external advanced level to still have big gap, make that the guanosine production cost is higher, become the important bottleneck of domestic flavour nucleotide industry of restriction and nucleotide drug development.
One of reason is that domestic guanosine strain improvement still adopts conventional physical chemistry mutafacient system, makes that the original product glycosides ability of bacterial classification is not high.And abroad on the molecular biology platform, adopt genetic engineering technique seed selection guanosine superior strain.For example CN1046558A reports, Japan Takede Chemical Industries Ltd replaces the promotor of the IMP dehydrogenase gene of bacillus micro-organism chromosomal DNA with a different promotor expressed, the genetic expression of this enzyme is enhanced, and the subtilis NA6242 bacterial strain that obtains can shake flask fermentation accumulation guanosine 24.0 grams per liters.For example CN101563448A reports again, Japan Ajincomoto Co., Inc introduces the bacillus amyloliquefaciens that produces purine nucleoside with purine analog resistant gene yeaS and belongs in the cell, make the expression that is enhanced of the purine nucleoside biosynthetic enzyme genes of bacillus amyloliquefaciens AJ1991 (pLF-YeaS) bacterial strain after the modification, improved inosine and guanosine throughput.
Former two is that domestic guanosine fermentation manufacturing technique level falls behind therefore, only being confined to culture medium prescription adjustment and fed-batch fermentation etc. is the static state operation method of foundation with the optimised process reference mark, ignored the time variation and the importance of metabolic regulation variation of cellular metabolism stream in the fermenting process to fermenting and influencing, make that the product glycosides level on the fermentor tank is not high, transformation efficiency is low.Domestic only have CN101487036A to report, adopts preservation strain subtilis TA208-IMPD, and stream adds the hydrolysis sugar liquid of cracking rice during the fermentation, ferments 50-60 hour, produce glycosides and reach 38 grams per liters, but transformation efficiency has only 20%.Other has CN101492484A to introduce a kind of synthetic circulation production process of guanosine-, adopt microfiltration membrane, ultra-filtration membrane and reverse osmosis membrane to extract the guanosine crystallization, wet thallus is used as feedstuff protein, the mother liquor behind the extraction guanosine is used as molten brilliant water and dialysis water cycle.Present domestic the have multiple dimensioned theory of bibliographical information employing biochemical industry process and the zymotechnique of method research guanosine, by adding the metabolic regulation agent in the fermention medium, the adjustment of combining with fermentation parameter, suppress the accumulation of amino acid, organic acid, ammonium ion, stop the migration of fermentation later stage guanosine metabolism stream, allow more carbon source flow to and produce the guanosine approach, improve the fermentation production rate of guanosine, but do not see the patent report of relevant this method so far.
Along with people are more and more higher to the requirement of high-grade food-flavoring comps, the market demand to flavour nucleotide novel food product fresheners such as (I+G) increases thus; In addition, also quite optimistic by the market outlook of the synthetic various medicines of guanosine and precursor, functional foodstuff.Therefore press for the whole fermentative production state of the art that promotes China's guanosine, strengthen the market competitiveness of China's guanosine, satisfy the growing needs of society.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing fermentation of bacillus production guanosine that improves the fermentation yield and the transformation efficiency of guanosine.
Technical solution of the present invention is:
A kind of method of utilizing fermentation of bacillus to produce guanosine is characterized in that: comprise the following steps: successively
(1) thalline activation
With subtilis CGMCC No.4587 (classification name: subtilis, Latin formal name used at school: Bacillus subtilis, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on January 27th, 2011) streak inoculation is in the nutrient broth agar test tube slant, line is transferred in agar eggplant bottle slant culture maturation then, prepares culture transferring;
(2) seed culture
Be equipped with uniform bacteria suspension with the ripe bacterium on the eggplant bottle inclined-plane is clay, bacteria suspension inserted seeding tank by the inoculum size of 1-10% carry out seed culture;
Or the test tube slant thalline after directly picking one ring activates, be inoculated in triangular flask, shake-flask culture;
(3) fermentation culture
Seed culture fluid inserts triangular flask, shake-flask culture; Or insert fermentor tank and carry out fermentor cultivation;
(4) guanosine extracts
The guanosine fermented liquid after filtration, concentrate, freezing and crystallizing obtains the guanosine crude product.
The guanosine crude product is through the refining guanosine elaboration that obtains.
Substratum when substratum when the nutrient broth agar test tube slant is cultivated in the step (1) and agar eggplant bottle slant culture is: glucose 5g/L, peptone 10g/L, yeast extract paste 10g/L, NaCl 5g/L, agar 20g/L, pH 7.0-7.2, even 32 ℃ of culture temperature, incubation time is 24-48 hour.
Substratum during step (2) seed culture is: glucose 10g/L, yeast extract paste 5g/L, peptone 5g/L, extractum carnis 5g/L; Sodium-chlor 2g/L, RNA0.25g/L, pH 7.0-7.2; Culture condition is during shake-flask culture: reciprocating type shaking table, amplitude 7cm, 108 rev/mins, 34 ℃ of frequencies, 10-18 hour; Culture condition during seed tank culture is: 150-180 rev/min of seeding tank mixing speed, air flow 1-2m
3/ hour, 34 ℃ of temperature, 10-15 hour.
During step (3) fermentation culture, substratum consists of: glucose 100g/L, yeast extract paste 4g/L, corn steep liquor 4g/L, K
2HPO
42g/L, MgSO
40.5g/L, MnCl
20.01g/L, FeSO
40.01g/L, KCl 2g/L, CaCO
320g/L, NH
4Cl 20g/L, pH7.0-7.2; The culture condition of shake-flask culture is: reciprocating type shaking table, amplitude 7cm, frequency 108 rev/mins, 36 ℃, 72 hours; The culture condition of fermentor cultivation is: 100-150 rev/min of fermentor tank mixing speed, air flow 1-2m
3/ hour, 36 ℃, 60-72 hour; Add somatomedin raw material-RNA during fermentation culture, when shake flask fermentation is cultivated the addition of RNA be disposable interpolation be equivalent to the nutrient solution gross weight 0.3%, the interpolation total amount of RNA is to be equivalent to 0.3% of nutrient solution gross weight during fermentor cultivation, ferment and added 0.03% in 0 hour, all the other press F (t)=F (0) * e 0.27% after setting the K value
(kt)Calculate the amount of per hour adding and add, the interpolation time first time wherein is controlled at thalli growth OD in the whole fermentation process
660Add for 1/5 o'clock of the highest number, per hour add once later on, finish until interpolation; The soya-bean oil or the bubble enemy that have added the froth breaking effect in the fermentor cultivation process.
Subtilis Bacillus subtilis JSIM-G518 is a strain metabolism of purine nucleotide mutant strain, purine nucleotides such as VITAMIN B4 there is special nutritional requirement, must in the scope of not obvious inhibition thalli growth, reduce the concentration of VITAMIN B4 in the fermention medium as far as possible.Addition by the restriction VITAMIN B4 makes the concentration of intracellular major control factor adenine kind Nucleotide remain on the concentration that can not cause feedback regulation, thereby helps the accumulation of guanosine.This concentration is to change along with incubation time, and cell proliferation increases, and the concentration of additive is along with becoming big.Nucleic acid RNA is the somatomedin raw material that cell the most easily absorbs.
Advantage of the present invention:
The present invention has adopted the metabolism of purine nucleotide mutant strain Bacillus subtilis JSIM-G518 of a bacillus subtilis to carry out the fermentative production of guanosine.The stabilization characteristics of genetics of this bacterial strain is blocked and strengthens about the key enzyme of guanosine biosynthetic pathway, and tunning is single, does not accumulate other ucleotides materials.
The present invention is according to the metabolic regulation theory of biological nucleic acid route of synthesis, special nutrition demand at Bacillussubtilis JSIM-G518, add nucleic acid RNA as growth promoter in the seed culture stage, improve the biomass of seed culture, helped the product accumulation of follow-up fermentation culture.
The present invention is main purpose in fermentation culture stage with the accumulation product according to the metabolic engineering theory, adopts exponential Function Model F (t)=F (0) exp (kt) (k ≠ 0), { F (t)=F (0) * e
(kt)Carry out the meticulous feed supplement of key growth factors nucleic acid RNA, this feed supplement mode is a time variation, between cell growth and product accumulation, keep running balance, thereby removing the biosynthetic metabolic regulation effect of guanosine to greatest extent, significantly improving guanosine fermentation yield and transformation efficiency.
Contain materials such as a large amount of thalline and albumen in the guanosine fermented liquid, can hinder the extraction of guanosine, influence purity, color and luster, the crystallization velocity of guanosine, reduce extract yield.The centrifugal settling method apparatus expensive, and energy consumption is very big; Membrane filtration is a very fast isolation technique of development in recent years, thalline and albumen clearance height, but at present ultra-filtration membrane and reverse osmosis membrane because material expensive, regeneration difficulty, filtering rate slow, easily stop up, and can't the heavy industrialization application.The ceramic membrane aperture is greater than ultra-filtration membrane and reverse osmosis membrane, and is simple to operate, and use cost is cheap, be easy to promote.The present invention removes thalline, flocculating settling with Plate Filtration and removes albumen, ceramic membrane filter removal of impurities, concentrating under reduced pressure and freezing and crystallizing etc. and separate extractive technique and combine, and the reasonable combination design by operation, extracts the guanosine elaboration from fermented liquid with high yield.
The invention will be further described below in conjunction with embodiment.
Subtilis CGMCC No.4587 (classification name: subtilis, Latin formal name used at school: Bacillus subtilis, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on January 27th, 2011).
Embodiment
Embodiment 1:
(1) thalline activation
In the nutrient broth agar test tube slant, line is transferred in agar eggplant bottle slant culture maturation then, prepares culture transferring with subtilis CGMCC No.4587 streak inoculation; Substratum when substratum when the nutrient broth agar test tube slant is cultivated and agar eggplant bottle slant culture is: glucose 5g/L, peptone 10g/L, yeast extract paste 10g/L, NaCl 5g/L, agar 20g/L, pH 7.0-7.2, even 32 ℃ of culture temperature, incubation time is 24-48 hour.
(2) seed culture
The inclined-plane refrigeration bacterial classification of subtilis Bacillus subtilis JSIM-G518, streak inoculation is in the nutrient broth agar test tube slant, slant medium consists of: glucose 5g/L, peptone 10g/L, yeast extract paste 10g/L, NaCl 5g/L, agar 20g/L, pH 7.2, cultivated 48 hours, and be maturation for 32 ℃.
On the clean platform of the behaviour of sterilisable chamber, picking one encircles the test tube slant thalline after the activation, is inoculated in the seed culture medium interior (500ml triangular flask, liquid amount 30ml) of the bacterium of having gone out.Seed culture medium consists of: glucose 10g/L, yeast extract paste 5g/L, peptone 5g/L, extractum carnis 5g/L, sodium-chlor 2g/L, RNA0.25g/L, 7.2,121 ℃, 1 kilogram pressure of pH, sterilization 15 minutes.
The seed culture condition is: reciprocating type shaking table, amplitude 7cm, frequency 108 rev/mins, 34 ℃, 10 hours.As growth OD
660Reach at 0.7 o'clock, i.e. culture transferring.
(3) fermentation culture
Seed culture fluid inserts with the inoculum size of volume ratio 10% (or 1%, 5%) in the fermention medium of the bacterium of having gone out (500ml triangular flask, liquid amount 20ml).Fermention medium consists of: glucose 100g/L, yeast extract paste 4g/L, corn steep liquor 4g/L, K
2HPO
42g/L, MgSO
40.5g/L, MnCl
20.01g/L, FeSO
40.01g/L, KCl 2g/L, CaCO
320g/L, NH
4Cl 20g/L, 7.2,121 ℃, 1 kilogram pressure of pH, sterilization 10 minutes; Add somatomedin raw material-RNA during fermentation culture, the addition of RNA be disposable interpolation be equivalent to the nutrient solution gross weight 0.3%.
The shake flask fermentation culture condition is: reciprocating type shaking table, amplitude 7cm, frequency 108 rev/mins, 36 ℃, 72 hours.After the fermentation ends, the guanosine output in the electrophoresis technique determining nutrient solution reaches 20.10g/L, transformation efficiency 20.10%.
Embodiment 2:
Step (1) is with embodiment 1.
Step (2) seed culture
The inclined-plane refrigeration bacterial classification of subtilis Bacillus subtilis JSIM-G518, streak inoculation is in the nutrient broth agar test tube slant, slant medium consists of: glucose 5g/L, peptone 10g/L, yeast extract paste 10g/L, NaCl 5g/L, agar 20g/L, pH cultivated 48 hours for 7.2,32 ℃.Line is transferred in agar eggplant bottle inclined-plane (substratum is formed identical with the test tube slant) then, cultivates 48 hours, and is maturation for 32 ℃.
Prepare the aseptic triangular flask of an interior glaze pearl, with sterilized water the ripe bacterium mud on the eggplant bottle inclined-plane is scraped and washed, the triangular flask of packing into, vibrating dispersion bacterium mud obtains uniform bacteria suspension; Bacteria suspension is pressed 100 liters of seeding tanks of inoculum size access of 10%, and the loading amount coefficient of seeding tank is 70% (V/V).Seed culture medium consists of: glucose 10g/L, yeast extract paste 5g/L, peptone 5g/L, extractum carnis 5g/L; Sodium-chlor 2g/L, RNA0.25g/L, 7.2,121 ℃, 1 kilogram pressure of pH, sterilization 15 minutes.
The seed tank culture condition is: 180 rev/mins of mixing speed, air flow 2m
3/ hour, temperature 34 ℃, 15 hours.As growth OD
660Reach at 0.7 o'clock, i.e. culture transferring.
(3) fermentation culture
Seed culture fluid inserts 100 liter fermentor tanks with the inoculum size of volume ratio 10%, and the loading amount coefficient of fermentor tank is 70% (V/V).
The fermentor cultivation condition is: 100~150 rev/mins of mixing speed, air flow 1~2m
3/ hour, 36 ℃, 65 hours.The bubble enemy (V/V) who adds fermentating liquid volume 0.001% in the fermentor cultivation process.Fermention medium consists of: glucose 100g/L, yeast extract paste 4g/L, corn steep liquor 4g/L, K
2HPO
42g/L, MgSO
40.5g/L, MnCl
20.01g/L, FeSO
40.01g/L, KCl 2g/L, CaCO
320g/L, NH
4Cl 20g/L, 7.2,121 ℃, 1 kilogram pressure of pH, sterilization 10 minutes.
The interpolation total amount of RNA is to be equivalent to 0.3% of nutrient solution gross weight in the fermentation culture process, ferments to add 0.03% in 0 hour, and all the other press F (t)=F (0) * e 0.27% after setting K value=0.12
(kt)Calculate the amount of per hour adding, be listed in the following table 1.The interpolation time adds after 8 hours for fermentation for the first time, per hour presses the amount of table 1 regulation later on and adds once, finishes until interpolation.
Nucleic acid RNA feed supplement timetable during table 1K=0.12
Ferment by above-mentioned fermentation parameter and supplying technics, put jar after the fermentation ends, the guanosine output in the electrophoresis technique determining fermented liquid is 32.55g/L, and transformation efficiency is 32.55%.
Embodiment 3:
1, collect guanosine fermented liquid 70 liters, send in the storage tank, chuck is heated to about 95 ℃, is incubated 15 minutes; Take advantage of heat to filter filter pressure 0.40Mpa with plate-and-frame filter press.Thalline uses as feedstuff protein, and filtrate is sent in the storage tank;
2, the natural flocculating agent ZTC-II (or in the chitosan, chitin, sodium alginate a kind of) (being made into the solution of weight concentration 1% in advance) that in filtrate, adds 0.2% volume, 32 ℃ of chuck insulations, flocculating settling was got supernatant liquor and is sent in the storage tank to remove albumen in 12 hours.
3, supernatant liquor is crossed ceramic membrane and handled 75 ℃ of membrane filtration temperature; 60 ℃ of reduced vacuum of filtrate are concentrated into 1/5 volume.
4, concentrated solution is regulated PH7.0 after, deliver in the crystallizer, stir crystallization 12h under 4 ℃ of conditions.
5, filter with plate-and-frame filter press, filter pressure 0.35Mpa obtains the wet crude product of guanosine.
6,, add water with the wet dissolving crude product of guanosine in 1: 50 ratio (weight ratio); Lysate adds the pharmaceutical powder gac of 5% volume, stirs decolouring 30 minutes under 95 ℃ of conditions.Take advantage of heat to filter filter pressure 0.30Mpa with plate-and-frame filter press.Contain assorted gac manipulation of regeneration, filtrate is sent in the concentration tank.
7, filtrate is concentrated into 1/10 volume, behind the adjusting PH7.0, delivers in the crystallizer, stir crystallization 12h under 4 ℃ of conditions.
8, filter with plate-and-frame filter press, filter pressure 0.35Mpa obtains the wet elaboration of guanosine.
9, the wet elaboration of guanosine with twin-roll scraper-type drying machine drying, is pulverized under 60 ℃ of conditions, sieves, and obtains the guanosine finished product, and product purity is 98%, and extract yield is 82%.