CN1322000C - Diabetin-1, 6-diphosphonie acid as well as preparation method and application - Google Patents
Diabetin-1, 6-diphosphonie acid as well as preparation method and application Download PDFInfo
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- CN1322000C CN1322000C CNB031007244A CN03100724A CN1322000C CN 1322000 C CN1322000 C CN 1322000C CN B031007244 A CNB031007244 A CN B031007244A CN 03100724 A CN03100724 A CN 03100724A CN 1322000 C CN1322000 C CN 1322000C
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- fructose
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- diphosphate
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a method for preparing fructose-1, 6-bisphosphatase, which is characterized in that a solution with fructose contacts immobilized yeast so that the fructose in the solution with fructose is converted to form the fructose-1, 6-bisphosphatase. The present invention also relates to fructose-1, 6-bisphosphatase prepared by using the method and applications of the fructose-1, 6-bisphosphatase.
Description
Technical field
The present invention relates to biochemical field, more particularly relate to fructose-1,6-diphosphate.The invention still further relates to the preparation method of fructose-1,6-diphosphate.The present invention also relates to the purposes of fructose-1,6-diphosphate.
Background technology
Fructose-1,6-bisphosphate (FDP) is an intermediate product of keeping needed by human in the metabolism of human body normal function, and the cytolemma that can see through the damaged cell enters into cell interior and directly improves energy, can improve the cellular energy metabolism, quicken the cellular-restoring normal function, rapidly the relieving fatigue state.
The sodium salt of fructose-1,6-diphosphate and calcium salt can have the effect of regulating some enzymic activitys, active cells, raising body adaptability to changes in the carbohydrate metabolism rapidly for cell provides energy.
On the pharmacology, exogenous FDP can't cross over cytolemma usually, but can act on cytolemma, by stimulating the activity of phosphofructokinase, poly-energy-rich phosphate pond in the cell, the interior ATP concentration of cell of increasing promotes stream in the potassium ion, recover the cell polarized state, thereby be of value under the states such as shock, ischemic, damage, body-internal-circulation, blood transfusion the cellular energy metabolism and to the utilization of glucose, repair promoting, improve function.
FDP can effectively prevent outer circulation to erythrocytic damage, and increases the generation of ATP in the body, improves the utilization of hypoxic cell to glucose, be of value to red corpuscle and discharge oxygen, so can increase the sections blood supply of ischemia lower limb, improve muscle function, alleviate in the rest and the post exercise limbs pain.
FDP also can be used for hypoglycemia, fetal anoxia, crapulent rescue, cardiac surgery's extracorporeal circulation assisting therapy.The diabetic subject uses FDP to have good curative effect, and the degree of depth jaundice that various hepatitis are caused, transaminase raise and hang down the albumen leukemia simultaneously also therapeutic action preferably.
Be used for healthy people, but the FDP Ginseng Extract, improve energy, improve eyesight.
This series products is the active drug of treatment cardiovascular diseases, has to utilize to improve myocardial ischemia, to rescuing urgent patients such as hemorrhagic shock, heart failure, liver and kidney failure effect is preferably arranged.Along with the expansion of clinical use, the domestic market sales volume can increase to tons up to a hundred from several tons.
Western developed country painstaking effort systemic disease sickness rate and onset diabetes rate China are high, and therefore FDP is at first in the listing of industrial powers such as Italy, the U.S. and Japan over year surplus in the of 10, and FDP obtained drugs approved by FDA in 1991, and international demand is very big.
At present, domestic heart failure patient, hypoglycemia patient, diabetic subject if there is 30% people to use the FDP treatment, estimate year usage quantity at the hundreds of ton in above-mentioned three kinds of patients more than several ten million people, and market potential is very big.
Since 1905, Britain scientist Harden-Young found after the important physiological function and meaning of FDP in human body, and Italian FOX Ka Ma company has at first successfully developed the FDP injection liquid, but does not see its relevant FDP preparation method's report.Fructose-1,6-diphosphate preparation method in the past adopts the phosphorylation of glucose mostly, and for example referring to Chinese patent application 93110528.5, this patent has been described the use dry yeast glucose and inorganic phosphorus are converted into FDP.The raw material sugar that this method is used is pure glucose, and the dry yeast activity is low and consumption is bigger, and generally speaking cost is higher.Again in Chinese patent application 98112858.0, be described to and use that head mold, colter are mould, Mucor is to containing glucose, K
2HPO
4, MgCl
2Solution ferment and obtain FDP.Similarly, the raw material sugar that this method is used is pure glucose, and cost is higher; And employed head mold, colter cellular component and some composition in the meta-bolites mould, Mucor are harmful.
Therefore, existence is for the demand of more effective, the safer method for preparing FDP.
The employed yeast of the application can be grown on simple and cheap substratum and be reached high cell concentration.The more important thing is that zymic cellular component and metabolite are nontoxic to human body.Among the present invention the yeast thalline has been carried out the immobilization processing, thus lower to the consumption of yeast thalline, and help serialization scale operation, shortened the process time, the constant product quality raising.Facility investment is less, and discharging of waste liquid is few, can reduce production costs significantly and protects environment.And the raw material that the present invention uses is fructose or fructose syrup, causes transformation efficiency to be higher than prior art.
Summary of the invention
First aspect of the present invention is about a kind of production method, and this method adopts yeast to come biosynthesizing FDP as bacterial classification.Described method mainly comprises with yeast and fructose and inorganic phosphorus effect, fructose is converted into fructose-1,6-diphosphate.
Second aspect of the present invention is about a kind of production method, and this method adopts yeast to come biosynthesizing FDP as bacterial classification.Described method mainly comprises two stages, and the fs is the preparation fructose syrup, and subordinate phase is that fructose is converted into fructose-1,6-diphosphate.
The 3rd aspect of the present invention is fructose-1,6-diphosphate.This fructose-1,6-diphosphate adopts yeast to be transformed from the fructose syrup in fructose soln or multiple source, and wherein fructose-1,6-diphosphate content is higher, does not contain harmful composition.
The 4th aspect of the present invention is the purposes about fructose-1,6-diphosphate.Fructose-1,6-diphosphate not only can add in the multiple medicine as medicine intermediate, and can be used as the composition of healthcare products, can also add in some makeup or the food.Fructose-1,6-diphosphate of the present invention also can add in the animal-feed.
In an embodiment of the method for the invention, fructose-1,6-diphosphate is to contact with fixed yeast by the solution that will contain fructose, fructose in the described solution that contains fructose is transformed obtain.Used at this, " contact " is meant after the suitable mixing of two kinds of working substances, places effect for some time under the certain condition." solution that contains fructose " comprises pure fructose soln, contains the slurries of fructose, for example uses the fructose syrup of glucose solution through glucose isomerase effect formation, perhaps contains the fermented liquid of fructose.These contain in the solution of fructose and contain: 1) fructose; 2) phosphate radical; 3) magnesium ion; And other composition, comprise other composition of having a mind to other composition of interpolation and existing naturally.
In the described solution that contains fructose, the content of fructose is the described 10-25wt% that contains the solution of fructose, is benchmark with the described gross weight that contains the solution of fructose; The content of phosphate radical is 0.3-0.5mol/L, is benchmark with the described cumulative volume that contains the solution of fructose; The content of magnesium ion is 0.01-0.03mol/L, is benchmark with the described cumulative volume that contains the solution of fructose.
The described solution that contains fructose can contain the slurries of fructose by at pure fructose soln, for example uses glucose solution through the fructose syrup that the glucose isomerase effect forms, and perhaps contains in the fermented liquid of fructose to replenish phosphate radical and magnesium ion preparation.
Phosphate radical is that inorganic phosphate ionization in water forms, and also can be that phosphoric acid salt dissolve generation in water, and these phosphoric acid salt can be Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, trimagnesium phosphate etc., also can be at least a or any at least two kinds mixture in them.
Magnesium ion is that magnesium salts dissolving forms, and example includes but not limited to magnesium chloride, sal epsom, trimagnesium phosphate etc., also can be at least a or at least two kinds mixture arbitrarily in them.
Described fixed yeast is preparation like this: 1) culturing yeast bacterium; 2) yeast is adsorbed onto on the resin, becomes fixed yeast.Its typical preparation method is: yeast is inoculated in to shake cultivates 5-8h in the bottle, per minute is with 130-140 vibration, move into the seeding tank enlarged culturing then, substratum is selected the Semen Maydis powder saccharification liquid that contains sugared 3.5-4.5% for use, pH value 6.2-6.4, and temperature is 30-33 ℃, cultivate 15-18h, collect fermented liquid, adsorb fermented liquid with anionite-exchange resin, preparation contains saccharomycetic resin.
Another embodiment of the method for the invention is: at first be that starch-containing material transforms fructose; Be that the solution that contains fructose contacts with fixed yeast then, will contain fructose in the solution of fructose and transform and obtain fructose-1,6-diphosphate.
Described " starch-containing material " is meant the material of rich in starch, such as but not limited to corn, cassava, potato, wheat, barley etc.These materials can exist with forms such as particle, meals.Be preferably fine form of powder.Their water content can have multiple variation.
Described fructose is preparation like this: 1) starch-containing material is converted into glucose; 2) glucose isomerization forms fructose.Wherein in step 1) the polysaccharide in the plurality of raw materials is converted into fructose, this process can adopt enzyme process, acid system or biological process, preferably adopts biological process.Wherein in step 2) in utilized glucose isomerase, also abbreviate isomerase sometimes among the present invention as.Can add this enzyme in solution or make immobilized enzyme and use.If the employing fixed glucose isomerase behind the dress post, is called immobilized enzyme isomery post or is called for short the isomery post.
Described acid system is meant acid-hydrolysis method, is that the mineral acid with hydrochloric acid and so on is hydrolyzed to starch.In rare starch suspension, acid hydrolysis almost is converted into glucose fully with starch.In actual process, be unpractical with rare starch suspension, this class acid hydrolysis is carried out containing under the condition of 20% starch suspension, pH2 and 140 ℃ usually.With yellow soda ash neutralizing hydrolysis thing (result forms inorganic salt), refining then glucose syrup.By glucose syrup being placed alkaline condition and hot environment, chemical conversion glucose is fructose.
Described enzyme process is meant and utilizes enzyme to obtain fructose syrup, can be divided into full-enzyme method and hybrid system.Full-enzyme method refers to that whole process all uses enzyme, and hybrid system refers to that certain stage uses enzyme.When being converted into dextrin and sugar, starch can use three kind of starch lytic enzymes, i.e. Alpha-starch lytic enzyme, glucoamylase, Starch debranching enzyme and other debranching factor.Can adopt the D-glucose isomerase to finish during for fructose syrup conversion of glucose.These methods can be used in combination with above-mentioned acid-hydrolysis method, i.e. hybrid system.Also can use immobilized enzyme.Immobilized enzyme is called water desmoenzyme or solid enzyme again, is meant water-soluble enzyme after physics or chemical process are handled, and becomes water-fast but still has the derivative of a kind of enzyme of enzymic activity; It acts on substrate with solid state shape in catalyzed reaction.
Described biological process is meant and utilizes organism to prepare fructose syrup.The microorganism of secreting amylase and the microorganism of secretion allomerase perhaps also can be able to be used engineering bacteria, starch-containing material is carried out combined action, make it be converted into the slurries that contain fructose.
Yeast is a classification in the Mycophyta biology, for example comprises yeast belong (Saccharomycesspp.), as yeast saccharomyces cerevisiae (S.cerevisiae), and Ka Ersibai yeast (S.carlsbergensis), Lu Shi yeast (S.rouxii); Genus kluyveromyces (Kluyveromyces spp.), Kluyveromyces fragilis (K.fragilis) for example, Kluyveromyces lactis (K.lactis); Mycocandida (Candida spp.), candida pseudotropicalis (C.pseudotropicalis) for example, candida tropicalis (C.tropicalis).
For saccharomycetic cultivation, can adopt the preparation of solid fermentation method and solution fermentation.
Solid fermentation method is to adopt solid medium culturing yeast bacterium, and equipment and investment are lower, but the personal expenditures height.Equipment used has the fermentation dish, boiler, and dryer connects bacterium equipment, analytical test instrument, stirrer etc.
Solution fermentation is to adopt liquid nutrient medium culturing yeast bacterium, and equipment and investment are higher, but personal expenditures is lower.Equipment used has fermentor tank, seeding tank, and boiler, dryer connects bacterium equipment, analytical test instrument, stirrer etc.
Saccharomycetic immobilization is meant and utilizes the means of physics or chemistry to be fixed to process on the upholder in yeast that the gained yeast is called fixed yeast, also is referred to as biological yeast immobilized enzyme among the present invention sometimes; Behind the dress post, the present invention is referred to as " immobilized enzyme column that contains fixed yeast ".The method of physics has absorption, embedding etc., and the method for chemistry comprises uses glue crosslinking agent etc.Upholder also can be called carrier, for example resin etc.In a word, need only these methods and upholder for the zymic biological activity, perhaps certain biological activity is influence not, perhaps has the acceptable influence, so all can adopt.So the zymic immobilization can have various alternatives, material therefor also can change to some extent, but only otherwise deviate from aim of the present invention, promptly utilizes fixed yeast to prepare fructose-1,6-diphosphate from fructose, so all is within the scope of the invention.
Yeast of the present invention not only comprises mentioned kind, also comprises use among the embodiment various types of, and other kind, as long as this primary yeast meets the needs of production, also promptly can efficiently fructose be converted into fructose-1,6-diphosphate.Certainly, the bacterial classification that each primary yeast is carried out mutagenesis screening, genetically engineered operation gained also may be used for the present invention.
Magnesium ion can promote the effect of isomerase, so in one embodiment of the invention, with the contain magnesium salts of step 1) products therefrom according to every cubic metre of about 0.1-0.8kg of adding, and adjustment pH is 7.5-8.2, by flowing through the isomery post that contains immobilized isomerase, carry out isomerization.
The described solution that contains fructose can be flowed through off and on and be contained the immobilized enzyme column of fixed yeast, also can flow through continuously and contain the immobilized enzyme column of fixed yeast.
With starch-containing material is the effect that the method for raw material production FDP can accurately design two stages when being used for technology, makes it that coordinative role take place, and is integrated into a complete process.
The method for preparing fructose-1,6-diphosphate of the present invention can further include makes with extra care the solution that transforms through fixed yeast.
Described treating process comprises: add activated carbon decolorizing, through filtering and impurity removing, through ion exchange column.It is 30-35 ℃ that wherein said resin anion(R.A) is adsorbed on temperature, carries out under the pH6.5-7.0, and through wash-out, the content of fructose-1,6-diphosphate is 2-3% in the elutriant after the absorption, and purity reaches more than 99%.
Preparation fructose-1 of the present invention, the method of 6-bisphosphate can further include and changes above-mentioned refined products into fructose-1, the dry product of 6-bisphosphate, its process is: gained elutriant process is concentrated 95% ethanol sedimentation, water is the dissolution precipitation thing once more, add ethanolic soln once more and obtain clear crystal, filter crystallization, drying obtains the dry product of fructose-1,6-diphosphate.Carry out the crystallization first time again, the dissolving crystallized material of water, crystallization is once more filtered, and drying makes the fructose-1,6-diphosphate finished product.
The invention has the advantages that:
1) raw material sources of this synthesis technique are extensive, meet agricultural products in China deep processing industry policy.Investment of production equipment is low, and lucrative investment reclaims.
2) owing to adopt the fixed yeast post to carry out serialization production, need not to purchase expensive device such as fermentor tank, yeast whizzer, flame filter press.The suitableeest growth of yeast pH value is lower than most bacterium, and insensitive to the microbiotic of energy bacteria growing inhibiting.Therefore, a large amount of yeast cultures can exempt from the pollution of bacterium rapidly of growing, because they are individual big, so yeast is than the easier results of bacterium and more cheap.Be used for industrial yeast at present and publilc health harm problem do not occur.
3) conversion rate of products height, steady quality: adopt the fixed yeast serialization to produce, stable production process is easy to control, the reaction conditions gentleness, transformation efficiency can reach more than 70%, and existing technology only can reach 50%.Owing to repeatedly adopt ion exchange technique, improved quality product in the technology.Wherein the thermotolerance of fructose-1,6-diphosphate is brought up to 60 ℃ by original 40 ℃ and is helped the storage of product and the raisings of stability thereof.
4) production cost is low: the application of immobilized yeast technology, improved the dry solids content of process stream greatly, reduce the evaporation program, reduced steam consumption, with existing technology relatively, can save production cost about 20%.
5) remarkable environmental benefit: continuity is produced the wherein raising of useful component, doubles approximately, and it is over half that same throughput reduces the fermented waste fluid quantity discharged, helps environment protection.
Thalline composition and metabolite that the prepared fructose-1,6-diphosphate of the present invention contains are less, and purity is higher, and thermostability is significantly increased, and can be used to prepare medicine, for example can make injection liquid, oral liquid, capsule.It is the first aid good medicine of Acute Myocardial Infarction, cardiac insufficiency, coronary heart disease, myocardial ischemia outbreak.And the prepared fructose-1,6-diphosphate impurity of the present invention is less, can by with other salinity effect, make sodium salt, calcium salt etc., be convenient to absorption of human body and utilization.Fructose-1,6-diphosphate of the present invention also can mix use with other material, for example aspartic acid sodium salt, taurine, amino acid, pearl powder, VITAMIN, calcium lactate etc. are used.Fructose-1,6-diphosphate of the present invention also can with plant milk extract, particularly some Chinese herbal medicine extracts are used in combination.Fructose-1,6-diphosphate of the present invention also can join in food or the beverage, replenishes the nutritional needs of healthy population or special population.Fructose-1,6-diphosphate of the present invention also can join in the makeup.Fructose-1,6-diphosphate of the present invention also can join in animal-feed and the medicine.When making the goods that contain fructose-1,6-diphosphate of the present invention, can add multiple additives, for example sweeting agent, antioxidant and sanitas.
Fructose-1,6-diphosphate of the present invention is particularly suitable for making the nutritious prod at sub-health population.Fructose-1, the 6-bisphosphate is an intermediate product of keeping needed by human in the metabolism of human body normal function, and the cytolemma that can see through the damaged cell enters into cell interior and directly improves energy, can improve the cellular energy metabolism, quicken the cellular-restoring normal function, rapidly the relieving fatigue state.
Embodiment
Used at this, " raw material " refers to the material that technology begins, in one embodiment of the invention, be meant " solution that contains fructose ", comprise pure fructose soln, the slurries that contain fructose are for example used the fructose syrup of glucose solution through glucose isomerase effect formation, perhaps contain the fermented liquid of fructose.These contain in the solution of fructose and contain: 1) fructose; 2) phosphate radical; 3) magnesium ion; And other composition, comprise other composition of having a mind to other composition of interpolation and existing naturally.In another embodiment of the invention, " raw material " comprises the material of various rich in starch, for example potato stem tuber, cassava and sweet potato, and the material in the seed of wheat, corn, barley and Chinese sorghum, the refuse that also comprises some technologies, as long as its rich in starch satisfies needs of the present invention and gets final product; It also refers to from the work in-process of other manufacturer's purchase, as long as can be converted into fructose syrup through certain technology.Above-mentioned is not determinate for example.
Used at this, " starch " is meant the linearity of D-glucose or the mixture of branch-like homopolymer.In plant, starch is as the storage thing of carbohydrate and form.In the different vegetable-derived materials, the type of starch and branch's situation be difference to some extent, and the technological process that is translated into fructose syrup should change as the case may be to some extent." starch " among the present invention also refers to take place the starch derivative of certain hydrolysis.
Used at this, " fructose syrup " is meant and ins all sorts of ways the direct material of feedstock conversion for the preparation fructose-1,6-diphosphate.According to the difference of technology, the fructose content in the described fructose syrup can change to some extent, and its potential of hydrogen, salts contg and other composition can change to some extent.Certain this variation is relevant with initial used starting material.Fructose syrup can be regulated its composition before being used for transforming, to satisfy the demand of subordinate phase technology.
Used at this, " fructose-1,6-diphosphate " is meant FDP, is also referred to as 1, the 6-hexose diphosphate, and English is Fructose-1,6-diphophate.Some people also uses neologisms " biological fructose " address.Various statements so just should be seen fructose-1,6-diphosphate of the present invention as long as their essence is meant fructose-1,6-diphosphate.The content of fructose-1,6-diphosphate can change in the finished product to some extent.Fructose-1,6-diphosphate of the present invention is to transform gained with organism, particularly is to transform gained with yeast, also will utilize organism to realize that the material transformation is called " biosynthesizing " sometimes.
Used at this, " isomerization " is meant glucose isomerase turned to fructose.
Used at this, " conversion " is meant that a kind of material becomes the process of another material through certain mechanism.Its connotation is equal to " transformation ", " variation ", " becoming " " becomes " etc.In the present invention, " starch becomes glucose ", " glucose becomes fructose ", " fructose becomes fructose-1,6-diphosphate " etc. all can be explained with above-mentioned various synonym sayings.And in the terms such as " organism conversions ", " conversion " has same connotation; In this case, be meant the transformation that utilizes organism to realize material.
Below be an employed raw material of embodiment, the concrete technology and relevant test data of the method for the invention, main explanation the method for the invention is utilized starch-containing material production fructose-1, the superiority of 6-bisphosphate, do not limit the included scope of the present invention, as those skilled in the art understood, the description here only is exemplary, and scope of the present invention only is subjected to the restriction of claims.And following narration has illustrated the superiority of another one embodiment of the present invention too, just directly uses the technical scheme that contains fructose soln.
One, main raw material: (by producing the 1.0t fructose-1,6-diphosphate)
1. high-grade maize: 3.5-3.8t
2. Ye Huamei: 0.45-0.47kg
3. saccharifying enzyme: 0.8-0.9kg
4. isomerase: 0.09-0.11kg
5. biological yeast: 0.1-0.12t
Two, the fructose-1,6-diphosphate synthesis technique is introduced:
1. at first be the preparation of raw material fructose:
The raw material corn quality requires to be starch content 〉=74%, impurity≤1.0%, moisture content≤14.0, unsound grain≤10.0, moldy kernel≤1.0.Get high-grade maize and sieve, the selection by winnowing removal of impurities is soaked 45-50h with the 0.3-0.5% sulfurous acid solution, handles through wet milling process and isolates zein, plumule, maize peel, obtains the W-Gum slurries.
Adjusting the corn slurries is degree Beaume 17-22, pH value 5.8-6.5, add the low calcium of NOVO company, low pH Ye Huamei, utilize liquefaction injector steam ejection liquefaction under temperature 105-110 ℃ condition, and keep 95-100 ℃, 1.5-2h, reach liquefaction DE value 12-15%, be cooled to 58-62 ℃ after 140 ℃ of high temperature go out enzyme, adjust pH is 3.8-4.8, adds NOVO product compounded saccharifying enzyme and carries out saccharification, 48-60h, reach the DE value more than 95%, add diatomite then, activated carbon carries out the liquid glucose degreasing, decolouring is handled, and reaches transparence 〉=99%, colourity≤0.005, enter the multiple bed of the resin of forming by positive resin and negative resin and carry out ion-exchange, remove the inorganic salt ash content, reach below the specific conductivity 10us/cm, enter vapo(u)rization system concentration and evaporate as for 40-45%, and add 0.1kg/m by 30%-33%
3The sal epsom of liquid glucose, the pH value is adjusted into 7.5-8.2, enter in the isomery post by the immobilized isomerase filling and carry out continuous isomerization, fructose content has been risen to more than 42%, and then add activated carbon and further decolour and enter the secondary ions exchange column after handling and carry out ion-exchange, specific conductivity reaches below the 1cu/cm, enter the fructose separation system then, by separation resin fill simulation moving-bed in carry out chromatographic separation, separation temperature 75-80 ℃, reach the above fructose soln of fructose content 〉=95% after the separation, its concentration is adjusted into 10-30%, pH value 6.5-7.5 is as the raw material for standby of producing fructose-1,6-diphosphate.
2. the preparation of immobilization biological yeast enzyme:
The preferred yeast bacterial classification, for example with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), candiyeast (Candida), torulopsis (Torulopsis), candiyeast bacterial classification roguings such as (Candida) is inoculated in to shake cultivates 4-5h in the bottle, per minute is with 130-140 vibration, move into then in the nutrient solution cultivate 10-15h after, collect thalline, isolate thalline with drier, add in the thalline after the solvent be used for broken wall treatment carries out broken wall treatment, adding pure water dilutes, activation thalline 3-4h, ion exchange column by forming by anionite-exchange resin then, utilize ion-exchange resin absorption that thalline is adsorbed on the resin matrix, so just be prepared into biological yeast immobilized enzyme.
3. fructose-1,6-diphosphate preparation:
Fructose soln is added the 0.4mol/L sodium phosphate, and 0.01mol/L sal epsom is under 40-45 ℃ of condition, press the 0.1-0.15Bv/h flow velocity from top to bottom by biological yeast immobilized enzyme column, carry out biological respinse, obtain containing the solution of fructose-1,6-diphosphate 8-10% concentration.
4. refining, the crystallization processes of fructose-1,6-diphosphate:
Above-mentioned solution is at first added the activated carbon removal of impurities of decolouring to be handled, handle the back and analyse post by the resinbed of forming by chlorine type anionite-exchange resin resin, fructose-1 like this, the 6-bisphosphate will be by resin absorption, other impurity are then stayed in the solution, discard, and resin absorption is full carries out gradient elution after closing, to increase fructose-1,6-diphosphate purity.
Adsorption conditions is: temperature is 30-35 ℃, pH6.5-7.0, fructose-1,6-bisphosphate content 5-7%, fructose behind the wash-out-1, the about 2-3% of 6-bisphosphate content, purity reaches more than 99%, and further evaporation concentration adds 95% ethanol and obtains white precipitate to 15-20% then, filter floating throw out, the precipitation that is dissolved in water adds ethanolic soln once more, clear crystal, filtration, crystallization, the dry fructose-1,6-diphosphate finished product that gets.
Three, culture of strains and extraction, immobilization
First group of test
(Saccharomyces cerevisiae) is inoculated in shake-flask culture 4-5h with yeast saccharomyces cerevisiae, per minute is with 130-140 vibration, the back moves into the seeding tank enlarged culturing, and substratum is selected the Semen Maydis powder saccharification liquid that contains sugared 3.5-4.5% for use, and the pH value is 6.2-6.4, temperature is 30-33 ℃, collect fermented liquid after cultivating 10-14h, adsorb fermented liquid with anionite-exchange resin, thalline then is attracted on the resin matrix, become immobilized yeast, survey and show that its bacteria containing amount is the 121.1mg/g dried resin.
Second group of test
Schizosaccharomyces pombe (Schizosaccharomyces pombe) is inoculated in to shake cultivates 5-6h in the bottle, per minute is with 130-140 vibration, the back moves into the seeding tank enlarged culturing, substratum is selected the Semen Maydis powder saccharification liquid of 3.5-4.5% for use, and the pH value is 6.2-6.4, and temperature is 30-33 ℃, after cultivating 12-15h, collect fermented liquid and adsorb fermented liquid with anionite-exchange resin, absorption is full close after, measuring the resin bacteria containing amount is the 113.8mg/g dried resin.
The 3rd group of test
Torulopsis (Torulopsis) is inoculated in to shake cultivates 5-6h in the bottle, per minute is with 130-140 vibration, the back moves into the seeding tank enlarged culturing, substratum is selected the Semen Maydis powder saccharification liquid that contains sugared 3.5-4.5% for use, pH value 6.2-6.4, and temperature is 30-33 ℃, cultivate 15-18h, collect fermented liquid, adsorb fermented liquid, survey and show that the resin bacteria containing amount is the 136.2mg/g dried resin with anionite-exchange resin.
The 4th group of test
Candiyeast (Candida) is inoculated in to shake cultivates 5-6h in the bottle, per minute is with 130-140 vibration, the back moves into the seeding tank enlarged culturing, substratum is selected the Semen Maydis powder saccharification liquid that contains sugared 3.5-4.5% for use, pH value 6.2-6.4, and temperature is 30-33 ℃, cultivate 15-18h, collect fermented liquid, adsorb fermented liquid, survey and show that the resin bacteria containing amount is the 135.2mg/g dried resin with anionite-exchange resin.
Four, product performance relatively
As can be seen from the above table, the fructose-1,6-diphosphate that novel process is made exceeds 3 percentage points of commercially available prod at content, and inorganic ash content content is starkly lower than the commercially available prod, and thermotolerance is far above the product of commercially available former explained hereafter.
Five, conversion rate of products contrast:
To drop into the 1.0t corn is base-material, the contrast of output fructose-1,6-diphosphate amount:
From above correlation data as can be seen, former technology average yield is 48.55%, and new synthesis process is 69.97%, and the more former technology of novel process exceeds about 20 percentage points.
Explain: former technology, i.e. traditional technology is to be direct material with glucose, the production technique of utilizing cereuisiae fermentum to transform.
Embodiment
For more particularly bright the present invention, provide following embodiment especially, but the present invention only is subjected to the restriction of claims.These embodiment are only used for illustrating, as understood by one of ordinary skill in the art, they can not be used for limiting the scope of the invention.Wherein embodiment 1-4 be relevant of the present invention be the technical scheme of raw material with the solution that contains fructose, embodiment 5 be relevant of the present invention be the technical scheme of raw material with starch-containing material.
Embodiment 1
Select yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) for use, carry out amplification culture, collect thalline behind the 12h, behind the fixed on ion exchange resin, get 20% fructose soln and add 0.38mol/LNa
2HPO
4, 0.015mol/L MgSO
4, accent pH is 6.8-7.1,40-42 ℃ with the 0.1Bv/h condition under by the fixed yeast post, measure that fructose-1,6-diphosphate content is 80.3g in the effluent liquid, by behind the above-mentioned flow process purifying fructose-1,6-diphosphate 71.3g.
Embodiment 2
Get candiyeast (Candida) and carry out amplification culture, behind the collection thalline fixed on ion exchange resin, get 21.2% fructose soln and add 0.36mol/L Na behind the 10h
2HPO
4, 0.02mol/LMgSO
4It is 6.5-7.0 that pH is transferred in the back, under the 38-40 ℃ of condition, by the fixed yeast post, measures that fructose-1,6-diphosphate content is 78.3g in the effluent liquid, by above-mentioned flow process after refining fructose-1,6-diphosphate 69.2g.
Embodiment 3
Get torulopsis (Torulopsis) and carry out amplification culture, behind the 18h, collect thalline, behind the fixed on ion exchange resin, get 19.5% fructose soln and add 0.42mol/L Na
2HPO
4, 0.013mol/L MgSO
4, at pH6.8, temperature 43-45 ℃ of condition passed through the fixed yeast post with 0.1Bv/h, measures fluid and gets fructose-1,6-diphosphate 89.1g, after making with extra care by above-mentioned flow process, gets fructose-1,6-diphosphate 78.8g.
Embodiment 4
Get candiyeast (Candida) and carry out amplification culture, behind the 18h, collect thalline, behind the fixed on ion exchange resin, get 19.5% fructose soln and add 0.42mol/L Na
2HPO
4, 0.013mol/L MgSO
4, at pH6.8, temperature 43-45 ℃ of condition passed through the fixed yeast post with 0.1Bv/h, measures fluid and gets fructose-1,6-diphosphate 79.5g, after making with extra care by above-mentioned flow process, gets fructose-1,6-diphosphate 68.0g.
Embodiment 5
With the corn is the raw material production fructose-1,6-diphosphate.
1). the preparation of raw material fructose:
The raw material corn quality requires to be starch content 〉=74%, impurity≤1.0%, moisture content≤14.0, unsound grain≤10.0, moldy kernel≤1.0.Get high-grade maize and sieve, the selection by winnowing removal of impurities is soaked 45-50h with the 0.3-0.5% sulfurous acid solution, handles through wet milling process and isolates zein, plumule, maize peel, obtains the W-Gum slurries.
Adjusting the corn slurries is degree Beaume 17-22, pH value 5.8-6.5, add the low calcium of NOVO company, low pH Ye Huamei, utilize liquefaction injector steam ejection liquefaction under temperature 105-110 ℃ condition, and keep 95-100 ℃, 1.5-2h, reach liquefaction DE value 12-15%, be cooled to 58-62 ℃ after 140 ℃ of high temperature go out enzyme, adjust pH is 3.8-4.8, adds NOVO product compounded saccharifying enzyme and carries out saccharification, 48-60h, reach the DE value more than 95%, add diatomite then, activated carbon carries out the liquid glucose degreasing, decolouring is handled, and reaches transparence 〉=99%, colourity≤0.005, enter the multiple bed of the resin of forming by positive resin and negative resin and carry out ion-exchange, remove the inorganic salt ash content, reach below the specific conductivity 10us/cm, enter vapo(u)rization system concentration and evaporate as for 40-45%, and add 0.1kg/m by 30%-33%
3The sal epsom of liquid glucose, the pH value is adjusted into 7.5-8.2, enter in the isomery post by the immobilized isomerase filling and carry out continuous isomerization, fructose content has been risen to more than 42%, and then add activated carbon and further decolour and enter the secondary ions exchange column after handling and carry out ion-exchange, specific conductivity reaches below the 1cu/cm, enter the fructose separation system then, by separation resin fill simulation moving-bed in carry out chromatographic separation, separation temperature 75-80 ℃, reach the above fructose soln of fructose content 〉=95% after the separation, its concentration is adjusted into 10-30%, pH value 6.5-7.5 is as the raw material for standby of producing fructose-1,6-diphosphate.
2). the preparation of immobilization biological yeast enzyme
Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) roguing is inoculated in to shake cultivates 4-5h in the bottle, per minute is with 130-140 vibration, move into then in the nutrient solution cultivate 10-15h after, collect thalline, isolate thalline with drier, add solvent (toluene for example in the thalline, Triton-X 100 etc.) carry out broken wall treatment after, adding pure water dilutes, activation thalline 3-4h, ion exchange column by being made up of anionite-exchange resin utilizes ion-exchange resin absorption that thalline is adsorbed on the resin matrix then, so just has been prepared into fixed yeast.
3). the fructose-1,6-diphosphate preparation:
The fructose soln of step 1) gained is added the 0.4mol/L sodium phosphate, 0.01mol/L sal epsom, under 40-45 ℃ of condition, press the 0.1-0.15Bv/h flow velocity from top to bottom by loading the chromatography column of fixed yeast resin, carry out biological respinse, obtain containing the solution of fructose-1,6-diphosphate 8-10% concentration.
4). refining, the crystallization processes of fructose-1,6-diphosphate:
With above-mentioned steps 3) gained solution at first adds the activated carbon removal of impurities of decolouring and handles, handle the back and analyse post by the resinbed of forming by chlorine type anionite-exchange resin resin, fructose-1 like this, the 6-bisphosphate will be by resin absorption, other impurity are then stayed in the solution, discard, and resin absorption is full carries out gradient elution after closing, to increase fructose-1,6-diphosphate purity.
Adsorption conditions is: temperature is 30-35 ℃, pH6.5-7.0, fructose-1,6-bisphosphate content 5-7%, fructose behind the wash-out-1, the about 2-3% of 6-bisphosphate content, purity reaches more than 99%, and further evaporation concentration adds 95% ethanol and obtains white precipitate to 15-20% then, filter floating throw out, the precipitation that is dissolved in water adds ethanolic soln once more, clear crystal, filtration, crystallization, the dry fructose-1,6-diphosphate finished product that gets.
Claims (13)
1. one kind prepares fructose-1, the method of 6-bisphosphate is characterized in that: this method comprises that the solution that will contain fructose contacts with fixed yeast, fructose in the described solution that contains fructose is transformed obtain fructose-1, the 6-bisphosphate, the wherein said solution that contains fructose comprises:
1). fructose;
2). phosphate radical;
3). magnesium ion.
2. the method for claim 1 is characterized in that the content of wherein said fructose is 10-25wt% at the described solution that contains fructose, is benchmark with the described gross weight that contains the solution of fructose in the described solution that contains fructose; The content of described phosphate radical is 0.3-0.5mol/L, is benchmark with the described cumulative volume that contains the solution of fructose; The content of described magnesium ion is 0.01-0.03mol/L, is benchmark with the described cumulative volume that contains the solution of fructose.
3. the method for claim 1 is characterized in that the described phosphate radical in the described solution that contains fructose is to be selected from least a of Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, trimagnesium phosphate and phosphoric acid or its combination of at least two kinds arbitrarily from adding in the solution that contains fructose; Described magnesium ion is to be selected from least a of magnesium chloride, sal epsom and trimagnesium phosphate or its combination of at least two kinds arbitrarily from adding in the solution that contains fructose.
4. method for preparing fructose-1,6-diphosphate, it is characterized in that: this method mainly comprises: the fs is with the conversion of starch-containing material to fructose; Subordinate phase is that the solution that will contain fructose contacts with fixed yeast, will contain fructose in the solution of fructose and transform and obtain fructose-1,6-diphosphate, and the wherein said solution that contains fructose comprises:
1). fructose;
2). phosphate radical;
3). magnesium ion.
5. as claim 1 or 4 described methods, it is characterized in that the preparation of wherein said fixed yeast comprises the steps:
1). the culturing yeast bacterium;
2). yeast is adsorbed onto on the resin, becomes fixed yeast.
6. method as claimed in claim 4 is characterized in that the preparation of described fructose comprises the steps:
1). starch-containing material is converted into glucose;
2). glucose isomerization forms fructose.
7. method as claimed in claim 6 is characterized in that described step 1) adopts acid-hydrolysis method and/or enzyme catalysis method; Described step 2) be that the isomerase immobilization is formed immobilized enzyme isomery post; According to every cubic metre of magnesium salts that contains that adds 0.1-0.8kg, and to adjust pH be 7.5-8.2, by the isomery post, carries out isomerization with the step 1) products therefrom.
8. as claim 1 or 4 described methods, it is characterized in that the described solution that will contain fructose contacts with fixed yeast, is the described solution that contains fructose to be flowed through off and on or continuously contain the immobilized enzyme column of fixed yeast.
9. as claim 4 or 6 described methods, it is characterized in that described starch-containing material is selected from least a or its at least two kinds the combination arbitrarily in corn, wheat, barley and the cassava.
10. as claim 1 or 4 described methods, it is characterized in that further can comprising: the solution that contains fructose-1,6-diphosphate that will transform gained is made with extra care.
11. as method as described in the claim 10, it is characterized in that described treating process comprises: the decolouring removal of impurities, adsorb with chlorine type resin anion(R.A), it is 30-35 ℃ that wherein said resin anion(R.A) is adsorbed on temperature, pH carries out under the 6.5-7.0, after the absorption through wash-out, fructose in the elutriant-1, the content of 6-bisphosphate is 2-3%, and purity reaches more than 99%.
12., it is characterized in that further can comprising: the dry product of preparation fructose-1,6-diphosphate as method as described in the claim 10.
13. as method as described in the claim 12, it is characterized in that described preparation fructose-1, the process of the dry product of 6-bisphosphate is: gained elutriant process is concentrated, 95% ethanol sedimentation, water be the dissolution precipitation thing once more, adds ethanolic soln once more and obtains clear crystal, filter, crystallization, drying obtain the dry product of fructose-1,6-diphosphate.
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| CN100395252C (en) * | 2006-06-12 | 2008-06-18 | 南京工业大学 | Method for continuously separating 1, 6-fructose diphosphate |
| CN101904856A (en) * | 2010-08-06 | 2010-12-08 | 南京南农高科兽药研究所有限公司 | Application of 1,6-diphosphofructose and derivative thereof in preparing animal medical health care products |
| CN109836464B (en) * | 2019-03-21 | 2021-02-19 | 张家港市华天药业有限公司 | Preparation method of fructose calcium diphosphate |
| CN109970825A (en) * | 2019-03-21 | 2019-07-05 | 张家港市华天药业有限公司 | A method of preparing magnesium fructose-diphosphate |
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