CN103266146B - Two enzyme immobilization coupling multistage membrane sepn prepare the method for medicinal dextran and fructose - Google Patents
Two enzyme immobilization coupling multistage membrane sepn prepare the method for medicinal dextran and fructose Download PDFInfo
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Abstract
The invention discloses the method that a kind of pair of enzyme immobilization coupling multistage membrane sepn prepares medicinal dextran and fructose, dextransucrase and dextranase are immobilized onto in sodium alginate micro ball jointly and become two enzyme immobilization bead; Take sucrose solution as substrate, in substrate, add two enzyme immobilization bead, form the reaction system of immobilized bi-enzyme and sucrose; Reaction product is after the Using Multistage Membranes systematic position be made up of ultra-filtration membrane and nanofiltration membrane, directly obtain medicinal dextran in the Ultra filtration membrane stage, and high-purity fructose solution is obtained in final stage nanofiltration membrane separation, enzyme contained in the trapped fluid of first step ultra-filtration membrane and macromolecule dextran then return the reaction system of immobilized bi-enzyme and sucrose, again participate in reaction, realize the recycle of enzyme.Cost of the present invention is lower, environmental protection, safe and efficient, simple to operate, achieve directed regulation and control and produce the medicinal dextran of high-quality target molecular weight, obtain the fructose by product of high purity, high added value simultaneously.
Description
Technical field
The invention belongs to bio-pharmaceuticals and biomass separation field, particularly relate to the method that a kind of pair of enzyme immobilization coupling multistage membrane sepn prepares medicinal dextran and fructose.
Background technology
Dextran is the exocellular polysaccharide produced by bacterium (as leuconostoc mesentroides (Leuconostoc mesenteroides)), and its main chain is formed by connecting with α (1 → 6) glycosidic link by D-Glucose unit.The dextran of different molecular weight has multiple use clinically, as plasma substitute, dredging capillary blood vessel, control thrombus etc." Chinese Pharmacopoeia " (2010 editions) have been included and have been comprised Dex-70(molecular-weight average about 70,000), Dex-40(molecular-weight average about 40,000), Dex-20(molecular-weight average about 20,000) three kinds of dextran and drug derivatives totally 12 kinds thereof.Domesticly at present prepare the technique that dextran adopts " strain fermentation-alcohol settling-hydrochloric acid hydrolysis-ethanol precipitation " mostly, product contains the impurity such as muriate, and molecular weight distribution is wider, cause, during Clinical practice, anaphylaxis occurs, bring greater risk to patient, limit promoting the use of of dextran.
Fructose is the sweetest natural sugar, for diabetics, fructose is again the sugar that a kind of safety coefficient is higher, and its glycemic index produced is about 20% of glucose, and fructose has the plurality of advantages that moisture retention etc. is applicable to foodstuffs industry production, is thus widely used at medicine, field of food.Fructose solubleness is very large, and namely decompose at 103 ~ 105 DEG C, adopt traditional hot-work method to obtain the technical difficulty of crystal diabetin very greatly, complex manufacturing, cost is naturally also high, and this is that fructose price is far away higher than the one of the main reasons of dextrose plus saccharose.
Dextransucrase (Dextransucrase, is called for short DSase, and the phylogenetic systematics number of enzyme is E.C.2.4.1.5) derives from the bacteriums such as Leuconostoc mesenteroides, can act on substrate Sucrose synthesis dextran.Domestic and international research shows, its molecular weight ranges of dextransucrase that Leuconostoc mesenteroides produces is 160 ~ 180kDa.Dextranase (Dextranase, be called for short Dase, the phylogenetic systematics of enzyme number is E.C.3.2.1.11) be the enzyme of α (1 → 6) glycosidic link in a species specificity, randomly inscribe dextran chain, apply the molecular weight that this enzyme significantly can reduce dextran.Foreign study shows, its molecular weight ranges of dextranase produced by thin beautiful Chaetomium is 70 ~ 90kDa.
Membrane separation technique is the Green production process in biomass separation field, can effective separation of realize target product under the prerequisite not introducing exogenous objectionable impurities.Contriver furthers investigate and shows in Using Multistage Membranes separation dextran, membrane separation technique can be separated and enrichment dextran preferably by the size of molecular weight, but because dextran molecule becomes chain or linear expansion in the solution, larger molecule can penetrate less filter membrane, adopts the molecular weight cut-off of filter membrane to demarcate molecular weight of product distribution range there is certain error so simple.And for the protein-based nonlinear molecule such as enzyme molecule, then there is not this shortcoming in membrane sepn, directly can demarcate molecular weight of product distribution range by the molecular weight cut-off of filter membrane.
Strain fermentation method and enzymatic clarification belong to biological process, and its reaction conditions is gentle, and power consumption is few, and particularly enzymatic clarification is extensively described as the one preferred technique of high-end biological products suitability for industrialized production.Current domestic existing bacterial classification or the dextransucrase fermentation sucrose of utilizing prepares dextran in advance, and then the method for the fermentation clear liquid added containing dextrase or dextranase, to prepare the dextran of different molecular weight, but its follow-up product separation is all more complicated, and use the traditional separation method such as filtered through gauze, ethanol precipitation to carry out purified product, may affect the safe handling of medicinal dextran, preparation method has larger room for improvement.In addition, the reaction of dextransucrase and sucrose can generate rapidly the dextran that molecular weight is greater than 1,000,000 at short notice, although this method is had outstanding performance in Sucrose conversion, the enzymolysis due to dextranase is random, so target product yield is not high.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of cost is lower, environmental protection, two enzyme immobilization coupling multistage membrane sepn safe and efficient, simple to operate prepare the method for medicinal dextran and fructose, produce the medicinal dextran of high-quality target molecular weight to realize directed regulation and control, obtain the fructose by product of high purity, high added value simultaneously.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: two enzyme immobilization coupling multistage membrane sepn prepare the method for medicinal dextran and fructose, dextransucrase and dextranase is jointly immobilized onto in sodium alginate micro ball and becomes two enzyme immobilization bead; Take sucrose solution as substrate, in substrate, add two enzyme immobilization bead, form the reaction system of immobilized bi-enzyme and sucrose; Reaction product is after the Using Multistage Membranes systematic position be made up of ultra-filtration membrane and nanofiltration membrane, directly obtain medicinal dextran in the Ultra filtration membrane stage, and high-purity fructose solution is obtained in final stage nanofiltration membrane separation, enzyme contained in the trapped fluid of first step ultra-filtration membrane and macromolecule dextran then return the reaction system of immobilized bi-enzyme and sucrose, again participate in reaction.
Dextransucrase (being produced by the Leuconostoc mesenteroides sucrose that ferments) and dextranase (commercialization enzyme) carry out enzyme immobilizatio by the enzyme activity ratio ratio of dextranase (dextransucrase with) 100:1 ~ 10:1 after mixing jointly; The whole volumetric molar concentration of sucrose solution is 0.01M ~ 0.8M; Using Multistage Membranes system comprises first step ultra-filtration membrane, second stage ultra-filtration membrane, third stage ultra-filtration membrane and the final stage nanofiltration membrane that molecular weight cut-off successively decreases step by step.
First step ultra-filtration membrane molecular weight cut-off is 70,000,50,000 or 30,000, and second stage ultra-filtration membrane molecular weight cut-off is 50,000 or 30,000, and third stage ultra-filtration membrane molecular weight cut-off is 30,000 or 10,000, and final stage nanofiltration membrane molecular weight cut-off is 200.In the trapped fluid of the Ultra filtration membrane acquisition of molecular weight cut-off 50,000,30,000 and 10,000, contained medicinal dextran is respectively Dex-70, Dex-40, Dex-20.
The reaction solution of reaction system is first through first step Ultra filtration membrane, one-level trapped fluid (containing two kinds of enzymes and macromolecule dextran) Returning reacting system, and one-level permeate is through second stage Ultra filtration membrane; Second entrapment liquid is derived and concentrate drying obtains medicinal dextran, and secondary permeate is through third stage Ultra filtration membrane; Three grades of trapped fluids are derived also concentrate drying and are obtained medicinal dextran, three grades of permeate are through final stage nanofiltration membrane concentrating and separating, final stage concentrated solution can be used for the industry such as food, feed as by product after drying, and final stage permeate (containing high-purity fructose) becomes the fructose syrup of 50 ~ 60% to can be used for the industry such as food, medicine through concentrating under reduced pressure.Can add new sucrose solution again in reaction system after a batch of reaction terminates and continue to utilize original immobilized enzyme to carry out the reaction of next batch, the two enzyme immobilization beads adding system each time can use the reaction time of more than 6.
Two enzyme immobilization bead and substrate contact reaction times 35 ~ 70h in reaction system, temperature of reaction 20 ~ 30 DEG C, pH scope 5.5 ~ 6.8.
First step ultra-filtration membrane top hole pressure is 0.03 ~ 0.15MPa, and second stage ultra-filtration membrane top hole pressure is 0.03 ~ 0.15MPa, and third stage ultra-filtration membrane top hole pressure is 0.05 ~ 0.30MPa, and final stage nanofiltration membrane top hole pressure is 1.5 ~ 3.5MPa.
The operation of first step ultra-filtration membrane is stopped when reaction solution remains 1/5 volume, second stage ultra-filtration membrane operation is stopped when second entrapment liquid reaches 1/6 volume of one-level permeate, stop the operation of third stage ultra-filtration membrane when three grades of trapped fluids reach 2/5 volume of secondary permeate, stop the operation of final stage nanofiltration membrane when final stage permeate reaches 6/7 volume of three grades of permeate.
Secondary or three grades of trapped fluids carry out concentrating under reduced pressure at 50 ~ 80 DEG C of temperature, then after pre-freeze, utilize vacuum lyophilization to obtain dry powder the finished product, and product water dissolubility is effective, and molecular size range, molecular weight distribution meet medicinal dextran requirement.
In reaction system, dextransucrase enzyme amount is 0.02U/mL ~ 0.1U/mL; Dextranase enzyme amount is determined according to two enzyme ratio.
Enzyme immobilizatio is that the sodium alginate soln being 2 ~ 4%w/w by dextransucrase and dextranase and strength of solution whole after pasteurization mixes, mixed solution to be statically placed in 4 DEG C of environment after degassed 2h vacuumize degassing 15 minutes again, then utilize asepsis injector that mixed solution is instilled in the aseptic calcium chloride solution of 2% to prepare two enzyme immobilization bead, two enzyme immobilization small spherical particles is after aseptic water washing, pull out after putting into the crosslinked 20 ~ 90min of glutaraldehyde solution of 0.1 ~ 0.7%v/v again, use aseptic water washing again, be stored in 4 DEG C of environment for subsequent use, the enzyme rate of recovery alive reaches 52.7 ~ 66.3%.
Prepare Problems existing for current dextran, contriver, in conjunction with immobilized enzyme method synthesis and the advantage of membrane sepn two kinds of technology, establishes the method that the present invention's pair enzyme immobilization coupling multistage membrane sepn prepares medicinal dextran and fructose.The whole technological process of this method may cause the material of medicinal dextran clinical response without the need to introducing in the traditional method such as ethanol, hydrochloric acid, belong to Green Manufacturing Technology; Membrane sepn is the new and high technology during biomass are separated, by changing the parameters such as reaction substrate concentration, enzyme amount, enzyme activity ratio, and change membrane module, control reaction times etc., the present invention directly can achieve the directional preparation of target molecular weight dextran, substrate conversion efficiency is high, product accurately can both carry out orientation regulation and control in molecular weight, molecular weight distribution etc., is convenient to industrial application; It is comparatively large that zymin accounts for production cost proportion, and the present invention utilizes enzyme immobilization technology can realize the multiple batches of recycle of enzyme to reduce production cost, and two enzyme immobilization is more conducive to the regulation and control of dextran molecule amount in product; Meanwhile, present invention obtains the fructose of high purity, high added value, make nanofiltration be that high-purity fructose production provides new approaches; And another kind of by product is less than the oligose composition of 10,000 primarily of molecular weight, is also taken as a kind of potential biomass material and is processed.
Accompanying drawing explanation
Fig. 1 is the process flow sheet that the two enzyme immobilization coupling multistage membrane sepn of the present invention prepares the method for medicinal dextran and fructose.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the invention will be further described.
Embodiment 1
Dextransucrase system ferments through Leuconostoc mesenteroides, and under fermentation liquor 12,000r/min, 4 DEG C of conditions after centrifugal 20min, get supernatant liquor and obtain, enzyme activity is detected by DNS method and obtains.Dextranase derives from commercialization zymin (A Manuo amano enzyme preparation commerce and trade (Shanghai) Co., Ltd.), by Hanes method in conjunction with practical situation of the present invention to condition determination slightly modified, measure the enzyme activity of dextranase.After measured, before non-immobilization, dextransucrase enzyme activity is 8.4U/mL, and the enzyme activity of dextranase is 22,000U/mL.
Two enzyme immobilization method be dextransucrase and dextranase with the ratio of enzyme activity for 10:1(fixes dextransucrase final concentration for 0.6U/mL) ratio mix with the sodium alginate soln after pasteurization, sodium alginate soln final concentration is 2.5%(w/v, g/100mL), mixed solution to be statically placed in 4 DEG C of environment after degassed 2h vacuumize degassing 15 minutes again, then utilize asepsis injector that mixed solution is instilled in the aseptic calcium chloride solution of 2% to prepare immobilized spherule particle, immobilized spherule particle is after aseptic water washing, put into 0.10%(v/v again, mL/mL) glutaraldehyde solution is pulled out after being cross-linked 90min, with aseptic water washing, get the enzyme activity that partial fixing bead measures its dextransucrase and dextranase respectively, it is for subsequent use that remaining is stored in 4 DEG C of environment.Enzyme activity determination result is dextransucrase is 0.39U/g, and dextranase is 0.041U/g.
Prepare the sucrose solution of 1M as substrate mother liquor with sterilized water, be then placed in the hopper of membrane sepn front end with the sucrose liquid 2.5L that sterilized water preparation final concentration is 0.4M, add immobilized spherule 400g anabolic reaction system.Temperature of reaction is 26 DEG C, and pH is 6.3.Starting first step molecular weight cut-off is the hyperfiltration membrane assembly of 70,000, mouth pressure is adjusted for 0.03 ~ 0.15MPa after running 58h in total reflux mode, and open successively second stage molecular weight cut-off be 50,000 hyperfiltration membrane assembly (top hole pressure is 0.03 ~ 0.15MPa), third stage molecular weight cut-off be 30,000 hyperfiltration membrane assembly (top hole pressure is 0.05 ~ 0.30MPa) and molecular weight cut-off be 200 nanofiltration membrane component (top hole pressure is 1.5 ~ 3.0MPa), start to collect target product.The operation of first step film is stopped when barrel solution is about 1/5 of original volume; The operation of second stage ultra-filtration membrane is stopped when second stage membrane sepn trapped fluid volume reaches 1/6 of first step permeate; When third stage membrane sepn trapped fluid volume reaches 2/5 of second stage permeate, stop the operation of third stage ultra-filtration membrane; Nanofiltration operation is stopped when the permeate volume of nanofiltration membrane separation reaches 6/7 of third stage ultrafiltration membrane permeate liquid volume.Measure barrel residual reaction liquid HPGPC and analyze, display Sucrose conversion reaches more than 89%.The trapped fluid of the second stage, third stage Ultra filtration membrane gained pre-freeze extremely less than-30 DEG C after concentrating under reduced pressure, it is being carried out to vacuum lyophilization acquisition dry powder dextran, carry out High Performance Gel Permeation chromatogram (HPGPC) to this product to detect, result shows, from the trapped fluid of second stage Ultra filtration membrane and the dextran weight-average molecular weight obtained is 65110, D values is 1.16; Third stage ultra-filtration membrane products obtained therefrom molecular weight is 34080, D value is 1.19.The permeate of nanofiltration membrane separation gained, after the syrup that concentrating under reduced pressure becomes 50 ~ 60%, utilizes high performance liquid chromatography (HPLC) to measure its purity, and result shows, and fructose purity reaches more than 95%.After nanofiltration membrane gained concentrated solution drying, pulverizing, pack is deposited.
Embodiment 2
The acquisition of dextransucrase and dextranase and enzyme activity determination are with embodiment 1.
Two enzyme immobilization method be dextransucrase and dextranase with the ratio of enzyme activity for 10:1(fixes dextransucrase final concentration for 0.8U/mL) ratio mix with the sodium alginate soln after pasteurization, sodium alginate soln final concentration is 4.0%(w/v, g/100mL), mixed solution to be statically placed in 4 DEG C of environment after degassed 2h vacuumize degassing 15 minutes again, then utilize asepsis injector that mixed solution is instilled in the aseptic calcium chloride solution of 2% to prepare immobilized spherule particle, immobilized spherule particle is after aseptic water washing, put into 0.4%(v/v again, mL/mL) glutaraldehyde solution is pulled out after being cross-linked 45min, with aseptic water washing, get the enzyme activity that partial fixing bead measures its dextransucrase and dextranase respectively, it is for subsequent use that remaining is stored in 4 DEG C of environment.Enzyme activity determination result is dextransucrase is 0.44U/g, and dextranase is 0.047U/g.
Prepare the sucrose solution of 1M as substrate mother liquor with sterilized water, be then placed in the hopper of membrane sepn front end with the sucrose liquid 2.5L that sterilized water preparation final concentration is 0.2M, add immobilized spherule 400g anabolic reaction system.Temperature of reaction is 25 DEG C, and pH is 6.6.Starting first step molecular weight cut-off is the hyperfiltration membrane assembly of 70,000, mouth pressure is adjusted for 0.03 ~ 0.15MPa after running 48h in total reflux mode, and open successively second stage molecular weight cut-off be 30,000 hyperfiltration membrane assembly (top hole pressure is 0.03 ~ 0.15M Pa), third stage molecular weight cut-off be 10,000 hyperfiltration membrane assembly (top hole pressure is 0.05 ~ 0.30MPa) and molecular weight cut-off be 200 nanofiltration membrane component (top hole pressure is 1.5 ~ 2.8MPa), start to collect target product.The operation of first step film is stopped when barrel solution is about 1/5 of original volume; The operation of second stage ultra-filtration membrane is stopped when second stage membrane sepn trapped fluid volume reaches 1/6 of first step permeate; When third stage membrane sepn trapped fluid volume reaches 2/5 of second stage permeate, stop the operation of third stage ultra-filtration membrane; Nanofiltration operation is stopped when the permeate volume of nanofiltration membrane separation reaches 6/7 of third stage ultrafiltration membrane permeate liquid volume.Measure barrel residual reaction liquid HPGPC and analyze, display Sucrose conversion reaches more than 91%.The trapped fluid of the second stage, third stage Ultra filtration membrane gained pre-freeze extremely less than-30 DEG C after concentrating under reduced pressure, it is being carried out to vacuum lyophilization acquisition dry powder dextran, carry out High Performance Gel Permeation chromatogram (HPGPC) to this product to detect, result shows, from the trapped fluid of second stage Ultra filtration membrane and the dextran weight-average molecular weight obtained is 37560, D values is 1.36; Third stage ultra-filtration membrane products obtained therefrom molecular weight is 18360, D value is 1.36.The permeate of nanofiltration membrane separation gained, after the syrup that concentrating under reduced pressure becomes 50 ~ 60%, utilizes high performance liquid chromatography (HPLC) to measure its purity, and result shows, and fructose purity reaches more than 95%.After nanofiltration membrane gained concentrated solution drying, pulverizing, pack is deposited.Embodiment 3
The acquisition of dextransucrase and dextranase and enzyme activity determination are with embodiment 1.
Two enzyme immobilization method be dextransucrase and dextranase with the ratio of enzyme activity for 100:1(fixes dextransucrase final concentration for 0.8U/mL) ratio mix with the sodium alginate soln after pasteurization, sodium alginate soln final concentration is 3.0%(w/v, g/100mL), mixed solution to be statically placed in 4 DEG C of environment after degassed 2h vacuumize degassing 15 minutes again, then utilize asepsis injector that mixed solution is instilled in the aseptic calcium chloride solution of 2% to prepare immobilized spherule particle, immobilized spherule particle is after aseptic water washing, put into 0.7%(v/v again, mL/mL) glutaraldehyde solution is pulled out after being cross-linked 40min, with aseptic water washing, get the enzyme activity that partial fixing bead measures its dextransucrase and dextranase respectively, it is for subsequent use that remaining is stored in 4 DEG C of environment.Enzyme activity determination result is dextransucrase is 0.42U/g, and dextranase is 0.041U/g.
Prepare the sucrose solution of 1M as substrate mother liquor with sterilized water, be then placed in the hopper of membrane sepn front end with the sucrose liquid 2.5L that sterilized water preparation final concentration is 0.1M, add immobilized spherule 400g anabolic reaction system.Temperature of reaction is 22 DEG C, and pH is 5.8.Starting first step molecular weight cut-off is the hyperfiltration membrane assembly of 70,000, mouth pressure is adjusted for 0.03 ~ 0.15MPa after running 42h in total reflux mode, and open successively second stage molecular weight cut-off be 30,000 hyperfiltration membrane assembly (top hole pressure is 0.03 ~ 0.15MPa), third stage molecular weight cut-off be 10,000 hyperfiltration membrane assembly (top hole pressure is 0.05 ~ 0.30MPa) and molecular weight cut-off be 200 nanofiltration membrane component (top hole pressure is 1.5 ~ 3.2MPa), start to collect target product.The operation of first step film is stopped when barrel solution is about 1/5 of original volume; The operation of second stage ultra-filtration membrane is stopped when second stage membrane sepn trapped fluid volume reaches 1/6 of first step permeate; When third stage membrane sepn trapped fluid volume reaches 2/5 of second stage permeate, stop the operation of third stage ultra-filtration membrane; Nanofiltration operation is stopped when the permeate volume of nanofiltration membrane separation reaches 6/7 of third stage ultrafiltration membrane permeate liquid volume.Measure barrel residual reaction liquid HPGPC and analyze, display Sucrose conversion reaches more than 91%.The trapped fluid of the second stage, third stage Ultra filtration membrane gained pre-freeze extremely less than-30 DEG C after concentrating under reduced pressure, it is being carried out to vacuum lyophilization acquisition dry powder dextran, carry out High Performance Gel Permeation chromatogram (HPGPC) to this product to detect, result shows, from the trapped fluid of second stage Ultra filtration membrane and the dextran weight-average molecular weight obtained is 67650, D values is 1.46; Third stage ultra-filtration membrane products obtained therefrom molecular weight is 38640, D value is 1.39.The permeate of nanofiltration membrane separation gained, after the syrup that concentrating under reduced pressure becomes 50 ~ 60%, utilizes high performance liquid chromatography (HPLC) to measure its purity, and result shows, and fructose purity reaches more than 94%.After nanofiltration membrane gained concentrated solution drying, pulverizing, pack is deposited.
Claims (7)
1. two enzyme immobilization coupling multistage membrane sepn prepares a method for medicinal dextran and fructose, it is characterized in that: dextransucrase and dextranase are jointly immobilized onto in sodium alginate micro ball and become two enzyme immobilization bead; Take sucrose solution as substrate, in substrate, add two enzyme immobilization bead, form the reaction system of immobilized bi-enzyme and sucrose; Reaction product is after the Using Multistage Membranes systematic position be made up of ultra-filtration membrane and nanofiltration membrane, directly obtain medicinal dextran in the Ultra filtration membrane stage, and high-purity fructose solution is obtained in final stage nanofiltration membrane separation, enzyme contained in the trapped fluid of first step ultra-filtration membrane and macromolecule dextran then return the reaction system of immobilized bi-enzyme and sucrose, again participate in reaction; Described dextransucrase and dextranase carry out enzyme immobilizatio by enzyme activity jointly than after 100:1 ~ 10:1 mixing; The whole volumetric molar concentration of described sucrose solution is 0.01M ~ 0.8M; Described Using Multistage Membranes system comprises first step ultra-filtration membrane, second stage ultra-filtration membrane, third stage ultra-filtration membrane and the final stage nanofiltration membrane that molecular weight cut-off successively decreases step by step; Described first step ultra-filtration membrane molecular weight cut-off is 70,000,50,000 or 30,000, and second stage ultra-filtration membrane molecular weight cut-off is 50,000 or 30,000, and third stage ultra-filtration membrane molecular weight cut-off is 30,000 or 10,000, and final stage nanofiltration membrane molecular weight cut-off is 200; Described enzyme immobilizatio is that the sodium alginate soln being 2 ~ 4%w/w by dextransucrase and dextranase and strength of solution whole after pasteurization mixes, mixed solution to be statically placed in 4 DEG C of environment after degassed 2h vacuumize degassing 15 minutes again, then utilize asepsis injector that mixed solution is instilled in the aseptic calcium chloride solution of 2% to prepare two enzyme immobilization bead, two enzyme immobilization small spherical particles is after aseptic water washing, pull out after putting into the crosslinked 20 ~ 90min of glutaraldehyde solution of 0.1 ~ 0.7%v/v again, use aseptic water washing again, be stored in 4 DEG C of environment for subsequent use.
2. the according to claim 1 pair of enzyme immobilization coupling multistage membrane sepn prepares the method for medicinal dextran and fructose, it is characterized in that: the reaction solution of described reaction system is first through first step Ultra filtration membrane, one-level trapped fluid Returning reacting system, one-level permeate is through second stage Ultra filtration membrane; Second entrapment liquid is derived and concentrate drying obtains medicinal dextran, and secondary permeate is through third stage Ultra filtration membrane; Three grades of trapped fluids are derived also concentrate drying and are obtained medicinal dextran, and three grades of permeate are through final stage nanofiltration membrane concentrating and separating, and final stage concentrated solution is after drying as by product, and final stage permeate becomes the fructose syrup of 50 ~ 60% through concentrating under reduced pressure.
3. the according to claim 2 pair of enzyme immobilization coupling multistage membrane sepn prepares the method for medicinal dextran and fructose, it is characterized in that: two enzyme immobilization bead and substrate contact reaction times 35 ~ 70h in described reaction system, temperature of reaction 20 ~ 30 DEG C, pH scope 5.5 ~ 6.8.
4. the according to claim 3 pair of enzyme immobilization coupling multistage membrane sepn prepares the method for medicinal dextran and fructose, it is characterized in that: described first step ultra-filtration membrane top hole pressure is 0.03 ~ 0.15MPa, second stage ultra-filtration membrane top hole pressure is 0.03 ~ 0.15MPa, third stage ultra-filtration membrane top hole pressure is 0.05 ~ 0.30MPa, and final stage nanofiltration membrane top hole pressure is 1.5 ~ 3.5MPa.
5. the according to claim 4 pair of enzyme immobilization coupling multistage membrane sepn prepares the method for medicinal dextran and fructose, it is characterized in that: stop the operation of first step ultra-filtration membrane when reaction solution remains 1/5 volume, second stage ultra-filtration membrane operation is stopped when second entrapment liquid reaches 1/6 volume of one-level permeate, stop the operation of third stage ultra-filtration membrane when three grades of trapped fluids reach 2/5 volume of secondary permeate, stop the operation of final stage nanofiltration membrane when final stage permeate reaches 6/7 volume of three grades of permeate.
6. the according to claim 5 pair of enzyme immobilization coupling multistage membrane sepn prepares the method for medicinal dextran and fructose, it is characterized in that: described secondary or three grades of trapped fluids carry out concentrating under reduced pressure at 50 ~ 80 DEG C of temperature, then after pre-freeze, utilize vacuum lyophilization to obtain dry powder the finished product.
7. the according to claim 6 pair of enzyme immobilization coupling multistage membrane sepn prepares the method for medicinal dextran and fructose, it is characterized in that: in described reaction system, dextransucrase enzyme amount is 0.02U/mL ~ 0.1U/mL.
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| CN107988284A (en) * | 2017-12-07 | 2018-05-04 | 中国科学院过程工程研究所 | A kind of method for producing Dextran 10 and products thereof and purposes |
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