CN102676615A - Double enzyme method for preparing medicinal dextran with controllable molecular weight - Google Patents
Double enzyme method for preparing medicinal dextran with controllable molecular weight Download PDFInfo
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Abstract
The invention relates to a double enzyme method for preparing medicinal dextran with controllable molecular weight. The medicinal dextran comprises dextran 70, dextran 40 and dextran 20. The preparation steps of the medicinal dextran comprise two steps of: firstly, synthesizing high molecular dextran: firstly, preparing the high molecular dextran by catalyzing sucrose by adopting dextran, wherein the conversion rate of the sucrose converted into the dextran reaches over 95 percent; secondly, controllably degrading the high molecular dextran: carrying out catalytic degradation on the high molecular dextran into the medicinal dextran as the medicinal dextran by adopting dextranase with high enzyme activity and realizing the controllable degradation of the dextran; and after the reaction is ended, removing impure protein in the products by adopting a simple filtering method and obtaining dextrans with different molecular weights by adopting graded ethanol precipitation. The double enzyme method has the advantages of greenness, environment friendliness, low cost, simplicity and convenience; and in addition, according to the double enzyme method, the yield of products can be greatly improved and the medicinal dextran with high quality is obtained.
Description
Technical field
The present invention relates to bio-pharmaceuticals and enzyme engineering field, specifically adopt the technology of the controlled medicinal Expex of dextransucrase and dextranase coproduce molecular weight.
Background technology
Expex (Dextran) is the polymkeric substance that some VISOSE dehydrations form; Mainly by glucose α-1; 6 glycosidic links are formed by connecting, by the dextransucrase (Dextransucrase of the bright string bacterial strain of goldbeater's skin shape (Leuconstoc mesenteriodes) generation; E.C.2.4.1.5) the synthetic Expex contains 95% α-1,6 glycosidic link, contains the branched structure of a small amount of other glycosidic link simultaneously.Because of it has multiple advantages such as safe, nontoxic, a plurality of fields such as medicine, industry, food have been widely used in.High molecular dextran can be used as pharmaceutical carrier, and gel chromatography filler commonly used.Commonly used clinically have medium molecular dextran 70, Dextran 40 and a Dextran 20, mainly as plasma substitute, is used for hemorrhagic shock, traumatic shock and burn shock etc.The Expex of weight-average molecular weight 6-8 kDa often is used to process Iron Dextran, is used to treat serious anemia.Weight-average molecular weight is lower than the Expex of 5 kDa often by sulfation, is used for antithrombotic.(Dextran 1, M for Dextran 10 1
W1000 Da) come to light recently, can avoid the immune transformation reactions of human body self that causes owing to macrodex and 40.The oligomeric isomaltose that molecular weight is littler then is used to prebiotics.
The at present domestic fermentation process that adopts prepares high molecular dextran more; Utilize hydrochloric acid hydrolysis to obtain the medicinal Expex of different molecular weight again; Because the Expex of producing contains quality and the quality that a large amount of muriates has had a strong impact on Expex; Anaphylaxis in clinical use, often occurs, can cause skin reaction, serious breathing and circulatory failure, even cause death.Owing to be acid-catalyzed hydrolysis, hydrolytic process is unordered simultaneously, and the product molecular weight distribution that obtains is not concentrated, and influences the quality of product.In addition, owing to utilize hydrochloric acid hydrolysis to need mal-conditions such as high temperature peracid, be unfavorable for environmental protection, low-carbon (LC).And adopt dextransucrase and dextranase enzyme process to prepare the pharmaceutical grade Expex; Just can reduce the influence of muriate to quality product; Because enzyme to the specificity hydrolysis of substrate, can obtain the high-quality Expex that MWD is concentrated, product reaches high yield simultaneously.In addition, the enzymatic degradation reaction conditions is gentle, energy consumption is low, thereby in having realized, the target of the green of small molecular weight polysaccharide production technique, environmental protection, low-carbon (LC).The strain fermentation that dextransucrase and dextranase are produced in the external at present existing direct employing of scholar respectively prepares medicinal Expex; But because direct fermentation, the metabolites kinds that bacterial strain produces is numerous and diverse, is unfavorable for the product separation purifying; Because productive rate is low, be unfavorable for industrialization production simultaneously.Also there is the scholar to adopt dextransucrase and the two medicinal sugared acid anhydrides of enzyme coproduce of dextranase in addition, but because productive rate lower (36%), thereby be unfavorable for industrialization production.
Summary of the invention
The present invention is for fear of the weak point that prior art exists, and a kind of method that adopts the controlled medicinal Expex of dextransucrase and the two enzyme coproduce molecular weight of dextranase is provided, and this method is green, environmental protection, low cost, easy not only; And make the dextran molecule amount for preparing controlled.
Technical solution problem of the present invention adopts following scheme:
Double-enzyme method prepares the controlled medicinal Expex of molecular weight, and this method is carried out as follows:
(1) dextransucrase synthetic macromolecule Expex,
Adopt sucrose solution 1000 mL of pH 5.4 0.02 mol/L acetic acid-sodium-acetate buffer preparation final volume concentration 12%, add 0.05g/L CaCl in the said damping fluid
2After the sucrose solution that configures placed 25 ℃ of insulation 10 min, adding dextransucrase, to make its final concentration be 2 IU/mL, places 25 ℃ of constant temperature, 120r/min reaction 4 hours, placed under 25 ℃ of conditions insulation again 20 hours; Stop the catalyzed reaction of dextransucrase then, reaction solution is used for the catalytic hydrolysis of next step dextranase;
Enzyme of said dextransucrase IU that lives is meant, is that 10% sucrose solution is a substrate with acetic acid-calcium acetate damping fluid preparation final volume concentration of pH5.4; Contain calcium acetate 0.05 g/L, Glacial acetic acid min. 99.5 0.02 mol/L in the said damping fluid; Under 25 ℃ of conditions, in every milliliter of substrate reactions liquid per hour catalysis sucrose produce the needed enzyme amount of 0.1 mg fructose;
(2) Expex enzymatic hydrolysis high molecular dextran
In dextransucrase catalysis synthetic reaction solution, adding dextranase to step 1), to make its final concentration be 0.23 U/mL, places stirring reaction 160min-180 min under 35 ℃ of conditions, stops enzymatic reaction then;
An enzyme of said dextranase U alive is meant; With the final volume concentration of the acetate buffer of pH 5.0 preparation is that 3% weight-average molecular weight is that the dextran solution of 70 kDa is a substrate; Under 35 ℃ of conditions, PM produces the needed dextranase amount of 1 μ mol reducing sugar;
(3) remove impurity
With above-mentioned steps 2) place 80 ℃ of insulations 3 hours through the reaction solution of Expex enzymatic hydrolysis, adopt multilayer filtered through gauze impurity subsequently, repeat to filter twice, adopt middling speed qualitative filter paper filtering supernatant subsequently; And then with supernatant heated and boiled 15 min after the above-mentioned filtration, treat supernatant cooling after, utilize the middling speed qualitative filter paper to filter again and remove impurity, limpid colourless filtrating is used for next step alcohol grading alcohol precipitation;
(4) alcohol grading alcohol precipitation and vacuum-drying
Adopt 4 grades of divisions, it is 39% that one-level is divided the ethanol volumetric concentration, under whipping appts; In step (3) gained filtrating, slowly add volumetric concentration 95% ethanol; Regularly measure ethanol volumetric concentration in the filtrating, when the ethanol volumetric concentration reaches 39%, stop alcohol precipitation, filtrating is placed 40 degree insulations 22 hours; Pour out supernatant then, in deposition, add an amount of ethanol again and take out macromole impurity; It is 43% that secondary is divided the ethanol volumetric concentration; Slowly add the ethanol of volumetric concentration 95% in the supernatant after one-level is divided, concrete operations are the same, and the ethanol volumetric concentration of waiting to filtrate reaches at 43% o'clock; Stop alcohol precipitation; The filtrating that alcohol precipitation is good places 34 degree insulations 22 hours, pours out supernatant then, in deposition, adds ethanol again and obtains the macrodex product; Three grades are divided alcohol concn is 47%; Slowly add the ethanol of volumetric concentration 95% in the supernatant after secondary is divided, concrete operations are the same, and the ethanol volumetric concentration of waiting to filtrate reaches at 47% o'clock; Stop alcohol precipitation; The worry liquid that alcohol precipitation is good places 40 degree insulations 12 hours, pours out supernatant, in deposition, adds ethanol again and obtains the Dextran 40 product; It is 57% that level Four is divided alcohol concn, slowly adds the ethanol of volumetric concentration 95% in the supernatant after three grades of divisions, and concrete operations are the same; The ethanol volumetric concentration of waiting to filtrate reaches at 57% o'clock, stops alcohol precipitation, and the worry liquid that alcohol precipitation is good places normal temperature; After 2 hours, add ethanol again and obtain the Dextran 20 product;
Above-mentioned ethanol is divided resulting product placed 80 ℃ of vacuum drying ovens respectively dry 4 hours, remove residual ethanol and moisture in the product, promptly obtain corresponding Expex product.
The preferred request for utilization of the present invention number is the reorganization dextransucrase in 200710134765.4 the open text of patent; This enzyme has enzymatic activity high; Average enzyme work reaches 40IU/mL, and catalysis sucrose inversion efficiently is an Expex, and the transformation efficiency of sucrose is up to more than 95%; The preferred request for utilization of the present invention simultaneously number is the dextranase in 201110117071.6 the open text of patent; This enzymatic hydrolysis high molecular dextran has stronger substrate specificity; This enzyme when the catalysis high molecular dextran, the preferential bigger sugared acid anhydride of catalyzed degradation molecular weight, thereby the enrichment of Dextran 10 in realizing; Be beneficial to the enzyme process beam system and be equipped with the controlled medicinal Expex of molecular weight, obtain higher product yield simultaneously; The dextranase that also can use the sour jujube spore penicillium bacterial strain fermentation that from soil, filters out to form, this bacterial strain GenBank accession number is HQ647326.
The present invention adopts dextransucrase and the controlled medicinal Expex of dextranase coproduce molecular weight, and method has following advantage:
(1) the present invention acts on the Expex of sucrose synthetic macromolecule earlier with sucrase, and transformation efficiency adds Expex enzyme liberating 160-180min afterwards again up to 95%, obtains serial Expex product, and this method is green, environmental protection, low cost, easy not only; And can increase substantially product yield, obtain high-quality medicinal Expex.
(2) the present invention can realize the controlled production of molecular weight of medicinal Expex, and through the control reaction conditions, regulating the catalyzed reaction time can directed preparation be main product with the Dextran 20.
Embodiment
Below through specific embodiment technical scheme of the present invention is done further and to be explained.
Embodiment 1
(1) dextransucrase synthetic macromolecule Expex comprises following A, two steps of B:
A: dextransucrase vitality test
Final concentration with acetic acid-calcium acetate damping fluid (containing calcium acetate 0.05 g/L, Glacial acetic acid min. 99.5 0.02 mol/L) sucrose solution of pH5.4 is formulated as 10%, adds an amount of dilution enzyme liquid standing and reacting 1h under 25 ℃ of conditions.Sampling 500 μ l added DNS reagent 375 μ l (diluting 10 times) after reaction was accomplished, and 5min is accurately boiled in heating in the boiling water bath, takes out then it is cooled to room temperature, added zero(ppm) water and were settled to 5.375ml.With unreacted contrast liquid as reference, with spectrophotometer at wavelength 520nm place the measuring light density value, calculate quantity of fructose according to the typical curve of fructose, finally calculate enzyme activity.Enzyme unit (IU) that lives is defined as under these conditions, in every milliliter of substrate reactions liquid per hour catalysis sucrose produce the needed enzyme amount of 0.1 mg fructose.Detected result shows that dextransucrase crude enzyme liquid vigor reaches 110IU/mL.
B: enzyme process synthetic macromolecule Expex processing condition
Adopt pH 5.4 0.02 mol/L acetic acid-sodium-acetate buffers (to add 0.05g/L CaCl
2) preparation 12% sucrose solution 5000 mL; After placing 25 ℃ of insulation 10 min; It is 2 IU/mL that the adding dextransucrase makes its final concentration; Add dextransucrase 10000 IU of total activity unit, place 25 ℃ of constant temperature, 120r/min reaction 4 hours, place again under 25 ℃ of conditions and be incubated 20 hours.After reaction finishes, place 70 ℃ of insulation 20 min to stop the catalyzed reaction of dextransucrase reaction solution.Reaction solution is used for the catalytic hydrolysis of next step dextranase.The reaction solution sampling detects, and adopts the weight-average molecular weight of performance liquid chromatography test sample to reach more than the 1000kDa, and the sucrose inversion rate is up to more than 95%.
(2) Expex enzymatic hydrolysis high molecular dextran comprises following A, two steps of B:
A: Expex enzyme activity determination
The vitality test of dextranase: the 3% macrodex kDa solution that the acetate buffer of 4ml 0.02mol/L pH 5.0 is prepared places 35 ℃ of insulation 10min; After adding the enzyme liquid 1ml insulation 1h of suitably dilution again; Utilize 3,5-dinitrosalicylic acid (DNS) method is measured the reducing sugar that generates.Produce the needed enzyme amount of 1 μ mol reducing sugar (glucose equivalent) with PM under these conditions and be defined as an enzyme activity unit (U).Detected result shows that dextranase crude enzyme liquid vigor reaches 500U/mL.
B: the catalyzed degradation of dextranase
In dextransucrase catalysis synthetic reaction solution, adding dextranase to above-mentioned step 1), to make its final concentration be 0.23 U/mL; Adding dextranase total activity unit is 1150U; Place stirring reaction 160min-180 min under 35 ℃ of conditions, after reaction finishes, reaction solution is placed 100 ℃ of water bath heat preservation 10 min or feeds water vapour 5min; Make Expex enzyme denaturation inactivation, to stop enzymatic reaction.
(3) remove impurity
With above-mentioned steps 2) place 80 ℃ of insulations 3 hours through the reaction solution of Expex enzymatic degradation; The reaction solution insulation is after 3 hours; Having a large amount of cotton-shaped metaproteins separates out; Adopt eight layers of filtered through gauze impurity (being mainly the dextransucrase of heat denatured) subsequently, above step repeats twice, adopts middling speed qualitative filter paper filtering supernatant subsequently.And then with supernatant heated and boiled 15 min behind the above-mentioned reacting liquid filtering, treat supernatant cooling after, utilize middling speed qualitative filter paper impurity screening again, limpid colourless filtrating is used for next step alcohol grading alcohol precipitation, i.e. the removal of foreign protein was divided into for two steps and carries out.
(4) alcohol grading alcohol precipitation and vacuum-drying
Adopt 4 grades of divisions, it is 39% that one-level is divided the ethanol volumetric concentration, under whipping appts; In step (3) gained filtrating, slowly add volumetric concentration 95% ethanol, regularly measure the temperature and the alcoholic strength of filtrating, and according to temperature and alcoholic strength value table look-up filtrate in the ethanol volumetric concentration;, the ethanol volumetric concentration stops alcohol precipitation when reaching 39%; Filtrating is placed 40 degree insulations 22 hours, pour out supernatant then, in deposition, add an amount of ethanol again and take out macromole impurity; It is 43% that secondary is divided the ethanol volumetric concentration; Slowly add the ethanol of volumetric concentration 95% in the supernatant after one-level is divided, concrete operations are the same, and the ethanol volumetric concentration of waiting to filtrate reaches at 43% o'clock; Stop alcohol precipitation; The filtrating that alcohol precipitation is good places 34 degree insulations 22 hours, pours out supernatant then, in deposition, adds ethanol again and obtains the macrodex product; Three grades are divided alcohol concn is 47%; Slowly add the ethanol of volumetric concentration 95% in the supernatant after secondary is divided, concrete operations are the same, and the ethanol volumetric concentration of waiting to filtrate reaches at 47% o'clock; Stop alcohol precipitation; The worry liquid that alcohol precipitation is good places 40 degree insulations 12 hours, pours out supernatant, in deposition, adds ethanol again and obtains the Dextran 40 product; It is 57% that level Four is divided alcohol concn, slowly adds the ethanol of volumetric concentration 95% in the supernatant after three grades of divisions, and concrete operations are the same; The ethanol volumetric concentration of waiting to filtrate reaches at 57% o'clock, stops alcohol precipitation, and the worry liquid that alcohol precipitation is good places normal temperature; After 2 hours, add ethanol again and obtain the Dextran 20 product;
Obtain three kinds of medicinal Expexs that molecular weight is different at last, detect through performance liquid chromatography, the molecular weight of three kinds of products is respectively 69376; 38251 and 21364 Da; RT is respectively 13.866 min, 14.390 min and 15.037 min, and the MWD coefficient is respectively 2.08,1.39,1.21; The overall yield of three kinds of products is 80.32%, and corresponding productive rate is 5.77%, 12.55%, 62%.When the reaction times is 160min-180min, can directed preparation Dextran 20 be main product.
Dextransucrase in the present embodiment is that application number is the reorganization dextransucrase in the open text of 200710134765.4 patent; Dextranase is that application number is the dextranase in the open text of 201110117071.6 patent.
Claims (1)
1. double-enzyme method prepares the controlled medicinal Expex of molecular weight, it is characterized in that this method is carried out as follows:
(1) dextransucrase synthetic macromolecule Expex,
Adopt sucrose solution 1000 mL of pH 5.4 0.02 mol/L acetic acid-sodium-acetate buffer preparation final volume concentration 12%, add 0.05g/L CaCl in the said damping fluid
2After the sucrose solution that configures placed 25 ℃ of insulation 10 min, adding dextransucrase, to make its final concentration be 2 IU/mL, places 25 ℃ of constant temperature, 120r/min reaction 4 hours, placed under 25 ℃ of conditions insulation again 20 hours; Stop the catalyzed reaction of dextransucrase then, reaction solution is used for the catalytic hydrolysis of next step dextranase;
Enzyme of said dextransucrase IU that lives is meant, is that 10% sucrose solution is a substrate with acetic acid-calcium acetate damping fluid preparation final volume concentration of pH5.4; Contain calcium acetate 0.05 g/L, Glacial acetic acid min. 99.5 0.02 mol/L in the said damping fluid; Under 25 ℃ of conditions, in every milliliter of substrate reactions liquid per hour catalysis sucrose produce the needed enzyme amount of 0.1 mg fructose;
(2) Expex enzymatic hydrolysis high molecular dextran
In dextransucrase catalysis synthetic reaction solution, adding dextranase to step 1), to make its final concentration be 0.23 U/mL, places stirring reaction 160min-180 min under 35 ℃ of conditions, stops enzymatic reaction then;
An enzyme of said dextranase U alive is meant; With the final volume concentration of the acetate buffer of pH 5.0 preparation is that 3% weight-average molecular weight is that the dextran solution of 70 kDa is a substrate; Under 35 ℃ of conditions, PM produces the needed dextranase amount of 1 μ mol reducing sugar;
(3) remove impurity
With above-mentioned steps 2) place 80 ℃ of insulations 3 hours through the reaction solution of Expex enzymatic hydrolysis, adopt multilayer filtered through gauze impurity subsequently, repeat to filter twice, adopt middling speed qualitative filter paper filtering supernatant subsequently; And then with supernatant heated and boiled 15 min after the above-mentioned filtration, treat supernatant cooling after, utilize the middling speed qualitative filter paper to filter again and remove impurity, limpid colourless filtrating is used for next step alcohol grading alcohol precipitation;
(4) alcohol grading alcohol precipitation and vacuum-drying
Adopt 4 grades of divisions, it is 39% that one-level is divided the ethanol volumetric concentration, under whipping appts; In step (3) gained filtrating, slowly add volumetric concentration 95% ethanol; Regularly measure ethanol volumetric concentration in the filtrating, when the ethanol volumetric concentration reaches 39%, stop alcohol precipitation, filtrating is placed 40 degree insulations 22 hours; Pour out supernatant then, in deposition, add an amount of ethanol again and take out macromole impurity; It is 43% that secondary is divided the ethanol volumetric concentration; Slowly add the ethanol of volumetric concentration 95% in the supernatant after one-level is divided, concrete operations are the same, and the ethanol volumetric concentration of waiting to filtrate reaches at 43% o'clock; Stop alcohol precipitation; The filtrating that alcohol precipitation is good places 34 degree insulations 22 hours, pours out supernatant then, in deposition, adds ethanol again and obtains the macrodex product; Three grades are divided alcohol concn is 47%; Slowly add the ethanol of volumetric concentration 95% in the supernatant after secondary is divided, concrete operations are the same, and the ethanol volumetric concentration of waiting to filtrate reaches at 47% o'clock; Stop alcohol precipitation; The worry liquid that alcohol precipitation is good places 40 degree insulations 12 hours, pours out supernatant, in deposition, adds ethanol again and obtains the Dextran 40 product; It is 57% that level Four is divided alcohol concn, slowly adds the ethanol of volumetric concentration 95% in the supernatant after three grades of divisions, and concrete operations are the same; The ethanol volumetric concentration of waiting to filtrate reaches at 57% o'clock, stops alcohol precipitation, and the worry liquid that alcohol precipitation is good places normal temperature; After 2 hours, add ethanol again and obtain the Dextran 20 product;
Above-mentioned ethanol is divided resulting product placed 80 ℃ of vacuum drying ovens respectively dry 4 hours, remove residual ethanol and moisture in the product, promptly obtain corresponding Expex product.
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Cited By (11)
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| CN103266149A (en) * | 2013-05-22 | 2013-08-28 | 广西壮族自治区分析测试研究中心 | Method for preparing pharmaceutical dextran and levulose by bi-enzyme coupled multistage membrane separation |
| CN103290080A (en) * | 2013-06-21 | 2013-09-11 | 合肥工业大学 | Method for preparing molecular weight-controllable medicinal dextran |
| CN103436572A (en) * | 2013-08-14 | 2013-12-11 | 江南大学 | Preparation method of tubular starch derivative |
| CN105177086A (en) * | 2015-10-21 | 2015-12-23 | 合肥工业大学 | Process for preparing crystalline fructose from dextransucrase through one-step enzymatic method |
| CN106526050A (en) * | 2015-09-15 | 2017-03-22 | 河北远征药业有限公司 | Method for determining content of phenol in iron dextran injection liquid |
| CN107201387A (en) * | 2017-07-26 | 2017-09-26 | 合肥工业大学 | A kind of preparation method of iron-dextrin |
| CN111206059A (en) * | 2020-01-21 | 2020-05-29 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for controlling dextran molecular weight by adopting composite gel microspheres |
| CN111662940A (en) * | 2020-06-11 | 2020-09-15 | 劲牌有限公司 | Method for directionally preparing dextran |
| CN112458130A (en) * | 2020-12-01 | 2021-03-09 | 陕西省微生物研究所 | Method for preparing dextran 40 by direct enzymolysis of dextran fermentation liquor |
| CN113201513A (en) * | 2021-06-22 | 2021-08-03 | 广西产研院生物制造技术研究所有限公司 | Heat-resistant dextran sucrase mutant and preparation method and application thereof |
| CN113567370A (en) * | 2021-07-23 | 2021-10-29 | 广西南宁市桃源兽药厂 | Method for detecting content of sugar anhydride in iron dextran injection |
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| CN103266149A (en) * | 2013-05-22 | 2013-08-28 | 广西壮族自治区分析测试研究中心 | Method for preparing pharmaceutical dextran and levulose by bi-enzyme coupled multistage membrane separation |
| CN103290080A (en) * | 2013-06-21 | 2013-09-11 | 合肥工业大学 | Method for preparing molecular weight-controllable medicinal dextran |
| CN103290080B (en) * | 2013-06-21 | 2016-06-01 | 合肥工业大学 | Prepare the method for the controlled medicinal dextran of molecular weight |
| CN103436572A (en) * | 2013-08-14 | 2013-12-11 | 江南大学 | Preparation method of tubular starch derivative |
| CN106526050B (en) * | 2015-09-15 | 2018-06-19 | 河北远征药业有限公司 | The content assaying method of phenol in a kind of iron dextran injection |
| CN106526050A (en) * | 2015-09-15 | 2017-03-22 | 河北远征药业有限公司 | Method for determining content of phenol in iron dextran injection liquid |
| CN105177086A (en) * | 2015-10-21 | 2015-12-23 | 合肥工业大学 | Process for preparing crystalline fructose from dextransucrase through one-step enzymatic method |
| CN107201387A (en) * | 2017-07-26 | 2017-09-26 | 合肥工业大学 | A kind of preparation method of iron-dextrin |
| CN107201387B (en) * | 2017-07-26 | 2021-01-15 | 合肥工业大学 | Preparation method of iron dextran |
| CN111206059A (en) * | 2020-01-21 | 2020-05-29 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Method for controlling dextran molecular weight by adopting composite gel microspheres |
| CN111662940A (en) * | 2020-06-11 | 2020-09-15 | 劲牌有限公司 | Method for directionally preparing dextran |
| CN112458130A (en) * | 2020-12-01 | 2021-03-09 | 陕西省微生物研究所 | Method for preparing dextran 40 by direct enzymolysis of dextran fermentation liquor |
| CN113201513A (en) * | 2021-06-22 | 2021-08-03 | 广西产研院生物制造技术研究所有限公司 | Heat-resistant dextran sucrase mutant and preparation method and application thereof |
| CN113567370A (en) * | 2021-07-23 | 2021-10-29 | 广西南宁市桃源兽药厂 | Method for detecting content of sugar anhydride in iron dextran injection |
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Application publication date: 20120919 |