CN116590252A - Method for preparing dextran sucrase - Google Patents
Method for preparing dextran sucrase Download PDFInfo
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- CN116590252A CN116590252A CN202310812273.5A CN202310812273A CN116590252A CN 116590252 A CN116590252 A CN 116590252A CN 202310812273 A CN202310812273 A CN 202310812273A CN 116590252 A CN116590252 A CN 116590252A
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- dextran sucrase
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- schizophyllan
- leuconostoc mesenteroides
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- 108010042194 dextransucrase Proteins 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000000855 fermentation Methods 0.000 claims abstract description 66
- 230000004151 fermentation Effects 0.000 claims abstract description 66
- 239000002609 medium Substances 0.000 claims abstract description 35
- 241000192130 Leuconostoc mesenteroides Species 0.000 claims abstract description 30
- WDQLRUYAYXDIFW-RWKIJVEZSA-N (2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-4-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-[[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxy-6-(hydroxymethyl)oxane-2,3,5-triol Chemical compound O[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)O1 WDQLRUYAYXDIFW-RWKIJVEZSA-N 0.000 claims abstract description 23
- 229920002305 Schizophyllan Polymers 0.000 claims abstract description 23
- 238000012258 culturing Methods 0.000 claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 239000006228 supernatant Substances 0.000 claims abstract description 10
- 230000000968 intestinal effect Effects 0.000 claims abstract description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 229930006000 Sucrose Natural products 0.000 claims description 16
- 239000005720 sucrose Substances 0.000 claims description 16
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 235000015278 beef Nutrition 0.000 claims description 9
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 9
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 241000192132 Leuconostoc Species 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 13
- 108090000790 Enzymes Proteins 0.000 abstract description 12
- 102000004190 Enzymes Human genes 0.000 abstract description 12
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 15
- 229920002307 Dextran Polymers 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 108010055629 Glucosyltransferases Proteins 0.000 description 2
- 102000000340 Glucosyltransferases Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010008132 Cerebral thrombosis Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 208000032456 Hemorrhagic Shock Diseases 0.000 description 1
- 201000001429 Intracranial Thrombosis Diseases 0.000 description 1
- 229940079172 Osmotic diuretic Drugs 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 101710184309 Probable sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010049771 Shock haemorrhagic Diseases 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000194022 Streptococcus sp. Species 0.000 description 1
- 102400000472 Sucrase Human genes 0.000 description 1
- 101710112652 Sucrose-6-phosphate hydrolase Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000001882 diuretic effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000002337 osmotic diuretic agent Substances 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 239000003058 plasma substitute Substances 0.000 description 1
- 235000013406 prebiotics Nutrition 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 125000000185 sucrose group Chemical group 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01005—Dextransucrase (2.4.1.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Bioinformatics & Cheminformatics (AREA)
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- General Engineering & Computer Science (AREA)
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- Biomedical Technology (AREA)
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for preparing dextran sucrase, which comprises the following steps: the method comprises the following steps: inoculating activated intestinal membranous leuconostoc mesenteroides into a fermentation culture medium, culturing for 10-15h, adding schizophyllan into the fermentation culture medium, continuously culturing for 20-25h, ending the fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution. According to the method for preparing the dextran sucrase, the intestinal-membrane-shaped leuconostoc mesenteroides is fermented and cultured for a period of time in the fermentation medium, then the schizophyllan is added, the fermentation and the culture are continued, and the added schizophyllan can improve the content and the enzyme activity of the free dextran sucrase in the final fermentation liquid, improve the yield of the dextran sucrase and reduce the production cost.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for preparing dextran sucrase.
Background
Dextran sucrases, also known as glucosyltransferases (glucosyltransferases) and dextran sucrases (glucobiosucrases), belong to the class of glycoside hydrolases which are capable of synthesizing dextran of different structures using sucrose as substrate. The dextran sucrase can be used in the fields of medicine, food and the like, such as preventing and treating dental caries, preparing plasma substitutes by enzymolysis of high molecular dextran, and the like. The enzyme is produced by Leuconostoc mesenteroides (Leuconostoc mesenteroides) and Streptococcus sp.
The dextrorotatory sucrase is mainly used for preparing the dextran at present, the dextran can improve microcirculation, eliminate intravascular erythrocyte aggregation, prevent thrombosis and osmotic diuretic effects, can be used for treating acute hemorrhagic shock, myocardial infarction, cerebral thrombosis, cerebral hyposupply, peripheral vascular diseases, diffuse intravascular coagulation and renal failure and other effects, and can be widely applied to the industries of medicine, food, chemical industry and the like. Some functional oligosaccharides can be synthesized by using immobilized dextran sucrase, and these sugars are often used widely in the food industry as prebiotics.
The dextran sucrase is prepared by fermenting Leuconostoc mesenteroides (Leuconostoc mesenteroides). Since the substrate of the reaction of the carbon source for culturing the leuconostoc mesenteroides and the dextransucrase is the same substance, namely sucrose, dextran is generated along with the occurrence of the dextransucrase in the culture process, the dextran is difficult to separate with the enzyme, the viscosity of the fermentation broth is high, free enzyme is difficult to obtain, and the enzyme activity is influenced, so that how to obtain the dextran sucrase with high activity is a problem to be solved in the field.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for preparing dextran sucrase, which adds schizophyllan in the fermentation process of leuconostoc mesenteroides and has high activity of the prepared dextran sucrase.
The invention adopts the following technical scheme:
a method for preparing dextran sucrase: the method comprises the following steps: inoculating activated intestinal membranous leuconostoc mesenteroides into a fermentation culture medium, culturing for 10-15h, adding schizophyllan into the fermentation culture medium, continuously culturing for 20-25h, ending the fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
Preferably, the schizophyllan has an average molecular weight of 10-50KD.
Preferably, the components of the fermentation medium are: sucrose 5-10%, beef extract 0.02-0.04%, disodium hydrogen phosphate 0.1-0.3%, dipotassium hydrogen phosphate 0.1-0.3%, magnesium sulfate 0.05-0.1%, and water in balance.
Preferably, the leuconostoc mesenteroides is leuconostoc mesenteroides Lm-1226.
Preferably, the volume of the leuconostoc mesenteroides is 2-5% of the fermentation medium.
Preferably, the pH of the fermentation medium is 7.5-8.0.
Preferably, the fermentation culture temperature is: 25-30 ℃.
Compared with the prior art, the invention has the beneficial effects that: the invention provides a method for preparing dextran sucrase, which comprises the steps of fermenting and culturing leuconostoc mesenteroides in a fermentation culture medium for a period of time, adding schizophyllan, and continuing fermenting and culturing, wherein the added schizophyllan can improve the content and the enzyme activity of free dextran sucrase in a final fermentation broth, improve the yield of the dextran sucrase and reduce the production cost.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.
Example 1
A method for preparing dextran sucrase: the method comprises the following steps: inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, wherein the volume of the leuconostoc mesenteroides Lm-1226 is 2% of that of the fermentation medium, the pH of the fermentation medium is 7.5, and the components of the fermentation medium are as follows: sucrose 5%, beef extract 0.02%, disodium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and water in balance; adding schizophyllan with the average molecular weight of 10KD into a fermentation culture medium after culturing for 10 hours at 25 ℃, wherein the concentration of the schizophyllan in the culture medium is 1mmoL/L, continuing to culture for 20 hours, ending fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
Example 2
A method for preparing dextran sucrase: the method comprises the following steps: inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, wherein the volume of the leuconostoc mesenteroides Lm-1226 is 3% of that of the fermentation medium, the pH of the fermentation medium is 7.7, and the components of the fermentation medium are as follows: 8% of sucrose, 0.03% of beef extract, 0.2% of disodium hydrogen phosphate, 0.2% of dipotassium hydrogen phosphate, 0.08% of magnesium sulfate and the balance of water; adding schizophyllan with the average molecular weight of 30KD into a fermentation culture medium after culturing for 12 hours at 28 ℃, continuously culturing for 23 hours, ending fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
Example 3
A method for preparing dextran sucrase: the method comprises the following steps: inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, wherein the volume of the leuconostoc mesenteroides Lm-1226 is 5% of that of the fermentation medium, the pH of the fermentation medium is 8.0, and the components of the fermentation medium are as follows: 10% of sucrose, 0.04% of beef extract, 0.3% of disodium hydrogen phosphate, 0.3% of dipotassium hydrogen phosphate, 0.1% of magnesium sulfate and the balance of water; adding schizophyllan with the average molecular weight of 50KD into a fermentation culture medium after culturing for 15 hours at 30 ℃, continuously culturing for 25 hours, ending fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
Comparative example 1
A method for preparing dextran sucrase comprising the steps of: inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, wherein the volume of the leuconostoc mesenteroides Lm-1226 is 2% of that of the fermentation medium, the pH of the fermentation medium is 7.5, and the components of the fermentation medium are as follows: sucrose 5%, beef extract 0.02%, disodium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and water in balance; continuously culturing at 25 ℃ for 30 hours, ending fermentation, centrifuging, taking supernatant, and obtaining the dextran sucrase liquid after ultrafiltration.
Comparative example 2
A method for preparing dextran sucrase comprising the steps of: inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, wherein the volume of the leuconostoc mesenteroides Lm-1226 is 2% of that of the fermentation medium, adjusting the pH of the fermentation medium to 7.5, and the components of the fermentation medium are as follows: 5% of sucrose, 0.02% of beef extract, 0.1% of disodium hydrogen phosphate, 0.1% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate and the balance of water, adding schizophyllan with the average molecular weight of 10KD into a fermentation culture medium, wherein the concentration of the schizophyllan in the culture medium is 1mmoL/L, culturing at 25 ℃ for 30 hours, ending fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase liquid.
Comparative example 3
A method for preparing dextran sucrase comprising the steps of: inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, wherein the volume of the leuconostoc mesenteroides Lm-1226 is 2% of that of the fermentation medium, the pH of the fermentation medium is 7.5, and the components of the fermentation medium are as follows: sucrose 5%, beef extract 0.02%, disodium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and water in balance; adding schizophyllan with average molecular weight of 5KD into a fermentation culture medium after culturing for 10 hours at 25 ℃, wherein the concentration of the schizophyllan in the culture medium is 1mmoL/L, continuing to culture for 20 hours, ending fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
Comparative example 4
A method for preparing dextran sucrase comprising the steps of inoculating activated leuconostoc mesenteroides Lm-1226 into a fermentation medium, the volume of the leuconostoc mesenteroides Lm-1226 being 2% of the fermentation medium, the pH of the fermentation medium being 7.5, the components of the fermentation medium being: sucrose 5%, beef extract 0.02%, disodium hydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.1%, magnesium sulfate 0.05%, and water in balance; adding schizophyllan with the average molecular weight of 60KD into a fermentation culture medium after culturing for 10 hours at 25 ℃, continuously culturing for 20 hours, ending fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
Taking 1mL of dextran sucrase solution in examples 1 to 3 and comparative examples 1 to 4 respectively, adding 2mL of sucrose solution with the mass concentration of 5% for mixing reaction for 30min, taking out 200 microlitres, adding 130 microlitres of DNS reagent, heating boiling water for 5min, cooling to room temperature, adding distilled water, measuring absorbance at the wavelength of 520nm, taking the reaction of the sterilized enzyme solution and a substrate as a reference, defining the enzyme amount required for catalyzing sucrose to produce 1 mu moL of fructose per minute as one enzyme activity unit, and detecting the enzyme activity of each group, wherein the results are shown in Table 1.
TABLE 1
| Group of | Enzyme activity (U/mL) |
| Example 1 | 34.2 |
| Example 2 | 32.6 |
| Example 3 | 33.4 |
| Comparative example 1 | 5.8 |
| Comparative example 2 | 12.7 |
| Comparative example 3 | 27.5 |
| Comparative example 4 | 29.4 |
As can be seen from Table 1, the activities of the dextran sucrase in examples 1 to 3 were higher, and the activities of the enzymes in comparative examples 1 to 4 were reduced to different extents. Wherein, in the comparative example 1, schizophyllan is not added, and the activity of the dextransucrase is the lowest. The timing of addition of schizophyllan was changed in comparative example 2, and the activity of dextransucrase was inferior to that of example 1. The molecular weight of the added schizophyllan is changed in the comparative example 3 and the comparative example 4, and the activity of the dextransucrase is better than other comparative examples, but the preparation process of the dextransucrase is inferior to that of the example 1, and the yield of the dextransucrase can be obviously improved by adding the schizophyllan with the average molecular weight of 10-50KD in the preparation process of the dextransucrase.
The dextran sucrase prepared in example 1 was used for the preparation of dextran as follows: the dextran sucrase solution in example 1 was mixed with a sucrose reaction system in a volume ratio of 1:3, the composition of the sucrose reaction system being: sodium acetate 0.70g/L, calcium chloride 0.025g/L, sucrose 230g/L, hydrochloric acid to adjust pH to 5.4, at 30 ℃, reacting for 1h, filtering the reaction liquid, ultrafiltering, precipitating with absolute ethanol, and vacuum freeze-drying to obtain dextran, wherein the molar conversion rate is calculated to be 80%, and the average molecular weight of the prepared dextran is 3000-4000kDa.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (7)
1. A method for preparing dextran sucrase: the method is characterized by comprising the following steps of: inoculating activated intestinal membranous leuconostoc mesenteroides into a fermentation culture medium, culturing for 10-15h, adding schizophyllan into the fermentation culture medium, continuously culturing for 20-25h, ending the fermentation culture, centrifuging, taking supernatant, and ultrafiltering to obtain the dextran sucrase solution.
2. The method for preparing dextran sucrase according to claim 1: characterized in that the schizophyllan has an average molecular weight of 10-50KD.
3. The method for preparing dextran sucrase according to claim 1: the method is characterized in that the fermentation medium comprises the following components: sucrose 5-10%, beef extract 0.02-0.04%, disodium hydrogen phosphate 0.1-0.3%, dipotassium hydrogen phosphate 0.1-0.3%, magnesium sulfate 0.05-0.1%, and water in balance.
4. The method for preparing dextran sucrase according to claim 1: the leuconostoc mesenteroides is leuconostoc mesenteroides Lm-1226.
5. The method for preparing dextran sucrase according to claim 1: the method is characterized in that the volume of the intestinal membranous leuconostoc is 2-5% of that of the fermentation medium.
6. The method for preparing dextran sucrase according to claim 1: characterized in that the pH of the fermentation medium is 7.5-8.0.
7. The method for preparing dextran sucrase according to claim 1: the method is characterized in that the fermentation culture temperature is as follows: 25-30 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243052A (en) * | 2013-05-22 | 2013-08-14 | 广西大学 | Breeding method for excellent strain capable of producing dextranum sucrase |
| CN103620048A (en) * | 2011-08-01 | 2014-03-05 | S.P.C.M.股份公司 | New method for manufacture of dextran, dextran solution obtained, and uses |
| CN105132390A (en) * | 2015-09-28 | 2015-12-09 | 合肥工业大学 | Method for preparing dextransucrase by using mixed fermentation |
| KR20170020258A (en) * | 2015-08-13 | 2017-02-22 | 서울대학교산학협력단 | Modified leuconostoc for high efficient production of glucansucrase |
-
2023
- 2023-07-04 CN CN202310812273.5A patent/CN116590252B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103620048A (en) * | 2011-08-01 | 2014-03-05 | S.P.C.M.股份公司 | New method for manufacture of dextran, dextran solution obtained, and uses |
| CN103243052A (en) * | 2013-05-22 | 2013-08-14 | 广西大学 | Breeding method for excellent strain capable of producing dextranum sucrase |
| KR20170020258A (en) * | 2015-08-13 | 2017-02-22 | 서울대학교산학협력단 | Modified leuconostoc for high efficient production of glucansucrase |
| CN105132390A (en) * | 2015-09-28 | 2015-12-09 | 合肥工业大学 | Method for preparing dextransucrase by using mixed fermentation |
Non-Patent Citations (2)
| Title |
|---|
| HEE KYOUNG KANG等: "Bioengineering of Leuconostoc mesenteroides glucansucrases that gives selected bond formation for glucan synthesis and/or acceptor-product synthesis", J AGRIC FOOD CHEM., pages 4148 - 4155 * |
| 贺凤等: "裂褶菌多糖的研究进展", 食用菌学报, pages 88 - 93 * |
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