CN117866996B - Preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution - Google Patents
Preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution Download PDFInfo
- Publication number
- CN117866996B CN117866996B CN202410282912.6A CN202410282912A CN117866996B CN 117866996 B CN117866996 B CN 117866996B CN 202410282912 A CN202410282912 A CN 202410282912A CN 117866996 B CN117866996 B CN 117866996B
- Authority
- CN
- China
- Prior art keywords
- enzyme
- fermentation
- solution
- fucosyltransferase
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01069—Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69)
-
- 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
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to the technical field of breast milk oligosaccharide production, in particular to a preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme liquid, which comprises the following steps: taking escherichia coli engineering bacteria expressing alpha-1, 2-fucosyltransferase, and fermenting and culturing to obtain fermentation liquor; centrifuging the fermentation liquor to obtain fermentation thalli and a first supernatant; mixing the fermentation thalli with a sodium citrate buffer solution, then carrying out homogenizing wall breaking treatment to release intracellular enzyme, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a crude enzyme solution; clarifying and filtering the crude enzyme solution to obtain an enzyme clear solution, adding NaCl and MgSO 4, and adjusting the pH value to 7.2-7.6 to obtain the high-activity alpha-1, 2-fucosyltransferase enzyme solution. The preparation method can obviously improve the enzyme activity of the alpha-1, 2-fucosyltransferase liquid, reduce the loss of the enzyme activity and is beneficial to improving the yield of 2' -FL synthesized by an enzyme method.
Description
Technical Field
The invention relates to the technical field of breast milk oligosaccharide production, in particular to a preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme liquid.
Background
Breast milk oligosaccharides (human milk oligosaccharides, HMOs) are the third largest solid component in breast milk, are important efficacy factors in breast milk, have important biological functions, and play an important role in the growth and development of newborns, and are next to lactose and fat. HMOs include a variety of oligosaccharide structures and 200 or more different isomers, with more than 100 HMOs structures being demonstrated. Wherein, 2'-fucosyllactose (2' -fucosyllactose,2 '-FL) accounts for about 31 percent (mole fraction) in HMOs, the content is 0.06-3.93 g/L, and the 2' -fucosyllactose is the oligosaccharide with the highest relative abundance in human milk. The 2' -FL has multiple functional activities, including regulation of intestinal flora, adhesion resistance to pathogenic bacteria, immunoregulation, promotion of development and repair of nervous system, and the like, and is an ideal ingredient of infant formula milk powder. Currently, synthetic methods for 2' -FL include chemical synthesis, whole cell synthesis, and enzymatic synthesis. The synthesis of 2' -FL by whole cells is based on the metabolic mechanism of the microorganism itself to synthesize GDP-fucose and exogenously expressed alpha-1, 2-fucosyltransferase, which is the main method for the industrial production of 2' -FL at present, but the reaction product of the whole cell synthesis requires a complicated purification process to obtain 2' -FL of higher purity.
In enzymatic synthesis, the group replacement of GDP-fucose and lactose is often catalyzed by alpha-1, 2-fucosyltransferase to produce 2' -FL and Guanosine Diphosphate (GDP). The specificity of the enzymatic synthesis of 2' -FL is higher, the reaction condition is mild and controllable, the time for the product to reach the highest concentration is short, and the components in the reaction system are relatively simple and easy to purify. However, the existing alpha-1, 2-fucosyltransferase has low activity and is easy to inactivate in the storage process, so that the yield of the enzymatic synthesis of 2' -FL is low.
Disclosure of Invention
Aiming at the technical problems that the prior alpha-1, 2-fucosyltransferase has weak activity and is easy to inactivate in the storage process, the invention provides a preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme liquid, which comprises the following steps: (1) Taking escherichia coli engineering bacteria expressing alpha-1, 2-fucosyltransferase, and fermenting and culturing to obtain fermentation liquor; centrifuging the fermentation liquor to obtain fermentation thalli and a first supernatant; (2) Mixing the fermentation thalli with a sodium citrate buffer solution, then carrying out homogenizing wall breaking treatment to release intracellular enzyme, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a crude enzyme solution; (3) Clarifying and filtering the crude enzyme solution to obtain an enzyme clear solution, adding NaCl and MgSO 4, and adjusting the pH value to 7.2-7.6 to obtain the high-activity alpha-1, 2-fucosyltransferase enzyme solution.
The engineering bacteria of the escherichia coli are escherichia coli (ESCHERICHIA COLI) K-12 MG1655 BLBYZT6, the escherichia coli K-12 MG1655 BLBYZT6 is preserved in China general microbiological culture Collection center (CGMCC) No.28317, the preservation date is 2023 and 08 and 31 days, and the preservation address is North Star XILU No. 1 and 3 in the Korean region of Beijing.
Further, the concentration of NaCl is 50 to 100mM, and the concentration of MgSO 4 is 25 to 50mM.
Further, in the step (1), fermentation culture comprises inoculating escherichia coli engineering bacteria into a seed culture medium, and culturing for 12-16 hours at 37 ℃ to obtain seed liquid; and inoculating the seed liquid into a fermentation culture medium, fermenting and culturing at 33-35 ℃, continuously supplementing the fermentation culture medium in the fermentation process, and fermenting for 48-56 hours.
Further, the seed medium comprises the following components in concentration: 8-14 g/L of compound peptone, 4-12 g/L of yeast powder and 8-12 g/L of sodium chloride; the compound peptone comprises tryptone and bovine bone peptone with the weight ratio of 2-3:1.
Further, the fermentation medium comprises the following components in concentration: 5-40 g/L of glucose, 5-40 g/L of lactose, 10-15 g/L of compound peptone, 20-30 g/L of yeast powder, 10-20 g/L of dipotassium hydrogen phosphate, 1-5 g/L of potassium dihydrogen phosphate and 5-15 mL/L of trace metal element solution; the trace metal element solution comprises the following components in concentration: ferrous sulfate 6g/L, manganese sulfate monohydrate 0.35g/L, zinc sulfate heptahydrate 2.22g/L, anhydrous copper sulfate 1.0g/L, and ammonium molybdate 0.11g/L.
Further, in the step (2), the mixing volume ratio of the fermentation cells and the sodium citrate buffer is 1:10.
Further, in the step (2), the operation temperature of the homogenizing wall breaking treatment is 8-10 ℃ and the operation pressure is 90-100 mpa.
Further, in the step (3), a needle filter of 0.2 to 0.45 μm is used for clarification filtration.
The invention has the beneficial effects that:
1. According to the preparation method of the high-activity alpha-1, 2-fucosyltransferase enzyme solution, fermentation thalli in escherichia coli K-12 MG1655 BLBYZT6 fermentation broth are homogenized, intracellular enzymes are released, then the intracellular enzymes are mixed with supernatant fluid of the original fermentation broth, naCl and MgSO 4 are added, and the enzyme activity of the alpha-1, 2-fucosyltransferase enzyme solution can be obviously improved.
2. According to the preparation method of the high-activity alpha-1, 2-fucosyltransferase enzyme solution, the prepared alpha-1, 2-fucosyltransferase enzyme solution is stored for 20 days at the temperature of 3 ℃, the enzyme activity can be kept about 70%, the loss of the enzyme activity of the enzyme solution can be obviously reduced, the enzyme activity shelf life of the enzyme solution is prolonged, and the yield of synthesizing 2' -FL by an enzyme method is improved.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The Escherichia coli K-12 MG1655 BLBYZT6 used in the following examples was obtained by subjecting an Escherichia coli K-12 MG1655 strain purchased by the applicant to ultraviolet light and nitrosoguanidine compound mutagenesis treatment, and was deposited with China general microbiological culture Collection center, accession number: CGMCC No.28317, preservation date: 2023, 31, 08, deposit institution address: other relevant information is disclosed in patent CN117089503, no. 3, the west way No. 1, the korean district North Star, beijing.
The compositions of the seed medium and fermentation medium used in the following examples were as follows:
(1) The seed medium comprises the following components in concentration: 8-14 g/L of compound peptone, 4-12 g/L of yeast powder and 8-12 g/L of sodium chloride; the compound peptone comprises tryptone and bovine bone peptone with the weight ratio of 2-3:1.
(2) The fermentation medium comprises the following components in concentration: 5-40 g/L of glucose, 5-40 g/L of lactose, 10-15 g/L of compound peptone, 20-30 g/L of yeast powder, 10-20 g/L of dipotassium hydrogen phosphate, 1-5 g/L of potassium dihydrogen phosphate and 5-15 mL/L of trace metal element solution; the trace metal element solution comprises the following components in concentration: ferrous sulfate 6g/L, manganese sulfate monohydrate 0.35g/L, zinc sulfate heptahydrate 2.22g/L, anhydrous copper sulfate 1.0g/L, ammonium molybdate 0.11g/L; the compound peptone comprises tryptone and bovine bone peptone with the weight ratio of 2-3:1.
Example 1
The preparation method of the alpha-1, 2-fucosyltransferase enzyme solution specifically comprises the following steps:
(1) Inoculating Escherichia coli K-12 MG1655 BLBYZT6 into a seed culture medium, and culturing at 37deg.C for 12 hr to obtain seed solution; then inoculating the seed solution into a fermentation medium, fermenting and culturing at 33 ℃, continuously supplementing the fermentation medium in the fermentation process, and fermenting for 48 hours to obtain a fermentation liquid; and centrifuging the fermentation liquor to obtain fermentation thalli and a first supernatant.
(2) Mixing the fermentation thalli with a sodium citrate buffer solution in a volume ratio of 1:10, then carrying out homogenizing wall breaking treatment, wherein the operating temperature of a homogenizer is 8 ℃, the operating pressure is 90Mpa, releasing intracellular enzyme, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a crude enzyme solution.
(3) The crude enzyme solution was filtered using a 0.45 μm needle filter to obtain an enzyme supernatant, and 50mM NaCl and 25mM MgSO 4 were added to adjust the pH to 7.2 to prepare an alpha-1, 2-fucosyltransferase enzyme solution.
Example 2
The preparation method of the alpha-1, 2-fucosyltransferase enzyme solution specifically comprises the following steps:
(1) Inoculating Escherichia coli K-12 MG1655 BLBYZT6 into a seed culture medium, and culturing at 37deg.C for 14 hr to obtain seed solution; then inoculating the seed liquid into a fermentation medium, fermenting and culturing at 34 ℃, continuously supplementing the fermentation medium in the fermentation process, and fermenting for 52 hours to obtain a fermentation liquid; and centrifuging the fermentation liquor to obtain fermentation thalli and a first supernatant.
(2) Mixing the fermentation thalli with a sodium citrate buffer solution in a volume ratio of 1:10, then carrying out homogenizing wall breaking treatment, wherein the operating temperature of a homogenizer is 9 ℃, the operating pressure is 95Mpa, releasing intracellular enzymes, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a crude enzyme solution.
(3) The crude enzyme solution was filtered using a 0.45 μm needle filter to obtain an enzyme supernatant, and 75mM NaCl and 35mM MgSO 4 were added to adjust the pH to 7.4 to prepare an alpha-1, 2-fucosyltransferase enzyme solution.
Example 3
The preparation method of the alpha-1, 2-fucosyltransferase enzyme solution specifically comprises the following steps:
(1) Inoculating Escherichia coli K-12 MG1655 BLBYZT6 into a seed culture medium, and culturing at 37deg.C for 16 hr to obtain seed solution; then inoculating the seed liquid into a fermentation medium, fermenting and culturing at 35 ℃, continuously supplementing the fermentation medium in the fermentation process, and fermenting for 56 hours to obtain a fermentation liquid; and centrifuging the fermentation liquor to obtain fermentation thalli and a first supernatant.
(2) Mixing fermentation thalli and sodium citrate buffer solution in a volume ratio of 1:10, then carrying out homogenizing wall breaking treatment, wherein the operating temperature of a homogenizer is 10 ℃, the operating pressure is 100Mpa, releasing intracellular enzyme, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain crude enzyme solution.
(3) The crude enzyme solution was filtered using a 0.2 μm needle filter to obtain an enzyme supernatant, and 100mM NaCl and 50mM MgSO 4 were added to adjust the pH to 7.6 to prepare an alpha-1, 2-fucosyltransferase enzyme solution.
Comparative example 1
Unlike example 1, only the first supernatant was used as a crude enzyme solution, and the treatment of step (3) was performed to obtain an alpha-1, 2-fucosyltransferase enzyme solution.
Comparative example 2
Unlike example 1, only the second supernatant was used as a crude enzyme solution, and the treatment of step (3) was performed to obtain an alpha-1, 2-fucosyltransferase enzyme solution.
Comparative example 3
Unlike example 1, only 50mM NaCl was added to the enzyme supernatant of step (3).
Comparative example 4
Unlike example 1, only 25mM MgSO 4 was added to the enzyme supernatant of step (3).
Comparative example 5
Unlike example 1, no NaCl and no MgSO 4 were added to the enzyme supernatant of step (3).
Test case
1. Method for measuring enzyme activity of alpha-1, 2-fucosyltransferase enzymatic solution
(1) GDP-fucose is used as a donor, and lactose is used as an acceptor substrate. 150. Mu.L of alpha-1, 2-fucosyltransferase enzyme solution was taken, 150. Mu.L of 50mM Tris-HCI buffer (pH 7.4) was added, 2mM GDP-L-fucose, 5mM lactose, 5mM ATP was further added, and after the mixed solution was incubated at 37℃for 1 hour, the reaction was stopped in ice bath for 10 minutes, centrifuged at 12000rpm for 10 minutes, and ultra-filtered by a desalting column.
(2) And (3) injecting the reaction solution after impurity removal into an Agilent 1200 high performance liquid chromatograph (USA) for detection, qualitatively analyzing with retention time, quantitatively analyzing with peak area, and determining the amount of lactose as a substrate. The catalytic activity of alpha-1, 2-fucosyltransferase was calculated from lactose standard curve, defining the amount of enzyme required for conversion to 1. Mu. Mol 2' -FL as one activity unit (U) in 1 minute at 37 ℃.
2. Object of detection and result
The enzyme activity was measured by directly measuring the alpha-1, 2-fucosyltransferase enzyme solutions prepared in examples 1to 3 and comparative examples 1to 5, and the results are shown in Table 1 (0 d). In addition, some of the alpha-1, 2-fucosyltransferase enzyme solutions prepared in examples 1to 3 and comparative examples 1to 5 were stored in a refrigerator at 3℃for 20 days, and the enzyme activities were measured every 5 days, and the results are shown in Table 1.
TABLE 1 enzyme Activity Change of different alpha-1, 2-fucosyltransferase enzyme solutions
According to the data in the table above, the enzyme activity of the alpha-1, 2-fucosyltransferase liquids prepared in examples 1-3 was significantly improved as compared with comparative examples 1-2. As can be seen, the intracellular material of E.coli K-12 MG1655 BLBYZT6 and the fermentation broth are mixed to help increase the activity of alpha-1, 2-fucosyltransferase.
Secondly, the enzyme activity of the alpha-1, 2-fucosyltransferase enzyme solution prepared in the examples 1-3 is obviously higher than that of the comparative examples 3-5, which shows that the preparation method of the alpha-1, 2-fucosyltransferase enzyme solution provided by the invention can obviously improve the enzyme activity of the alpha-1, 2-fucosyltransferase enzyme solution.
In addition, the prepared alpha-1, 2-fucosyltransferase liquid is preserved for 20 days at 3 ℃, the enzyme activity of the alpha-1, 2-fucosyltransferase liquid prepared in examples 1-3 can still be kept about 70%, but the enzyme activity of the alpha-1, 2-fucosyltransferase liquid prepared in comparative examples 3-5 is reduced to below 70% at 5 days and reduced to about 50% at 10 days, which shows that the preparation method of the alpha-1, 2-fucosyltransferase liquid provided by the invention can obviously reduce the loss of the enzyme activity of the enzyme liquid, prolong the enzyme activity shelf life of the enzyme liquid and help to improve the yield of 2' -FL synthesized by an enzymatic method.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (8)
1. A method for preparing high-activity alpha-1, 2-fucosyltransferase enzyme solution, which is characterized by comprising the following steps: (1) Taking escherichia coli engineering bacteria expressing alpha-1, 2-fucosyltransferase, and fermenting and culturing to obtain fermentation liquor; centrifuging the fermentation liquor to obtain fermentation thalli and a first supernatant; (2) Mixing the fermentation thalli with a sodium citrate buffer solution, then carrying out homogenizing wall breaking treatment to release intracellular enzyme, centrifuging to obtain a second supernatant, and mixing the first supernatant and the second supernatant to obtain a crude enzyme solution; (3) Clarifying and filtering the crude enzyme solution to obtain an enzyme clear solution, adding NaCl and MgSO 4, and adjusting the pH value to 7.2-7.6 to obtain a high-activity alpha-1, 2-fucosyltransferase enzyme solution; the concentration of NaCl is 50-100 mM, and the concentration of MgSO 4 is 25-50 mM;
The engineering bacteria of the escherichia coli are escherichia coli (ESCHERICHIA COLI) K-12 MG1655 BLBYZT6, the escherichia coli K-12 MG1655 BLBYZT6 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.28317, the preservation date is 2023 and 31 months, and the preservation address is North Star Xili No. 1 and 3 in the Chaoyang area of Beijing city.
2. The method of claim 1, wherein the fermentation culture comprises inoculating an engineering bacterium of escherichia coli into a seed culture medium, and culturing for 12-16 hours at 37 ℃ to obtain a seed solution; and inoculating the seed liquid into a fermentation culture medium, fermenting and culturing at 33-35 ℃, continuously supplementing the fermentation culture medium in the fermentation process, and fermenting for 48-56 hours.
3. The method of claim 2, wherein the seed medium comprises the following concentrations of components: 8-14 g/L of compound peptone, 4-12 g/L of yeast powder and 8-12 g/L of sodium chloride; the compound peptone comprises tryptone and bovine bone peptone with the weight ratio of 2-3:1.
4. The method of claim 2, wherein the fermentation medium comprises the following concentrations of components: 5-40 g/L of glucose, 5-40 g/L of lactose, 10-15 g/L of compound peptone, 20-30 g/L of yeast powder, 10-20 g/L of dipotassium hydrogen phosphate, 1-5 g/L of potassium dihydrogen phosphate and 5-15 mL/L of trace metal element solution.
5. The method of claim 4, wherein the trace metal element solution comprises the following concentrations of components: ferrous sulfate 6g/L, manganese sulfate monohydrate 0.35g/L, zinc sulfate heptahydrate 2.22g/L, anhydrous copper sulfate 1.0g/L, and ammonium molybdate 0.11g/L.
6. The method of claim 1, wherein the mixing volume ratio of the fermentation broths to the sodium citrate buffer is 1:10.
7. The preparation method of claim 1, wherein the operation temperature of the homogenizing wall breaking treatment is 8-10 ℃ and the operation pressure is 90-100 mpa.
8. The method of claim 1, wherein the clarifying filtration is performed using a 0.2-0.45 μm pin filter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410282912.6A CN117866996B (en) | 2024-03-13 | 2024-03-13 | Preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410282912.6A CN117866996B (en) | 2024-03-13 | 2024-03-13 | Preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117866996A CN117866996A (en) | 2024-04-12 |
| CN117866996B true CN117866996B (en) | 2024-06-04 |
Family
ID=90585100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410282912.6A Active CN117866996B (en) | 2024-03-13 | 2024-03-13 | Preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117866996B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1433467A (en) * | 1999-12-21 | 2003-07-30 | 协和发酵工业株式会社 | Modified α1,2-fucosyltransferase gene, and method for producing α1,2-fucosyltransferase and fucose-containing sugars |
| CN103045526A (en) * | 2012-12-18 | 2013-04-17 | 江南大学 | A strain of recombinant Escherichia coli and its method for preparing phospholipase A1 |
| CN117089503A (en) * | 2023-10-17 | 2023-11-21 | 保龄宝生物股份有限公司 | Escherichia coli K-12 MG1655 BLBYZT6 and application thereof |
-
2024
- 2024-03-13 CN CN202410282912.6A patent/CN117866996B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1433467A (en) * | 1999-12-21 | 2003-07-30 | 协和发酵工业株式会社 | Modified α1,2-fucosyltransferase gene, and method for producing α1,2-fucosyltransferase and fucose-containing sugars |
| CN103045526A (en) * | 2012-12-18 | 2013-04-17 | 江南大学 | A strain of recombinant Escherichia coli and its method for preparing phospholipase A1 |
| CN117089503A (en) * | 2023-10-17 | 2023-11-21 | 保龄宝生物股份有限公司 | Escherichia coli K-12 MG1655 BLBYZT6 and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| One-pot multienzyme (OPME) synthesis of human blood group H antigens and a human milk oligosaccharide (HMOS) with highly active Thermosynechococcus elongatus α1–2-fucosyltransferase;Chao Zhao等;Chem Commun (Camb).;20160311;第第52卷卷(第第20期期);第3页第3段 * |
| 李斌等.食品酶学与酶工程 第3版.北京：中国农业大学出版社,2021,第172页. * |
| 李红等.食品化学 第2版.北京：中国纺织出版社,2022,第315页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117866996A (en) | 2024-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2690469B2 (en) | L-ascorbic acid-containing biomass | |
| US4321325A (en) | Process for producing L-threonine | |
| CN109609580B (en) | Fermentation medium and fermentation method of riboflavin | |
| WO2000009660A1 (en) | Novel microorganisms and method for producing l-threonine using the same | |
| Yoon et al. | Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor | |
| CN117866996B (en) | Preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution | |
| CN108410918B (en) | Tryptophan fermentation medium and tryptophan fermentation method | |
| CN109266578B (en) | Escherichia coli ACThr1032 and application thereof in fermentation production of L-threonine | |
| Luan et al. | Factors governing lactic acid formation in long term cultivation of hybridoma cells | |
| CN107058414B (en) | Method for preparing L-alanine | |
| CN112501221A (en) | Method for improving conversion rate of threonine and saccharic acid | |
| CN116179356B (en) | Method for high-density heterotrophic culture of chlamydomonas reinhardtii and application thereof | |
| CN106591401B (en) | Fermentation promoter for increasing yield of gentamicin C1a and addition method thereof | |
| Takagi et al. | Effects of shifts up and down in osmotic pressure on production of tissue plasminogen activator by Chinese hamster ovary cells in suspension | |
| CN111172094A (en) | Yeast extract and preparation method thereof | |
| CN113817794B (en) | Fermentation method of gibberellic acid GA3 ∈ 7.5g/L | |
| CN112011419A (en) | A kind of nutrient solution rich in caproic acid producing bacteria and preparation method and application thereof | |
| JPS60180597A (en) | Production of l-threonine by fermentation | |
| CN113755548A (en) | Method for improving fermentation level of polymyxin B | |
| CN113957115B (en) | Fermentation method for high yield of gibberellic acid | |
| CN112195206A (en) | Amino acid fermentation process using liquid caustic soda to replace part of liquid ammonia | |
| CN110923275A (en) | Fermentation and extraction process of glutamic acid | |
| CN117660307A (en) | Method for cell culture and kit for cell culture | |
| CN118064651B (en) | Method for maintaining steady state of cell perfusion culture system | |
| Babich et al. | Innovative technologies of bioconvertion of renewable raw materials in etanol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |