CN117866996A - 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
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 82
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 82
- 101710098620 Alpha-1,2-fucosyltransferase Proteins 0.000 title claims abstract description 43
- 230000000694 effects Effects 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 59
- 238000000855 fermentation Methods 0.000 claims abstract description 51
- 230000004151 fermentation Effects 0.000 claims abstract description 51
- 239000006228 supernatant Substances 0.000 claims abstract description 33
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000011780 sodium chloride Substances 0.000 claims abstract description 14
- 241001052560 Thallis Species 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 13
- 241000588724 Escherichia coli Species 0.000 claims abstract description 10
- 230000003834 intracellular effect Effects 0.000 claims abstract description 8
- 239000001509 sodium citrate Substances 0.000 claims abstract description 8
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims abstract description 8
- 241000894006 Bacteria Species 0.000 claims abstract description 7
- 239000007853 buffer solution Substances 0.000 claims abstract description 6
- 238000001914 filtration Methods 0.000 claims abstract description 5
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 14
- 235000019319 peptone Nutrition 0.000 claims description 14
- 150000001875 compounds Chemical class 0.000 claims description 10
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 9
- 239000001963 growth medium Substances 0.000 claims description 9
- 239000008101 lactose Substances 0.000 claims description 9
- 241001646716 Escherichia coli K-12 Species 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 229910021654 trace metal Inorganic materials 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 5
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 241000283690 Bos taurus Species 0.000 claims description 4
- 210000000988 bone and bone Anatomy 0.000 claims description 4
- 239000012137 tryptone Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000011609 ammonium molybdate Substances 0.000 claims description 3
- 235000018660 ammonium molybdate Nutrition 0.000 claims description 3
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 claims description 3
- 229940010552 ammonium molybdate Drugs 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 239000011790 ferrous sulphate Substances 0.000 claims description 3
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 3
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 3
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 claims description 3
- 235000010633 broth Nutrition 0.000 claims 1
- 210000004251 human milk Anatomy 0.000 abstract description 7
- 235000020256 human milk Nutrition 0.000 abstract description 7
- 150000002482 oligosaccharides Chemical class 0.000 abstract description 6
- 229920001542 oligosaccharide Polymers 0.000 abstract description 5
- 230000001105 regulatory effect Effects 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 8
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- LQEBEXMHBLQMDB-UHFFFAOYSA-N GDP-L-fucose Natural products OC1C(O)C(O)C(C)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C3=C(C(N=C(N)N3)=O)N=C2)O1 LQEBEXMHBLQMDB-UHFFFAOYSA-N 0.000 description 4
- LQEBEXMHBLQMDB-JGQUBWHWSA-N GDP-beta-L-fucose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C3=C(C(NC(N)=N3)=O)N=C2)O1 LQEBEXMHBLQMDB-JGQUBWHWSA-N 0.000 description 4
- 238000004977 Hueckel calculation Methods 0.000 description 4
- 229940062827 2'-fucosyllactose Drugs 0.000 description 3
- HWHQUWQCBPAQQH-UHFFFAOYSA-N 2-O-alpha-L-Fucosyl-lactose Natural products OC1C(O)C(O)C(C)OC1OC1C(O)C(O)C(CO)OC1OC(C(O)CO)C(O)C(O)C=O HWHQUWQCBPAQQH-UHFFFAOYSA-N 0.000 description 3
- HWHQUWQCBPAQQH-BWRPKUOHSA-N 2-fucosyllactose Chemical compound O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@H]([C@H](O)CO)[C@H](O)[C@@H](O)C=O HWHQUWQCBPAQQH-BWRPKUOHSA-N 0.000 description 3
- SNFSYLYCDAVZGP-UHFFFAOYSA-N UNPD26986 Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(OC(O)C(O)C2O)CO)OC(CO)C(O)C1O SNFSYLYCDAVZGP-UHFFFAOYSA-N 0.000 description 3
- QGWNDRXFNXRZMB-UUOKFMHZSA-N GDP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O QGWNDRXFNXRZMB-UUOKFMHZSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- QGWNDRXFNXRZMB-UHFFFAOYSA-N guanidine diphosphate Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O QGWNDRXFNXRZMB-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 235000013350 formula milk Nutrition 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- -1 nitrosoguanidine compound Chemical class 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
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- 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
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- 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)
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- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01069—Galactoside 2-alpha-L-fucosyltransferase (2.4.1.69)
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- 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
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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 enzyme clear solution, adding NaCl and MgSO 4 And regulating 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 solution of alpha-1, 2-fucosyltransferaseEnzyme activity, reduced loss of enzyme activity, and improved yield of 2' -FL synthesized by 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 next to lactose and fat, have important biological functions, and play an important role in the growth and development of newborns. 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' -FL) accounts for about 31 percent (mole fraction) in HMOs, and the content of the 2'-fucosyllactose 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 enzyme clear solution, adding NaCl and MgSO 4 And regulating 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, escherichia coli K-12 MG1655 BLBYZT6 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 28317, a preservation date of 2023 and 31 months, and a preservation address of North Chen Xiyu No. 1 and 3 in the Korean region of Beijing city.
Further, the concentration of NaCl is 50-100 mM, mgSO 4 The concentration is 25-50 mM.
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. the invention provides a preparation method of high-activity alpha-1, 2-fucosyltransferase enzyme solution, which comprises homogenizing fermentation thalli in escherichia coli K-12 MG1655 BLBYZT6 fermentation liquor, releasing intracellular enzyme, mixing with supernatant of the original fermentation liquor, and adding NaCl and MgSO 4 Can obviously improve the enzyme activity of alpha-1, 2-fucosyltransferase enzyme solution.
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 the 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, north chen west way 1, the region of korea in beijing city.
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 is usedFiltering with 0.45 μm needle filter to obtain enzyme supernatant, adding 50mM NaCl and 25mM MgSO 4 The pH value is regulated to 7.2, and the alpha-1, 2-fucosyltransferase enzyme solution is prepared.
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 give an enzyme supernatant, and 75mM NaCl and 35mM MgSO were added 4 The pH value is regulated to 7.4, and the alpha-1, 2-fucosyltransferase enzyme solution is prepared.
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 give an enzyme supernatant, and 100mM NaCl and 50mM MgSO were added 4 The pH value is regulated to 7.6, and the alpha-1, 2-fucosyltransferase enzyme solution is prepared.
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 was added to the enzyme supernatant of step (3) 4 。
Comparative example 5
Unlike example 1, no NaCl or MgSO was added to the enzyme supernatant of step (3) 4 。
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 1 to 3 and comparative examples 1 to 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 1 to 3 and comparative examples 1 to 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 were 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 (9)
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 enzyme clear solution, adding NaCl and MgSO 4 Adjusting the pH value to 7.2-7.6 to prepare high-activity alpha-1, 2-fucosyltransferase enzyme solution;
the engineering bacteria of the escherichia coli are escherichia coli @Escherichia coli) K-12 MG1655 BLBYZT6, escherichia coli K-12 MG1655 BLBYZT6 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 28317, a preservation date of 2023, 08 and 31 days, and a preservation address of North Chen Xiyu No. 1, 3 in the Chaoyang region of Beijing city.
2. The method according to claim 1, wherein the concentration of NaCl is 50 to 100mM, mgSO 4 The concentration is 25-50 mM.
3. 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.
4. A method of preparation as claimed in claim 3 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.
5. A method of preparation as claimed in claim 3 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.
6. The method of claim 5, 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.
7. The method of claim 1, wherein the mixing volume ratio of the fermentation broths to the sodium citrate buffer is 1:10.
8. 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.
9. The method of claim 1, wherein the clarifying filtration is performed using a 0.2-0.45 μm pin filter.
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| 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 |
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| 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 |
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