CN1641037A - Method for preparing egg yolk oligosaccharide using N-glycosidase E - Google Patents
Method for preparing egg yolk oligosaccharide using N-glycosidase E Download PDFInfo
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Abstract
The present invention provides the method of extracting yolk N-oligose with N-glycosidase F expressed efficiently on the surface of Saccharomyces cerevisiae cell and through the enzymic hydrolysis of glucoprotein and glucopeptide in egg yolk. The technological process includes the steps of: defatting yolk as material, hydrolyzing proteinase, denaturation, ecentrifuging to obtain the supernatant, enzymolysis with N-glycosidase F, ecentrifuging to obtain the supernatant, ultrafiltering, desalting, ion column exchanging, and decompression drying to obtain the yolk oligose product. The present invention has the innovation of utilizing PNGase F expressed on the surface of Saccharomyces cerevisiae in preparing acid N-glycochain. Research shows that the yolk N-oligose product may be used in inhibiting rotavirus infection and remitting influenza virus. In addition, the complete N-glycochain may find its wide application in sugar science research.
Description
Technical field: the present invention relates to bio-pharmaceuticals, specifically about utilizing enzyme process from egg yolk, to extract the method for sialyloligosaccharide.
Background technology: studies have shown that, sialyloligosaccharide plays important effect in cell recognition, virus infection and vital movement such as neutralize a toxin, the sialic acid residues of its sugar-chain end is the identification binding site of multiple pathogenic agent and toxin infected cell, as influenza virus and the rotavirus that causes infantile diarrhea; Simultaneously, liver is cleaned up Study on Mechanism to be shown, can remove the hepatic lectin of allos mixture in the blood, be asialoglycoprotein receptor (ASGR), it can be discerned and remove sialic galactose residue, and therefore having the sialyloligosaccharide recombinant protein medicine can obviously improve medicine in stability in blood and transformation period; Japan scientist (L.R.Juneja etc.) the also proof of feeding in the experiment of mouse is added a certain proportion of yolk sialyloligosaccharide in the daily ration, can obviously improve the intelligence of young mouse
[1]Therefore oligosaccharides, especially sialyloligosaccharide have very big application and development potentiality in industries such as medicine, food.At present, in materials such as human milk, cow's milk, egg, successfully isolate the free sialyloligosaccharide component, and finish structure analysis and its biological activity test; The research of sialyloligosaccharide chemosynthesis also is one of focus of sugared study on the synthesis; Utilize the hydrazinolysis method successfully from egg yolk, to isolate the yolk sialyloligosaccharide
[2], but owing to be subjected to the restriction of raw material and cost, more than resulting sialyloligosaccharide just be used for Analysis and Identification, Japanese Taiyo Kagaku company prepares the glycopeptide product (Sunsial that has sialyloligosaccharide with the protease hydrolysis method
TM) be widely used in types of functionality food
[3], utilize acid-hydrolysis method from yolk, to prepare sialic technology and dropped into application
[4], industrial biotechnology key lab of China Southern Yangtze University utilizes acid-hydrolysis method to prepare the hydrolysis clear liquid of high sialic acid content from yolk
[5]But end at present, yet there are no and utilize egg yolk to carry out the report of scale preparation sialyloligosaccharide.
Egg is the food with better nutritivity value that people generally acknowledge, basis as origin of life, the a large amount of types of functionality compound of contain in the egg, products such as the yolk antibody (IgY) of extraction preparation, Yelkin TTS have been widely used in industries such as medicine, foods and cosmetics at present from egg.Studies have shown that in recent years, sialic acid content in the egg yolk can be up to 0.2%, but also containing the free glycopeptide that has sialyloligosaccharide of 2.8umol/egg yolk (8.0mg/egg yolk, the yolk dry weight is 9.6 grams), egg yolk is a material storehouse with the acid oligosaccharides of high-content.Simultaneously, in the preparation of yolk antibody, it is produced in the waste liquid also has a large amount of glycoprotein, free glycopeptide is not loss also, and albumen is beneficial to and carries out follow-up enzymolysis operation, with the raw material of this waste liquid as yolk oligose extraction preparation not by denaturing treatment, raw materials cost is very cheap, is suitable for carrying out the industrialization operation.
Summary of the invention: the objective of the invention is to utilize at the N-of brewing yeast cell surface expression Glycosylase F (PNGase F), the N-sugar chain (N-glycans) that is had on the glycoprotein that contained in the egg yolk and the glycopeptide is carried out enzymolysis prepare yolk oligose.Yolk oligose involved in the present invention mainly is to be present in the N-sugar chain that glycoprotein and glycopeptide carry in the egg yolk, the primary structure of described acidic oligosaccharide is that N connects double antenna sialyloligosaccharide chain (N-linked disialylbiantennary glycan chain), and its primary structure formula is as follows:
N-Glycosylase F (Peptide-N
4-(the asparigiene amidase of N-acetyl-β-D-glucosaminyl); EC3.5.1.52), be made up of 314 amino acid, molecular weight is 34779Da.Can intactly downcut the N-glucosides from glycopeptide and glycoprotein surface, its action site is the valence bond structure between N-acetyl grape amine residue and the l-asparagine.The used N-Glycosylase of the present invention F expresses the N-Glycosylase F gene recombination that derives from meningitis purulence bacillus (Flavobacteriummeningoseptium) to yeast saccharomyces cerevisiae (S.cerevisiae) cell surface, the enzyme activity determination result is 20U/l fermented liquid (1 enzyme live unit definition be that per minute can complete hydrolysis 1 μ mol ribonuclease B).Technical scheme of the present invention is achieved in that the present invention is a kind of mass-producing operation that is suitable for carrying out, and the technical process that utilizes biological enzyme to extract the preparation egg yolk oligose is as follows:
Yolk raw material---→ degreasing---→ protease hydrolysis---→ denaturing treatment---→---→ N-Glycosylase F enzymolysis---→---→ ultrafiltration (molecular weight is held back and is 10kD)---→ desalination---→ ion exchange column---→ drying under reduced pressure---→ yolk oligose product---→ product detection---→ packing---→ finished product of getting supernatant liquor after centrifugal of getting supernatant liquor after centrifugal
Process explanation:
1, indication yolk raw material of the present invention is mainly:
A. the yolk in the new fresh hen egg;
B. commercial yolk powder;
C. the tankage waste liquid produced of yolk antibody.
In the above raw material, raw material A and raw material C yield are higher, and from cost consideration, we think that raw material C is best starting material.
2, degreasing process involved in the present invention mainly contains organic solvent degreasing (being suitable for yolk powder) and low-temperature centrifugation degreasing method (being suitable for liquid starting material), considers that from environmental protection and operational safety the present invention recommends to use the latter.The condition of low-temperature centrifugation degreasing is 4 ℃, 3000*g, 1hr.
3, the used proteolytic enzyme of the present invention is papoid or stomach en-.
4, denaturing treatment is meant that (90 ℃, 20min) processing makes the proteolytic enzyme deactivation, and increases the enzymolysis efficiency of N-Glycosylase F in heating.
5, indication N-Glycosylase F enzymolysis process of the present invention is to utilize at the N-of yeast saccharomyces cerevisiae surface expression Glycosylase F, directly adds hydrolyzed solution and carries out enzymolysis, or utilize the method for immobilized cell, hydrolyzed solution to cross post and carry out enzymolysis.Enzymatic hydrolysis condition is 25-42 ℃, 2-48hr, and top condition is 30-37 ℃, 20-36hr.
6, the ultrafiltration technology that the present invention relates to is that molecular weight cut-off is 10000 membrane filtration, and working pressure is 0.4Mpa, and service temperature is 40-55 ℃, and optimal temperature conditions is 45-50 ℃.
7, desalinating process involved in the present invention is for to carry out desalination by desalting column or the nanofiltration of Sephadex G25, and specific conductivity is lower than 200 μ s/cm after the desalination, and wherein, spissated effect has also been played in nanofiltration in desalination, can lower the burden of subsequent technique.
8, indication product inspection method of the present invention is meant purity and assay, available thin layer chromatography (TLC) or high-performance thin layer chromatography (HPTLC) (HPTLC) carry out purity detecting, acidic oligosaccharide can be determined the content of acidic oligosaccharide by the sialic acid content that the Resorcinol method is measured, and the mensuration of yolk oligose total content can utilize after the acid hydrolysis content of mensuration reducing sugar to determine.The reducing sugar test instrument is the automatic reducing sugar test instrument of producing in biological center, Shandong Scientific Research Academy of SGD-III type.
Innovative point of the present invention is for utilizing the PNGase that efficiently expresses on the yeast saccharomyces cerevisiae surface, by glycoprotein and glycopeptide in the enzymatic hydrolysis egg yolk, extract preparation yolk N-oligosaccharides, especially extract the technological line of preparation N-sialyloligosaccharide, it can utilize cheap biomaterial, and a large amount of the extraction prepares the oligosaccharides that some has important biomolecule function and application value, compares with synthesis method, oligosaccharides manufacturing cost of the present invention reduces greatly, helps the industrialization operation.
Example 1: utilize yolk powder to prepare yolk oligose
1.1 the degreasing of yolk powder
1.1.1 acetone extract:
Get yolk powder 2000 grams (available from Dalian biochemical pharmacy company), add 10 liters of acetone, be heated to backflow (57-58 ℃), reflux after 2 hours, stop heating, static 30 minutes, abandoning supernatant (orange-yellow) repeated aforesaid operations 2 times.
1.1.2 methanol extraction:
After acetone extract finishes, extract as stated above three times with the methyl alcohol of equal volume.
1.1.3 the preparation of de-fatted egg yolk powder (DEY, dilapidated egg yolk):
After extraction finished, 45 ℃ of dried overnight with the drying products weighing results were: 813 grams, place standby as for dry sections temperature DEY.
1.2 protease treatment DEY
800 gram DEY of preparation are dissolved in the sodium phosphate buffer (pH7.0) of 8000ml 0.01mol/l, and add 32 gram papoids (Fisher Scientific Ltd.), 65 ℃ of enzyme digestion reactions are after 4 hours under whipped state, and 90 ℃ are evaporated to 2000ml.
Enzyme hydrolyzate centrifugal treating (3000g, 30 minutes, room temperature) after concentrating, supernatant liquor are with 0.22 μ m membrane filtration, and collected volume is 1220ml, places in the sterilized 2000ml triangular flask standby.
1.3 PNGase F enzymolysis
1.3.1 the preparation of PNGase F
The brewing yeast cell of culturing cell surface expression PNGase F (Saccharomyces cereviae, this recombination yeast bacterial classification makes up for this seminar) 2000ml, behind the abduction delivering, centrifugal collection wet thallus 26 grams, standby.
1.3.2 enzymatic hydrolysis condition
Add sodium acetate 1.09 grams in the 1220ml of above-mentioned collection hydrolyzed solution, SDS 1.2 grams are transferred pH to 5.5 with spirit acid.Under rotation concussion condition (80rpm), 37 ℃, enzymolysis 24 hours.
1.3.3 enzymolysis solution aftertreatment
After enzymolysis finishes, and the hydrolyzed solution centrifugal treating (4 ℃, 6000g, 10min), it is standby to collect the about 1250ml of supernatant liquor.
1.4 oligosaccharides separates
1.4.1 uf processing
With stirring cup type ultra-fine filter, selecting molecular weight cut-off for use is 10000 filter membrane, and nitrogen pressure is filtered, and filtering layer is collected filtered solution with deionized water wash (30ml/ time) 4 times, is total to 1300ml, and 70 ℃ are evaporated to 200ml, and 4 ℃ of placements are standby.
1.4.2 nanofiltration concentrates, desalination
In nanofiltration process, detect to see through the specific conductivity of liquid by DS-11 type conductivity meter, and constantly spend ionized water and wash, reduce to 180 μ s/cm until specific conductivity, collect the concentrated solution after the nanofiltration, 50ml altogether, 4 ℃ of placements are standby.
14.3 ion exchange column separates oligosaccharides
Select Japan Cao Da DEAE-Toyopearl 650M anionite-exchange resin for use, 5mM Tris-HCl damping fluid (pH 8.0) balanced exchange post carries out gradient elution (0-0.1M), flow velocity 30ml/min with NaCl solution, the 10ml/ collection tube, HPTLC detects, and collects 10-18 pipe (detecting positive) at last, merges, press the described method of 1.4.2, carry out the nanofiltration desalination once more, the liquid specific conductivity is constant in 150 μ s/cm until seeing through, and collects altogether and obtains 50ml desalination and concentration liquid.
1.4.4 decompression, lyophilize
With Rotary Evaporators (RE 52A, Shanghai Yarong Biochemical Instrument Plant) earlier with 50ml desalination and concentration liquid under 60 ℃ of conditions, be evaporated to the 20ml volume, utilize lyophilize to remove out residual moisture again, obtain 2.1 gram dry products altogether.
1.5 product detected result
Resorcinol method detected result: the product sialic acid content is 0.19 gram; The reducing sugar detected result is: the product reducing sugar content is 93% (is benchmark with glucose).
Example 2: utilize new fresh hen egg to prepare yolk oligose
2.1 the preparation of de-fatted egg yolk
Get 100 new fresh hen eggs, get yolk after the fragmentation, obtain 1.6 liters of egg yolk liquids, this egg yolk liquid ice bath is refrigerated to 4 ℃, the plasma water (4 ℃) that adds 5 times of volumes, not 1 hour (in the entire operation process, solution temperature is not higher than 4 ℃) of vigorous stirring, (4 ℃ of centrifugal treating, 4000g, 1hr) after, collect supernatant liquor, totally 9.2 liters, be de-fatted egg yolk solution.
2.2 protease hydrolyzed
In the preparation yolk solution, and add 30 gram papoids (Fisher Scientific Ltd.), 65 ℃ of enzyme digestion reactions are after 4 hours under whipped state, and 90 ℃ are evaporated to 2000ml.
Enzyme hydrolyzate centrifugal treating (8000g, 30 minutes, 4 ℃) after concentrating, supernatant liquor are with 0.22 μ m membrane filtration, and collected volume is 1850ml, places in the sterilized 2000ml triangular flask standby.
2.3 PNGase F enzymolysis
2.3.1 the preparation of PNGase F
Cultivate recombinant Saccharomyces cerevisiae cell 1500ml, behind the abduction delivering, centrifugal collection wet thallus 20 grams, standby.
2.3.2 enzymatic hydrolysis condition.
Add sodium acetate 1.65 grams in the 1850ml of above-mentioned collection hydrolyzed solution, SDS 1.85 grams are transferred pH to 5.5 with spirit acid.Under rotation concussion condition (80rpm), 37 ℃, enzymolysis 24 hours.
2.3.3 enzymolysis solution aftertreatment
After enzymolysis finishes, and the hydrolyzed solution centrifugal treating (4 ℃, 6000g, 10min), it is standby to collect the about 1800ml of supernatant liquor.
2.4 oligosaccharides separates
2.4.1 uf processing
With stirring cup type ultra-fine filter, selecting molecular weight cut-off for use is 10000 filter membrane, and nitrogen pressure is filtered, and filtering layer is collected filtered solution with deionized water wash (30ml/ time) 3 times, is total to 1900ml, and 70 ℃ are evaporated to 400ml, and 4 ℃ of placements are standby.
2.4.2 nanofiltration concentrates, desalination
In nanofiltration process, detect to see through the specific conductivity of liquid by DS-11 type conductivity meter, and constantly spend ionized water and wash, reduce to 180 μ s/cm until specific conductivity, collect the concentrated solution after the nanofiltration, 30ml altogether, 4 ℃ of placements are standby.
2.4.3 ion exchange column separates oligosaccharides
Select Japan Cao Da DEAE-Toyopearl 650M anionite-exchange resin for use, 5mM Tris-HCl damping fluid (pH 8.0) balanced exchange post carries out gradient elution (0-0.1M), flow velocity 30ml/min with NaCl solution, the 10ml/ collection tube, HPTLC detects, and collects 10-18 pipe (detecting positive) at last, merges, press the described method of 2.4.2, carry out the nanofiltration desalination once more, the liquid specific conductivity is constant in 150 μ s/cm until seeing through, and collects altogether and obtains 50ml desalination and concentration liquid.
2.4.4 lyophilize
Utilize lyophilize to remove out residual moisture, obtain 3.3 gram yolk oligose products altogether.
2.5 product detects
Resorcinol method detected result: the product sialic acid content is 0.31 gram; The reducing sugar detected result is: the product reducing sugar content is 91% (is benchmark with glucose).
Example 3: utilize yolk antibody to prepare waste liquid and prepare yolk oligose
3.1 raw material degreasing
Get 9 liters of stock liquids (academy of agricultural sciences, Shandong Province poultry institute provides), 1000 gram ice cubes, 1 hour (in the entire operation process, solution temperature is not higher than 4 ℃) of vigorous stirring, centrifugal treating (4 ℃, 4000g, 1hr) after, collect supernatant liquor, totally 9.6 liters, be de-fatted egg yolk solution.
3.2 protease hydrolyzed
In the preparation yolk solution, and add 20 gram papoids (Fisher Scientific Ltd.), 65 ℃ of enzyme digestion reactions are after 4 hours under whipped state, and 90 ℃ are evaporated to 2000ml.
Enzyme hydrolyzate centrifugal treating (8000g, 30 minutes, 4 ℃) after concentrating, supernatant liquor are with 0.22 μ m membrane filtration, and collected volume is 1920ml, places in the sterilized 2000ml triangular flask standby.
3.3 PNGase F enzymolysis
3.3.1 the preparation of PNGase F
Cultivate recombinant Saccharomyces cerevisiae cell 1000ml, behind the abduction delivering, centrifugal collection wet thallus 13 grams, standby.
3.3.2 enzymatic hydrolysis condition
Add sodium acetate 1.70 grams in the 1900ml of above-mentioned collection hydrolyzed solution, SDS 1.90 grams are transferred pH to 5 with spirit acid: 5.Under rotation concussion condition (80rpm), 37 ℃, enzymolysis 24 hours.
3.3.3 enzymolysis solution aftertreatment
After enzymolysis finishes, and the hydrolyzed solution centrifugal treating (4 ℃, 6000g, 10min), it is standby to collect the about 1850ml of supernatant liquor.
3.4 oligosaccharides separates
3.4.1 uf processing
With stirring cup type ultra-fine filter, selecting molecular weight cut-off for use is 10000 filter membrane, and nitrogen pressure is filtered, and filtering layer is collected filtered solution with deionized water wash (50ml/ time) 3 times, is total to 2000ml, and 70 ℃ are evaporated to 300ml, and 4 ℃ of placements are standby.
3.4.2 nanofiltration concentrates., desalination
In nanofiltration process, detect to see through the specific conductivity of liquid by DS-11 type conductivity meter, and constantly spend ionized water and wash, reduce to 180 μ s/cm until specific conductivity, collect the concentrated solution after the nanofiltration, 30ml altogether, 4 ℃ of placements are standby.
3.4.3 ion exchange column separates oligosaccharides
Select Japan Cao Da DEAE-Toyopearl 650M anionite-exchange resin for use, 5mM Tris-HCl damping fluid (pH 8.0) balanced exchange post carries out gradient elution (0-0.1M), flow velocity 30ml/min with NaCl solution, the 10ml/ collection tube, HPTLC detects, and collects 10-18 pipe (detecting positive) at last, merges, press the described method of 2.4.2, carry out the nanofiltration desalination once more, the liquid specific conductivity is constant in 150 μ s/cm until seeing through, and collects altogether and obtains 50ml desalination and concentration liquid.
3.4.4 lyophilize
Utilize lyophilize to remove out residual moisture, obtain 1.45 gram yolk oligose products altogether.
3.5 product detects
Resorcinol method detected result: the product sialic acid content is 0.13 gram; The reducing sugar detected result is: the product reducing sugar content is 94% (is benchmark with glucose).
By above example as can be seen, the present invention utilizes reorganization PNGase F, adopts different yolk raw materials to carry out the preparation of yolk oligose, and has obtained the mixture of the yolk N-oligosaccharides of higher degree, proves that preparation method of the present invention is feasible.From the report analysis of having delivered, the content of yolk N-acidic oligosaccharide is greater than 0.5%, but the yolk oligose that the present invention prepares in yolk powder only accounts for about 0.1% of raw material, and yield is lower, utilizes the analysis of SDS protein electrophoresis as can be known, the solubility of commercially available yolk powder is relatively poor, influence the treatment effect of proteolytic enzyme, thereby greatly reduced the yield of product, and the price of yolk powder higher (30 yuan/kilogram), utilize the product cost of this feedstock production higher, be unfavorable for the industrialization operation; And raw material C is the waste material that yolk antibody is produced, price very cheap (raw materials used price is about 0.5 yuan/liter), the product cost of preparation is lower, be suitable for carrying out industrialization, but the shortcoming of this raw material is the Antibody Preparation technology difference of each producer, raw materials quality is difference slightly, need do certain pre-treatment before the preparation, to guarantee the stability of subsequent technique; Raw material B is new fresh hen egg, moderate cost, and also the oligosaccharides resource do not have any loss, the product yield height, if can fully utilize these raw material resources, the potentiality of its industrialization development are very big.
Innovative point of the present invention is to utilize the F at the PNGase of yeast saccharomyces cerevisiae surface expression, and the enzyme hydrolyzing prepares the acid N-sugar chain in the yolk glycoprotein, and result of study shows, these goods can suppress rotavirus the infant infected control infant's symptom of diarrhea; Simultaneously, the non-reducing end of this oligosaccharides also can be used as the adsorption site of influenza virus, slows down virus infecting human body; In addition, present sugared synthetic technology also can't obtain complete N-sugar chain, and the complete N-sugar chain product that utilizes the present invention to prepare also has broad application prospects in the research of glycoscience.
Reference:
[1]Yamamoto，T.，et?al，Oyotosbitsu?Kagaku?9，15-18。
[2]Koketsu，M.，et?al，Journal?of?Food?Science?58,743-747
[3]Koketsu，M.，et?al，1995c，Journal?of?Agricultural?and?Food?Chemistry?43，858-861
[4]European?Patent，Publication?number：0474410A2
[5] Chou Haiying etc., food science and technology, 2003, No2:28-33
Claims (6)
1, a kind of N-of utilization Glycosylase F prepares the method for yolk oligose, it is characterized in that: its technical process is to get supernatant liquor → ultrafiltration (molecular weight is held back and is 10kD) → desalination → ion exchange column → drying under reduced pressure → yolk oligose product after getting supernatant liquor → N-Glycosylase F enzymolysis → centrifugal after yolk raw material → degreasing → protease hydrolysis → denaturing treatment → centrifugal.
2, the N-of utilization Glycosylase F according to claim 1 prepares the method for yolk oligose, it is characterized in that: described N-Glycosylase F hydrolysis is to utilize directly to add hydrolyzed solution at the N-of brewing yeast cell surface expression Glycosylase F and carry out enzymolysis.
3, the N-of utilization Glycosylase F according to claim 1 prepares the method for yolk oligose, it is characterized in that: described N-Glycosylase F hydrolysis is a method of utilizing immobilized cell, and hydrolyzed solution is crossed post and carried out enzymolysis.
4, the N-of utilization Glycosylase F according to claim 1 prepares the method for yolk oligose, it is characterized in that: described yolk raw material is the yolk in the new fresh hen egg.
5, the N-of utilization Glycosylase F according to claim 1 prepares the method for yolk oligose, it is characterized in that: described yolk raw material is commercially available yolk powder.
6, the N-of utilization Glycosylase F according to claim 1 prepares the method for yolk oligose, it is characterized in that: described yolk raw material is the waste liquid in the yolk antibody preparation process preferably.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103906840A (en) * | 2011-06-07 | 2014-07-02 | 艾比欧公司 | In vivo de-glycosylation of recombinant proteins by co-expression with PNGase F |
| CN104974994A (en) * | 2014-04-02 | 2015-10-14 | 复旦大学 | Glycosidase for deglycosylation of N-glycoproteins and application thereof |
| WO2022255289A1 (en) * | 2021-05-31 | 2022-12-08 | Ｋｈネオケム株式会社 | Method for producing glycopeptide |
-
2004
- 2004-10-13 CN CN 200410035917 patent/CN1641037A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103906840A (en) * | 2011-06-07 | 2014-07-02 | 艾比欧公司 | In vivo de-glycosylation of recombinant proteins by co-expression with PNGase F |
| CN103906840B (en) * | 2011-06-07 | 2018-01-30 | 艾比欧公司 | Recombinant protein with PNGase F by co-expressing deglycosylation in vivo |
| US11673926B2 (en) | 2011-06-07 | 2023-06-13 | Ibio, Inc. | In vivo de-glycosylation of recombinant proteins by co-expression with PNGase F |
| CN104974994A (en) * | 2014-04-02 | 2015-10-14 | 复旦大学 | Glycosidase for deglycosylation of N-glycoproteins and application thereof |
| CN104974994B (en) * | 2014-04-02 | 2019-02-26 | 复旦大学 | One kind being used for the deglycosylated glycosidase of N- glycoprotein and its application |
| WO2022255289A1 (en) * | 2021-05-31 | 2022-12-08 | Ｋｈネオケム株式会社 | Method for producing glycopeptide |
| JP7261366B1 (en) * | 2021-05-31 | 2023-04-19 | Ｋｈネオケム株式会社 | Method for producing glycopeptide |
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