CN101906213B - Novel protein glycosylation grafting method - Google Patents

Novel protein glycosylation grafting method Download PDF

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CN101906213B
CN101906213B CN2010102374561A CN201010237456A CN101906213B CN 101906213 B CN101906213 B CN 101906213B CN 2010102374561 A CN2010102374561 A CN 2010102374561A CN 201010237456 A CN201010237456 A CN 201010237456A CN 101906213 B CN101906213 B CN 101906213B
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protein
albumen
crowded
polysaccharide
stir
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CN101906213A (en
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齐军茹
杨晓泉
卓秀英
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South China University of Technology SCUT
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South China University of Technology SCUT
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Abstract

The invention discloses a novel protein glycosylation grafting method. The method comprises the following steps of: (1) adding buffer solution into a crowding reagent and stirring the mixture to obtain solution; and (2) adding protein and glucan into the solution obtained by the step (1), stirring the mixture for 2 hours, adding NaN3 into the mixture, placing the mixture for 24 hours at the temperature of 5 DEG C, stirring the mixture for 12 to 48 hours at the temperature of between 50 and 70 DEG C again and quickly cooling the mixture to the temperature of less than 25 DEG C to obtain a glycosylation protein product. The method has the advantages of greatly improving functional characters such as water solubility, emulsibility, antioxidation and the like of a graft, along with convenient operation and high reaction efficiency; and the product has industrial and large-scale application prospect.

Description

A kind of method of novel protein glycosylation grafting
Technical field
The invention belongs to field of food, relate to protein modified method, particularly a kind of method of novel protein glycosylation grafting.
Background technology
Protein and polysaccharide are to coexist as most important two types of biomacromolecules in the food emulsification system, are the principal elements that influences food configuration and matter structure.Protein reduces IT and how in colloidal dispersion, to serve as emulsifying agent because of on liquid liquid or liquid-gas interface, forming adsorption layer; Polysaccharide is then used always and is made stablizer owing to its good thickening and water retention characteristic, has given the performance of the function when system is different from both exist singlys thus.The protein of covalent bonds and polysaccharide form grafts; Not only keep proteinic surfactivity but also had the hydrophilicity of polysaccharide; Mixture contrast with protein polysaccharide weak interaction forms has higher adaptability to envrionment conditions (temperature, pH value, ionic strength etc.).
Through spontaneous Maillard reaction proteinic epsilon-amino group is combined with the reductibility carbonyl of polysaccharide is terminal, can obtain having the protein glycosylation grafting thing of superior functionality character.Up to the present, effectively the preparation method still is a dry heating method, and its principle is to be in non-accumulative response behaviour through the amino in the protein under lower water-activity, so so that reactive group and polysaccharide generation covalent attachment to be provided.But dry heating method has long reaction time, and the Maillard level of response can not be controlled the defective that waits self.In foodstuffs industry, Food science man often hope that reaction occurs in aqueous phase freely, and take place in traditional aqueous solution also can make the protein molecular sex change and produce protein aggregation when the Maillard reaction generates glycosylated protein.Therefore, for extremely, polysaccharide and proteic Maillard are reflected in the aqueous solution effectively carry out up till now.
" macromole crowded " (macromolecular crowding) is completely new concept in the life science, and scientist advised strongly that factor was the same studies biomacromolecule as conventional factor " macromole is crowded " environment and pH, ionic strength and solution composition etc. in recent years.When having the biomacromolecule of high density in the system, molecular repulsion volume theoretical (excluded volume effect) is followed in the reaction between the macromole, promotes reaction to the direction that the solute TV reduces, and promptly moves towards bonding position; In addition, the albumen molecular configuration tends towards stability under the crowded environment of macromole, and protein denaturation degree and aggregation extent will alleviate.Thus; This project proposes polysaccharide and protein are in the crowded environment of macromole as biomacromolecule; Albumen can be in non-sex change or few accumulative response behaviour, also will in the aqueous solution, carry out towards forming covalently bound reaction path with the Maillard reaction of polysaccharide.The present invention with " macromole crowded " environmental applications in the life science in generating the protein-polysaccharide functional macromolecule; Molecular repulsion volume according under the crowded environment of macromole is theoretical; Albumen and polysaccharide in the aqueous solution will preferentially carry out towards the bonded direction, make in this environment albumen take place to assemble simultaneously or the probability of sex change reduces greatly, and as the macromole of " crowded reagent "; Can be the inertia superpolymer, also can be to participate in the polysaccharide that reacts simultaneously.
Summary of the invention
The purpose of this invention is to provide a kind of novel protein glycosylation grafting method.The present invention simulates the crowded system of macromole of life science, and the crowded reagent in the system can be inertia superpolymer or VISOSE.That this method has is easy to operate, reaction times weak point, stable performance, products therefrom do not produce advantages such as brown stain; Product has the application prospect of favorable industrialization, mass-producing, and this method has overcome existing protein and sugared grafting method is difficult to realize industrialized defective.
The object of the invention is realized through following technical proposals:
A kind of method of novel protein glycosylation grafting may further comprise the steps:
(1) in crowded reagent, adds buffered soln, stir, obtain solution;
(2) albumen and VISOSE are added in the solution that step (1) obtains, stir 2h after, add NaN 3, place 24h at 5 ℃, again 50~70 ℃ stir 12~48h after, be cooled to rapidly and be lower than 25 ℃, obtain the glycosylated protein product;
The mass fraction of said crowded reagent is 10~40 parts,
Proteic mass fraction is 1~10 part,
The mass ratio of albumen and polysaccharide is 1: 3~1: 6.
Said albumen is the heavy albumen of soy acid, soybean protein isolate, whey-protein, beta-lact oglobulin or wheat-gluten.
Said crowded reagent is polymer inert polymer or polysaccharide.
Said crowded reagent is the polymer inert polymer, for example; Ficol1 70 or PEG2000.
Said crowded reagent is polysaccharide, for example: Dextran 70, Dextran 10 or Dextran 40.
Said buffered soln is the phosphate buffered saline buffer of pH 5.0~8.0
A kind of method of novel protein glycosylation grafting may further comprise the steps:
(1) with albumen and crowded agent dissolves in buffered soln, stir 2h after, add NaN 3,
(2) place 24h at 5 ℃, again 50~70 ℃ stir 12~48h after, be cooled to rapidly and be lower than 25 ℃, obtain the glycosylated protein product;
The mass ratio of albumen and crowded reagent is 1: 3~1: 6.
Said albumen is the heavy albumen of soy acid, soybean protein isolate, whey-protein, beta-lact oglobulin or wheat-gluten.
Said crowded reagent is polysaccharide.For example: Dextran 70, Dextran 10 or Dextran 40.
Said buffered soln is the phosphate buffered saline buffer of pH 5.0~8.0.
The glycosylated protein product of method preparation of the present invention.
The application of described glycosylated protein product in preparation modified protein or foodstuff additive.
Described glycosylated protein product not purifying perhaps utilizes the gel filtration chromatography purifying to remove unreacted albumen and VISOSE and inertia superpolymer.
The principle of the invention is following:
Through spontaneous Maillard reaction proteinic epsilon-amino group is combined with the reductibility carbonyl of polysaccharide is terminal, can obtain having the protein glycosylation covalence graft thing of superior functionality character.Therefore in reaction process, select for use the reactive grafting degree as detecting index.It is the content of free amino group in the OPA method assaying reaction liquid that graft(ing)degree adopts the OPA method; The degree that reflects graft modification; Its principle be according to the free amino group in OPA and the protein in the presence of beta-mercaptoethanol; The compound that generates has absorption at the 340nm place, utilizes spectrophotometry can calculate the free amino group content in the reaction solution in view of the above, with the degree of reactive grafting modification.
The present invention adopts " macromole crowd environment " as the reaction system that generates glycosylated protein, through crowded theory in the life science further is applied to the preparation of protein glycosylation grafting thing to the stable scientific phenomena of protein structure.The crowded environment of macromole can make protein molecular in the aqueous solution, be in non-gathering or less accumulative response behaviour equally, simultaneously, promotes the reaction of albumen and polysaccharide to carry out towards covalently bound direction.
The relative prior art of the present invention has following advantage and beneficial effect:
(1) in the present invention; The covalence graft of Sunlover 10 and polysaccharide;, only in aqueous phase solution, under the temperature of gentleness, carry out as catalyzer without any need for chemical reagent, " macromole crowd environment " of simulation in the life science carries out proteinic glycosylation; Speed of response is very fast, and the product color is creamy white;
(2) can free stirring reaction in the water react, overcome the uneven shortcoming of reaction in the xeothermic reaction, farthest polysaccharide fully contacts with protein;
(3) compare with other graft-modification methods, the present invention has the application prospect of mass-producing, and industry is workable.
Embodiment:
Embodiment 1:
Accurately take by weighing 0.5g Sunlover 10,1.5g VISOSE, be dissolved in the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 6.5, behind the stirring at room 2h, add 3 NaN 3(0.02%w/w) anticorrosion, place 24h at 5 ℃, 60 ℃ of constant temperature, stirring velocity is to stir 30h under the 500r/min condition.Reaction is cooled to the room temperature termination reaction rapidly after finishing, and lyophilize obtains the glycosylated protein product.
Comparative example 1-1:
Accurately take by weighing the 0.5g Sunlover 10, be dissolved in the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 6.5, behind the stirring at room 2h, add 3 NaN 3(0.02%w/w) anticorrosion, place 24h at 5 ℃, 60 ℃ of constant temperature, stirring velocity is to stir 30h under the 500r/min condition.Reaction is cooled to the room temperature termination reaction rapidly, lyophilize after finishing.
Comparative example 1-2:
Accurately take by weighing 0.01g Sunlover 10,0.03g VISOSE, be dissolved in the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 6.5, behind the stirring at room 2h, add 3 NaN 3(0.02%w/w) anticorrosion, place 24h at 5 ℃, 60 ℃ of constant temperature, stirring velocity is to stir 30h under the 500r/min condition.Reaction is cooled to the room temperature termination reaction rapidly, lyophilize after finishing.
Adopt the OPA method to measure the graft(ing)degree of gained glycosylation Sunlover 10.The performance comparison result of embodiment 1 and comparative example's 1 binding substances is as shown in table 1:
Table 1
Can find out from table 1; In the embodiment of the invention 1 with VISOSE not only as crowded reagent but also high as reaction polysaccharide gained glycosylated protein graft(ing)degree; Colours white because system reactant concentration is too low, does not make polysaccharide effectively combine with albumen among the comparative example 1-2; Graft(ing)degree is lower, and reaction effect is not obvious; Association reaction can't take place in thermal treatment (comparative example 1-1) the back explanation protein itself that Sunlover 10 carries out under the similarity condition.
Embodiment 2:
1gPEG2000 is placed reaction unit as crowded reagent, add the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 7.0, stir 2h; Make it abundant aquation dissolving; 0.2g Sunlover 10 and 0.6g VISOSE add in this solution that contains crowded reagent, fully stir 2h after, add NaN 3(0.02%w/w) anticorrosion, behind 5 ℃ of placement 24h, 50 ℃ are stirred 48h, and reaction is cooled to 24 ℃ rapidly after finishing, and obtains the glycosylated protein product, removes unreacted albumen and VISOSE and inertia superpolymer PEG2000 through the gel filtration chromatography purifying.
Comparative example 2-1:
1g PEG2000 is placed reaction unit as crowded reagent, add the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 7.0, stir 2h; Make it abundant aquation dissolving; 0.2g Sunlover 10 adds in this solution that contains crowded reagent, fully stir 2h after, add NaN 3(0.02%w/w) anticorrosion, behind 5 ℃ of placement 24h, 50 ℃ are stirred 48h, and reaction is cooled to 24 ℃ rapidly after finishing, and obtains the glycosylated protein product, removes unreacted albumen and VISOSE and inertia superpolymer PEG2000 through the gel filtration chromatography purifying.
Comparative example 2-2:
Reaction unit adds the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 7.0, stirs 2h, and 0.2g Sunlover 10 and 0.6g VISOSE join in this solution, fully stir 2h after, adding NaN 3(0.02%w/w) anticorrosion, behind 5 ℃ of placement 24h, 50 ℃ are stirred 48h, and reaction is cooled to 24 ℃ rapidly after finishing, and obtains the glycosylated protein product, removes unreacted albumen and VISOSE through the gel filtration chromatography purifying.
Table 2
Figure BSA00000205751300051
Can find out from the performance comparison of table 2 embodiment 2 and comparative example's binding substances; The embodiment of the invention 2 is higher for crowded reagent place gets the glycosylated protein graft(ing)degree with PEG2000, colours white, among the comparative example 2-2 owing to do not add the crowded reagent of inertia in the system; Do not form the crowded environment of macromole; Polysaccharide is effectively combined with albumen, and graft(ing)degree is lower, and reaction effect is not obvious; Association reaction can't take place in thermal treatment (comparative example 2-1) the back explanation protein itself that Sunlover 10 carries out under the similarity condition.
Embodiment 3:
Accurately take by weighing 0.6g Sunlover 10,3.6g VISOSE, be dissolved in the buffer solution of sodium phosphate of 20mL 0.01mol/L pH 7.5, behind the stirring at room 2h, add 3 NaN 3(0.02%w/w) anticorrosion, place 24h at 5 ℃, 70 ℃ of constant temperature, stirring velocity is to stir 12h under the 500r/min condition.Reaction is cooled to the room temperature termination reaction rapidly, lyophilize after finishing.
Embodiment 4:
1.5gPEG2000 is placed reaction unit as crowded reagent, add the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 6.0, stir 2h; Make it abundant aquation dissolving; 0.5g Sunlover 10 and 2.5g VISOSE add in this solution that contains crowded reagent, fully stir 2h after, add NaN 3(0.02%w/w) anticorrosion, behind 5 ℃ of placement 24h, 60 ℃ are stirred 36h, and reaction is cooled to 20 ℃ rapidly after finishing, and obtains the glycosylated protein product, removes unreacted albumen and VISOSE and inertia superpolymer PEG2000 through the gel filtration chromatography purifying.
Embodiment 5:
1.5g Ficol170 is placed reaction unit as crowded reagent, add the buffer solution of sodium phosphate of 10mL 0.01mol/L pH 5.5, stir 2h; Make it abundant aquation dissolving; 0.5g Sunlover 10 and 3.0g VISOSE add in this solution that contains crowded reagent, fully stir 2h after, add NaN 3(0.02%w/w) anticorrosion, behind 5 ℃ of placement 24h, 70 ℃ are stirred 12h, and reaction is cooled to 19 ℃ rapidly after finishing, and obtains the glycosylated protein product, removes unreacted albumen and VISOSE and Ficol170 through the gel filtration chromatography purifying.

Claims (7)

1. the method for a novel protein glycosylation grafting is characterized in that, may further comprise the steps:
(1) in crowded reagent, adds buffered soln, stir, obtain solution;
(2) albumen and polysaccharide are added in the solution that step (1) obtains, stir 2h after, add NaN 3, place 24h at 5 ℃, again 50~70 ℃ stir 12~48h after, be cooled to rapidly and be lower than 25 ℃, obtain the glycosylated protein product;
The mass fraction of said crowded reagent is 10~40 parts,
Proteic mass fraction is 1~10 part,
The mass ratio of albumen and polysaccharide is 1: 3~1: 6;
Said buffered soln is the phosphate buffered saline buffer of pH 5.0~8.0.
2. method according to claim 1 is characterized in that, said albumen is the heavy albumen of soy acid, soybean protein isolate, whey-protein, beta-lact oglobulin or wheat-gluten.
3. method according to claim 1 is characterized in that, said crowded reagent is Ficol1 70, PEG2000 or polysaccharide.
4. the method for a novel protein glycosylation grafting is characterized in that, may further comprise the steps:
(1) with albumen and crowded agent dissolves in buffered soln, stir 2h after, add NaN 3,
(2) place 24h at 5 ℃, again 50~70 ℃ stir 12~48h after, be cooled to rapidly and be lower than 25 ℃, obtain the glycosylated protein product;
The mass ratio of albumen and crowded reagent is 1: 3~1: 6;
Said buffered soln is the phosphate buffered saline buffer of pH 5.0~8.0;
Said crowded reagent is polysaccharide.
5. method according to claim 4 is characterized in that, said albumen is the heavy albumen of soy acid, soybean protein isolate, whey-protein, beta-lact oglobulin or wheat-gluten.
6. glycosylated protein product according to each described method preparation of claim 1~5.
7. the application of the described glycosylated protein product of claim 6 in preparation modified protein or foodstuff additive.
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CN102308904A (en) * 2011-07-20 2012-01-11 华南理工大学 Method for rapidly preparing glycopeptides
CN103445282B (en) * 2013-07-19 2014-12-31 华南理工大学 Preparation method of corn peptide glycosylation product nano particles embedding lipid-soluble vitamins
CN106916319A (en) * 2017-04-01 2017-07-04 南京中医药大学 The ginkgo protein preparation method and applications of glucan graft modification
CN107151333B (en) * 2017-05-17 2020-03-10 朱吴喆 Glucan grafting modification process of wheat gluten protein
CN109105618A (en) * 2018-06-08 2019-01-01 河南蜀正园食品有限公司 Rich in flavones albumen and its preparing the application in health food
CN110591102B (en) * 2019-09-25 2021-08-27 临沂大学 Preparation method of peanut protein isolate-glucan graft polymer
CN113637063B (en) * 2021-08-17 2024-07-02 华南理工大学 Preparation process of dextran aldolization sodium caseinate
CN113651972A (en) * 2021-09-02 2021-11-16 浙江工商大学 Preparation method of graft modified fish protein-sugar coupling compound

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