CN103880938B - The preparation method of source, a kind of ocean metal chelating peptide - Google Patents
The preparation method of source, a kind of ocean metal chelating peptide Download PDFInfo
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- CN103880938B CN103880938B CN201410078706.XA CN201410078706A CN103880938B CN 103880938 B CN103880938 B CN 103880938B CN 201410078706 A CN201410078706 A CN 201410078706A CN 103880938 B CN103880938 B CN 103880938B
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- metal chelating
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
The present invention relates to the preparation method of source, a kind of ocean metal-chelating protein peptide, relate more specifically to a kind of metal chelating peptide utilizing compound protease and flavor protease enzymolysis fish-skin, fish scale albumen to prepare, with source, ocean fish-skin or fish scale albumen for raw material, adopt compound protease and flavor protease is composite carries out enzymolysis to it, separation and purification, lyophilize obtain metal chelating peptide, and aminoacid sequence is: gpagvkg.Research shows, polypeptide chelate calcium, due to the chelating system of its uniqueness and transporting mechanism, is easily absorbed, safety non-toxic, price are low, can supplement amino acid and calcium simultaneously, and become first-selection of replenishing the calcium.
Description
Technical field
The present invention relates to a kind of metal (Ca, Fe, Zn) chelating peptide, related more specifically to a kind of metal chelating peptide utilizing compound protease and flavor protease enzymolysis fish-skin, fish scale albumen to prepare, belonged to biological technical field.
Background technology
Marine protein source biologically active peptides has high nutritive value, edible safety and physiological hygiene function, promoting all to play an important role in the growth of body, growth and diseases prevention and treatment etc., can be used as functional foodstuff or foodstuff additive and even pharmaceutical applications in people's daily life.
Calcium deficiency is global nutrition problem, and our people is due to based on vegetable diet, and the phenomenon of calcium deficiency is more serious, the important topic become in China's dietary nutrition research of therefore replenishing the calcium.Traditional inorganic calcium salt, as calcium carbonate, calcium phosphate, calcium chloride etc., have certain destruction to supporting one's family, biological value is lower; Organic calcium salt, as citrate of lime, calcium lactate, calisanin etc., although calcium absorptivity increases, the molten mistake of its solubleness great Yi, and price is high.Research shows, polypeptide chelate calcium, due to the chelating system of its uniqueness and transporting mechanism, is easily absorbed, safety non-toxic, price are low, can supplement amino acid and calcium simultaneously, and become first-selection of replenishing the calcium.
Iron particularly plays an important role to infant and growing of children to human health.Although macro-molecular protein also can be combined with iron ion, also there is the relative molecular mass problem be difficult to by intestinal mucosa comparatively greatly in these macro-molecular proteins itself.And research finds, amino acid, polypeptide chelate iron can improve the specific absorption of iron ion greatly.
Although occurring in nature zinc source is very abundant, the Absorption And Metabolism of zinc in human body is by the restriction of all factors, and zn deficiencie has become the public health problem of China and many developing countries.Research finds that amino acid and little peptide have the effect promoting zinc-iron alloy solution, and the composite metal of amino acid or peptide is as higher and have no side effect than inorganic salt in the biology utilization ratio of Fe, Zn.Therefore, research and develop this biological state zinc-protein zymolyte chelated zinc, tool is of great significance.
Therefore, how to obtain the peptide with metal chelating activity, just become and prepare the urgent research direction of novel metal component extender.
Summary of the invention
In order to solve the problem, the invention provides the preparation method of source, a kind of ocean metal-chelating protein peptide, metal (Ca, Fe, Zn) sequestering activity be realized efficiently.
For achieving the above object, the present invention adopts following technical scheme:
A kind of metal chelating peptide, be the peptide be made up of 7 amino acid, the aminoacid sequence of described peptide is: gpagvkg.
Described metal is Ca, Fe or Zn.
A preparation method for metal chelating peptide, with source, ocean fish-skin or fish scale albumen for raw material, adopt compound protease and flavor protease is composite carries out enzymolysis to it, separation and purification, lyophilize obtain metal chelating peptide.
Enzymatic hydrolysis condition is: concentration of substrate 5%, and enzymolysis pH is 7.0, temperature 49 DEG C, enzymolysis time are 7 hours, enzyme-substrate weight proportion is 1:25; Described enzyme is compound protease and flavor protease, and the composite ratio of weight of two kinds of enzymes is compound protease: flavor protease=2:1.
The concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARL DEAE-650M anion-exchange chromatography to be separated, the phosphoric acid buffer of elutriant 0.02 mol/L, pH 9.0 that to be concentration gradient be containing 0-0.5 M NaCl, flow velocity is 0.5 mL/min, and elution peak is measured under 214 nm; Collect the peak with the highest metal chelating activity, then be separated by Sephadex G-25 gel filtration chromatography, elutriant is deionized water, and flow velocity is 0.3 mL/min, and elution peak measures under 214 nm; Collect the peak with the highest metal chelating activity, recycling RP-HPLC-C18 RPLC is further separated, the separation condition of reversed-phase HPLC is as gradient eluent with 10-90% acetonitrile solution, flow velocity is 1mL/min, the self-contained volume ratio of elutriant is that the mixed solution of 10% acetonitrile and 90% water starts, and the mixed solution to volume ratio 90% acetonitrile and 10% water terminates, and carries out gradient elution, collected volume, than the elution peak being 30% acetonitrile and 60% water place, obtains highly purified metal chelating peptide.
The present invention possesses the action site with metal ion-chelant based on polypeptide, compound that can be stable with its formation, and polypeptide-metallo-chelate has unique chelating system and transporting mechanism, easily absorbed, can be supplemented simultaneously the theoretical basis of amino acid and metal, to come from fish-skin, fish scale albumen for starting material, controlled by the cutting condition of compound protease and flavor protease, cutting preparation has the peptide of high metal (Ca, Fe, Zn) sequestering activity, and metal chelating activity is realized efficiently.The present invention is that the application of ocean source protein provides new approaches.
Accompanying drawing explanation
The CLC-HPLC-C18 color atlas of Fig. 1 purifying ocean source protein metal chelating peptide.
Embodiment
embodiment 1
Preparation method is as follows:
The ocean source protein that this technology adopts comes from laboratory self-control, and enzyme believes Bioisystech Co., Ltd (Chinese Tianjin) purchased from Novi.Adopt experiment of single factor, respectively four enzymolysis factors are investigated, be respectively concentration of substrate (1%, 3%, 5% and 7%), hydrolysis temperature (40,50,55 and 60 DEG C), enzyme composite than (compound protease: flavor protease=1:1,1:2,1:3 and 2:1 w/w), enzyme-substrate proportioning (1:100,1:50,1:25 and 1:20 w/w) and enzymolysis time (1,3,5,7,9 hours).Taking 5.0 grams of ocean source proteins is dissolved in 100ml distilled water, then uses 2mol/L NaOH by its pH regulator to 7.0.First this solution water-bath is heated to 49 DEG C, the ratio being then 1:25 according to enzyme-substrate proportioning again adds the enzyme of respective amount, and the mass ratio of compound protease and flavor protease is 2:1, and enzymolysis time is 7 hours.Then go out enzyme 10 minutes in boiling water bath, and centrifugal 10 minutes of 10000rpm again after cooling, collects supernatant liquor for subsequent use.
By supernatant liquor TOYOPEARL DEAE-650M anion-exchange chromatography (long 50 cm, external diameter 1.6cm) be separated, elutriant is the phosphoric acid buffer of the 0.02 mol/L pH 9.0 of the NaCl of concentration gradient 0-0.5M, flow velocity is 0.5 mL/min, collects each peak sample and measures metal (Ca, Fe, Zn) sequestering activity.
What be separated TOYOPEARL DEAE-650M anion-exchange chromatography has the highest metal (Ca, Fe, Zn) elution peak of sequestering activity carries out next step separation again, with Sepadex G-25 gel filtration chromatography (long 100 cm, external diameter 2.0 cm) collect there is the highest metal (Ca, Fe, Zn) peak of sequestering activity, recycling preparative RP-HPLC-C18 RPLC is separated further again, the separation condition of reversed-phase HPLC is with 10-90%(v/v) acetonitrile solution is as gradient eluent, flow velocity is 1 mL/min, collect each peak and carry out metal (Ca, Fe, Zn) chelating vitality test, the mixed solution of self-contained 10% acetonitrile of elutriant and 90% water (v/v) starts, mixed solution to 90% acetonitrile and 10% water (v/v) terminates, carry out gradient elution, collect the elution peak at 30% acetonitrile and 60% water (v/v) place, namely elution time is the elution peak at 17.031 minutes places, obtain highly purified metal chelating peptide.
Lyophilize obtains metal of the present invention (Ca, Fe, Zn) chelating peptide.
Adopt o-cresolphthalein colorimetry, measure metal chelating peptide to the sequestering action of calcium ion.By the CaCl of 1 mL 5 mmol/L
2add in tool plug test tube with the phosphate buffered saline buffer (pH 8.0) of 2 mL 0.2 mol/L, then add 1 mL white protein peptide solution, be placed in thermostatically heating shaking bath 37 DEG C of incubation 2h, centrifugal 10 min of 10000 r/min normal temperature after taking out.Get 1 mL supernatant liquor, add o-cresolphthalein nitrite ion 5 mL, shake up.Measure light absorption value in spectrophotometer 570 nm place after placing 10 min, numerical value is substituted in typical curve and calculate solubility calcium binding capacity.
The making of typical curve: the accurate Ca working fluid of label taking (10 ug/ mL) 0,0.2,0.4 respectively, 0.6,0.8,1.0 mL in 10 mL test tubes, add deionized water 1.0 respectively, 0.8,0.6,0.4,0.2,0 mL, adds o-cresolphthalein nitrite ion 5 mL, shake up, after placing 10 min, measure light absorption value in spectrophotometer 570 nm place.With solubility calcium content (ug/ mL) for X-coordinate, light absorption value is that ordinate zou is figure, obtains typical curve formula and is: y=0.0974x-0.0402, R
2=0.9996.
Adopt phenanthroline colorimetry, measure metal chelating peptide to the sequestering action of iron ion.0.05 g sample is placed in l00 mL beaker, adds 2 mL concentrated hydrochloric acids, after sample dissolves completely, be settled in l00 mL volumetric flask with distilled water.Accurate absorption 5 mL sample liquid, in 50 mL volumetric flasks, adds HCl solution 1 mL of l mol/L, the oxammonium hydrochloride l mL of 10%, and then 0.12% phenanthroline 1 mL adds 10% sodium-acetate 5 mL, be diluted with water to scale, shake up.Make reference liquid with the blank reagent solution not adding iron, measure absorbancy at 510 nm wavelength places, numerical value is substituted in typical curve and calculates iron binding capacity.
The making of typical curve: standardized solution 0,2.0,4.0,6.0,8.0,10.0 mL drawing 10 ug/mL iron, be placed in 50 mL volumetric flasks respectively, add HCl solution 1 mL of l mol/L, the oxammonium hydrochloride l mL of 10%, 0.12% phenanthroline 1 mL, then add 10% sodium-acetate 5 mL, be diluted with water to scale, shake up.Make reference liquid with the blank reagent solution not adding iron, measure absorbancy at 510 nm wavelength places, drawing standard curve, obtaining typical curve formula is: y=0.1717x+0.003, R
2=0.9994.
Adopt EDTA volumetry, measure metal chelating peptide to the sequestering action of zine ion.Weigh inner complex 100 mg in 100 mL small beakers, add water 50 mL, adds 6 mol/L salt acid numbers and drip.After shaking up, in water-bath, heating makes it to dissolve completely, is settled to l00 mL after cooling, therefrom draws l0 mL in triangular flask, flat 3 parts, adds NH
3-NH
4cI damping fluid (pH l0) l0 mL, chromium black T indicator is appropriate, then uses 0.0l mol/L Na
2eDTA drop is to blue; Record consumes the milliliter number of EDTA, calculates inner complex zinc content.
Application TOYOPEARL DEAE-650M anion-exchange chromatography, Sephadex G-25 molecular sieve, RP-HPLC RPLC etc. are separated means of purification, realize the high efficiency separation purifying of the metal-chelating protein peptide of remarkable activity.
Protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) is utilized to measure the overall amino acid sequence of metal chelating peptide.
The Specific metal chelating peptide that purifying obtains has very high metal (Ca, Fe, Zn) sequestering activity, and as can be seen from Table 1, compared with enzymolysis solution, the metal chelating of chelating peptide makes a concerted effort there has been large increase.
The metal chelating of the Specific metal chelating peptide of table 1 purifying is made a concerted effort
Protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) is utilized to measure the aminoacid sequence of Specific metal chelating peptide to the Specific metal chelating peptide of purifying.The aminoacid sequence of described metal chelating peptide is: gpagvkg.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> University of Fuzhou
The preparation method of source, <120> ocean metal chelating peptide
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> metal chelating peptide
<400> 1
Gly Pro Ala Gly Val Lys Gly
1 5
Claims (3)
1. a metal chelating peptide, is characterized in that: the aminoacid sequence of described peptide is: gpagvkg.
2. a kind of metal chelating peptide according to claim 1, is characterized in that: described metal is Ca, Fe or Zn.
3. the preparation method of an a kind of metal chelating peptide as claimed in claim 1, it is characterized in that: with source, ocean fish-skin or fish scale albumen for raw material, adopt compound protease and flavor protease is composite carries out enzymolysis to it, separation and purification, lyophilize obtain metal chelating peptide; The condition of described enzymolysis is: concentration of substrate 5%, and enzymolysis pH is 7.0, temperature 49 DEG C, enzymolysis time are 7 hours, enzyme-substrate weight proportion is 1:25; Described enzyme is compound protease and flavor protease, and the composite ratio of weight of two kinds of enzymes is compound protease: flavor protease=2:1; The concrete steps of described separation and purification are: first enzymolysis product utilizes TOYOPEARL DEAE-650M anion-exchange chromatography to be separated, the phosphoric acid buffer of elutriant 0.02 M, pH 9.0 that to be concentration gradient be containing 0-0.5 M NaCl, flow velocity is 0.5 mL/min, and elution peak is measured under 214 nm; Collect the peak with the highest metal chelating activity, then be separated by Sephadex G-25 gel filtration chromatography, elutriant is deionized water, and flow velocity is 0.3 mL/min, and elution peak measures under 214 nm; Collect the peak with the highest metal chelating activity, RP-HPLC-C18 RPLC is utilized further to be separated again, the separation condition of reversed-phase HPLC is as gradient eluent with 10-90% acetonitrile solution, flow velocity is 1mL/min, the self-contained volume ratio of elutriant is that the mixed solution of 10% acetonitrile and 90% water starts, the mixed solution being 90% acetonitrile and 10% water to volume ratio terminates, carry out gradient elution, collected volume, than the elution peak being 30% acetonitrile and 60% water place, obtains highly purified metal chelating peptide.
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| CN104012652A (en) * | 2014-06-30 | 2014-09-03 | 广西南宁至简至凡科技咨询有限公司 | Cranberry collagen polypeptide Mg yoghurt |
| CN104430857A (en) * | 2014-08-06 | 2015-03-25 | 广西南宁至简至凡科技咨询有限公司 | Mulberry collagen polypeptide Mg yoghurt |
| CN104430873A (en) * | 2014-08-06 | 2015-03-25 | 广西南宁至简至凡科技咨询有限公司 | Blueberry collagen polypeptide Mg sour milk |
| CN104430858A (en) * | 2014-08-06 | 2015-03-25 | 广西南宁至简至凡科技咨询有限公司 | Mulberry collagen polypeptide Zn sour milk |
| CN104928337B (en) * | 2015-04-15 | 2020-08-04 | 浙江海洋学院 | Navodon septentrionalis fish skin zinc chelating peptide |
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| WO1996003651A1 (en) * | 1994-07-25 | 1996-02-08 | Boehringer Mannheim Gmbh | Peptides marked with metal chelates |
| US7429472B2 (en) * | 2003-01-31 | 2008-09-30 | Promega Corporation | Method of immobilizing a protein or molecule via a mutant dehalogenase that is bound to an immobilized dehalogenase substrate and linked directly or indirectly to the protein or molecule |
| CN1871233B (en) * | 2003-08-29 | 2011-07-13 | 沃拉克有限公司 | Novel chelating agents and chelates and their use |
| CN101139398A (en) * | 2007-09-27 | 2008-03-12 | 南京农业大学 | Preparation method of heavy metal mercury monoclonal antibody |
| CN102286105B (en) * | 2011-06-14 | 2013-12-25 | 中国农业大学 | Sesame protein source metal chelating peptide and peptide trace element chelate and application thereof |
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