CN103788193B - A kind of method utilizing pair enzyme synergetic hydrolysis to prepare metal chelating peptide - Google Patents
A kind of method utilizing pair enzyme synergetic hydrolysis to prepare metal chelating peptide Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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Abstract
The invention provides a kind of metal (Ca, Fe, Zn) chelating peptide, be specifically related to a kind of method utilizing pair enzyme synergetic hydrolysis to prepare metal chelating peptide, with source, ocean fish-skin, 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 the aminoacid sequence obtaining peptide is: kngedg.Amino acid and little peptide have and promote the effect of 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.
Description
Technical field
The present invention relates to a kind of metal (Ca, Fe, Zn) chelating peptide, related more specifically to a kind of method utilizing pair enzyme synergetic hydrolysis to prepare metal chelating peptide, 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 a kind of method utilizing pair enzyme synergetic hydrolysis to prepare metal chelating peptide, metal (Ca, Fe, Zn) sequestering activity is 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 6 amino acid, the aminoacid sequence of described peptide is: kngedg.
Described metal is Ca, Fe or Zn.
A preparation method for metal chelating peptide, with source, ocean fish-skin, 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 TOYOPEARLDEAE-650M anion-exchange chromatography to be separated, the phosphoric acid buffer of elutriant to be concentration gradient be 0.02mol/L, pH9.0 containing 0-0.5mol/LNaCl, flow velocity is 0.5mL/min, and elution peak is measured under 214nm, collect the peak with the highest metal chelating activity, then be separated by SephadexG-25 gel filtration chromatography, elutriant is deionized water, and flow velocity is 0.3mL/min, and elution peak measures under 214nm, collect the peak with the highest metal chelating activity, half preparation RP-HPLC-C18 RPLC is utilized to be separated further again, separation condition is that 0-30% acetonitrile solution is as elutriant gradient elution by volume ratio, flow velocity is 4mL/min, collect the peak with the highest metal chelating activity, recycling RP-HPLC-C18 RPLC is further 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 is than the elution peak at 5% acetonitrile and 85% water place, obtain 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:
(1) optimization of ocean source protein enzymatic hydrolysis condition
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 2:1w/w), enzyme-substrate proportioning (1:100,1:50,1:25 and 1:20w/w) and enzymolysis time (1,3,5,7,9 hours).Take certain mass protein dissolution in distilled water, then with 2mol/LNaOH by its pH regulator to 7.0.First this solution water-bath is heated to and needs temperature, then add the enzyme of respective amount again by different enzyme-substrate proportionings, start reaction according to the predetermined reaction times.Then go out enzyme 10 minutes again in boiling water bath, centrifugal 10 minutes of 10000rpm again after cooling.After supernatant collection, respectively metal (Ca, Fe, Zn) sequestering activity is measured, to determine optimum enzymolysis condition.The enzymatic hydrolysis condition obtaining the enzymolysis solution with maximum metal sequestering activity is: concentration of substrate 5%, and enzymolysis pH is 7.0, temperature 49 DEG C, enzymolysis time are 7 hours, enzyme-substrate proportioning is 1:25(w/w); Described enzyme is compound protease and flavor protease, and the composite ratio of two kinds of enzymes is compound protease: flavor protease=2:1(w/w).
Taking 5.0 grams of ocean source proteins is dissolved in 100ml distilled water, then with 2mol/LNaOH 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.
(2) separation of enzymolysis product, purifying
By supernatant liquor TOYOPEARLDEAE-650M anion-exchange chromatography (long 50cm, external diameter 1.6cm) be separated, elutriant is the phosphoric acid buffer of the 0.02mol/LpH9.0 of the NaCl of concentration gradient 0-0.5M, flow velocity is 0.5mL/min, collects each peak sample and measures metal (Ca, Fe, Zn) sequestering activity.
What be separated TOYOPEARLDEAE-650M anion-exchange chromatography has the highest metal (Ca, Fe, Zn) elution peak of sequestering activity carries out next step separation again, collect by SepadexG-25 gel filtration chromatography and there is the highest metal (Ca, Fe, Zn) peak of sequestering activity, recycling analysis mode 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 1mL/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 5% acetonitrile and 85% water (v/v) place, namely elution time is the elution peak at 7.083 minutes places, obtain highly purified metal chelating peptide.
Lyophilize obtains metal of the present invention (Ca, Fe, Zn) chelating peptide.
(3) test of metal chelating activity
1) adopt o-cresolphthalein colorimetry, measure metal chelating peptide to the sequestering action of calcium ion.By the CaCl of 1mL5mmol/L
2add in tool plug test tube with the phosphate buffered saline buffer (pH8.0) of 2mL0.2mol/L, then add 1mL white protein peptide solution, be placed in thermostatically heating shaking bath 37 DEG C of incubation 2h, the centrifugal 10min of 10000r/min normal temperature after taking out.Get 1mL supernatant liquor, add o-cresolphthalein nitrite ion 5mL, shake up.Measure light absorption value in spectrophotometer 570nm place after placing 10min, numerical value is substituted in typical curve and calculate solubility calcium binding capacity.
The making of typical curve: the accurate Ca working fluid (10ug/mL) 0,0.2,0.4 of label taking respectively, 0.6,0.8,1.0mL is in 10mL test tube, add deionized water 1.0 respectively, 0.8,0.6,0.4,0.2,0mL, adds o-cresolphthalein nitrite ion 5mL, shake up, after placing 10min, measure light absorption value in spectrophotometer 570nm 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.
2) adopt phenanthroline colorimetry, measure metal chelating peptide to the sequestering action of iron ion.0.05g sample is placed in l00mL beaker, adds 2mL concentrated hydrochloric acid, after sample dissolves completely, be settled in l00mL volumetric flask with distilled water.Accurate absorption 5mL sample liquid, in 50mL volumetric flask, adds the HCl solution 1mL of lmol/L, the oxammonium hydrochloride lmL of 10%, 0.12% phenanthroline 1mL, then adds 10% sodium-acetate 5mL, be diluted with water to scale, shake up.Make reference liquid with the blank reagent solution not adding iron, measure absorbancy at 510nm wavelength place, numerical value is substituted in typical curve and calculates iron binding capacity.
The making of typical curve: draw the standardized solution 0 of 10ug/mL iron, 2.0,4.0,6.0,8.0,10.0mL, be placed in 50mL volumetric flask respectively, add the HCl solution 1mL of lmol/L, the oxammonium hydrochloride lmL of 10%, 0.12% phenanthroline 1mL, then add 10% sodium-acetate 5mL, be diluted with water to scale, shake up.Make reference liquid with the blank reagent solution not adding iron, measure absorbancy at 510nm wavelength place, 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 100mg in 100mL small beaker, add water 50mL, adds 6mol/L salt acid number and drip.After shaking up, in water-bath, heating makes it to dissolve completely, is settled to l00mL after cooling, therefrom draws l0mL in triangular flask, flat 3 parts, adds NH
3-NH
4cI damping fluid (pHl0) l0mL, chromium black T indicator is appropriate, then uses 0.0lmol/LNa
2eDTA drop is to blue; Record consumes the milliliter number of EDTA, calculates inner complex zinc content.
Application TOYOPEARLDEAE-650M anion-exchange chromatography, SephadexG-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 (AppliedBiosystemsModel476A, PerkinElmerCo.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 (AppliedBiosystemsModel476A, PerkinElmerCo.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: kngedg.
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.
SEQUENCELISTING
<110> University of Fuzhou
<120> mono-kind utilizes two enzyme synergetic hydrolysis to prepare the method for metal chelating peptide
<130>1
<160>1
<170>PatentInversion3.3
<210>1
<211>6
<212>PRT
<213> metal chelating peptide
<400>1
LysAsnGlyGluAspGly
15
Claims (2)
1. a metal chelating peptide, is characterized in that: the aminoacid sequence of described peptide is: kngedg.
2. a kind of metal chelating peptide according to claim 1, is characterized in that: described metal is Ca, Fe or Zn.
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| CN104928337B (en) * | 2015-04-15 | 2020-08-04 | 浙江海洋学院 | Navodon septentrionalis fish skin zinc chelating peptide |
| CN107759685B (en) * | 2017-10-26 | 2020-08-11 | 浙江海洋大学 | Sturgeon fishbone gelatin iron chelating peptide and preparation method thereof |
| CN107936113B (en) * | 2017-12-12 | 2020-11-10 | 浙江海洋大学 | Mullet scale iron chelating peptide and preparation method thereof |
| CN110684814A (en) * | 2019-08-13 | 2020-01-14 | 浙江海洋大学 | Preparation method of tuna skin collagen peptide iron chelate |
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Non-Patent Citations (3)
| Title |
|---|
| 带鱼下脚料蛋白水解螯合物制备及生特活性研究;钟明杰;《中国海洋大学论文集》;20090603;第38页倒数第2行至第39页第2行，12行 * |
| 带鱼下脚料酶解小肽亚铁螯合物结构鉴定及其生物活性研究;林慧敏;《中国博士学位论文全文数据库》;20121015;全文 * |
| 罗非鱼下脚料复合酶水解物锌螯合盐的制备;李同刚等;《包装与食品机械》;20130601;第31卷(第3期);全文 * |
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