CN103467572B - Antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins - Google Patents
Antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins Download PDFInfo
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Abstract
The invention provides an antifreeze polypeptide prepared by utilizing alkaline protease to carry out enzymolysis on collagens from fish skins. A preparation method is characterized by using the collagens from fish skins as raw materials, adopting alkaline protease to carry out enzymolysis on the collagens from fish skins and then carrying out separation and purification, thus obtaining a purified specific antifreeze polypeptide. The amino acid complete sequence of the antifreeze polypeptide is la-Asp-Gly-Gln-Thr-Gly-Gln-Arg-Gly-Glu-Lys-Gly-Pro-Ala-Gly-Val-Lys-Gly-Asp-Ala. By adopting the antifreeze polypeptide, the research ideas and methods of antifreeze proteins (polypeptides) existing at home and abroad are broken through, limitation of the quantity of the antifreeze proteins purified form natural organisms and considerations of international FDA (food and drug administration) for security of transgenic antifreeze proteins in food application are overcome, an efficient antifreeze polypeptide based on food sources is obtained, and a theoretical foundation is laid for developing the antifreeze polypeptide based on food sources and exploring extensive application of the antifreeze polypeptide to food and medicines.
Description
Technical field
The present invention relates to a kind of antifreeze peptide, related more specifically to a kind of antifreeze peptide utilizing Sumizyme MP enzymolysis collagen of fish skin to prepare, belonged to biological technical field.
Background technology
Food and medicine product suffers the problem of ice-crystal growth and recrystallization more and more to receive the concern of people repeatedly due to the fluctuation change of envrionment temperature in low-temperature storage and transportation.The lifting repeatedly of temperature makes ice crystal constantly grow, freeze thawing and recrystallization, havoc biological cells and tissues structure and lose the due quality of product.Worldwide scientist is faced with serious challenge: how to control ice-crystal growth and recrystallization, and the ice-crystal growth realized on low temperature cold chain controls, and is the key point restricting numerous food and medicine product quality.
Antifreeze protein, also claims " ice structural protein ", is that a class is attached to ice crystal surface and suppresses the growth of ice crystal and the active protein of recrystallization.Antifreeze protein controls ice-crystal growth because it has, reduce cell injury and keep the feature of product original weave construction, quality and quality and outstanding meaning and become hot research theme.Same research also shows, the Activity of Antifreeze segment of antifreeze protein be only present in local specific polypeptides hinge domain and be not overall protein in action, even sublimed antifreeze protein, Activity of Antifreeze is often not high, still needs to probe into its active territory and improves freeze proof efficiency further.So, how to obtain the high reactivity antifreeze peptide of the compact construction of food source, just become the research direction that antifreeze protein is urgent.
Summary of the invention
In order to solve the problem, the invention provides a kind of antifreeze peptide utilizing Sumizyme MP enzymolysis collagen of fish skin to prepare, Activity of Antifreeze is realized efficiently.
A kind of antifreeze peptide of the present invention is the polypeptide be made up of 20 amino acid.The aminoacid sequence of described polypeptide is: Ala-Asp-Gly-Gln-Thr-Gly-Gln-Arg-Gly-Glu-Lys-Gly-Pro-Ala-Gly-Val-Lys-Gly-Asp-Ala.
The preparation method of described antifreeze peptide is as follows:
Take collagen of fish skin as raw material, adopt Sumizyme MP to carry out enzymolysis to it, separation and purification, lyophilize obtain antifreeze peptide; Enzymatic hydrolysis condition is: enzymolysis pH is 9.0, temperature 45 C, enzymolysis time are 30 minutes, enzyme-substrate proportioning is 1:20(w/w); Described enzyme is Sumizyme MP.
The concrete steps of described separation and purification are: first enzymolysis product utilizes Sephadex G-50 gel chromatography to be separated, and elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm; Collect and have the peak of best Activity of Antifreeze, then be separated with Sulfopropyl-Sepadex C-25 cation-exchange chromatography, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5 M, flow velocity is 0.5mL/min; Collect the peak with best Activity of Antifreeze, RP-HPLC-C18 RPLC is utilized further to be separated again, the separation condition of reversed-phase HPLC is as elutriant gradient elution with 10%-90% (v/v) acetonitrile solution, flow velocity is 1mL/min, collect the elution peak at 10% acetonitrile and 90% water (v/v) place, obtain described antifreeze peptide.
In order to realize such scheme, the concrete steps that the present invention takes are as follows:
(1) optimization of gelatin enzymatic hydrolysis condition
The enzyme that this technology adopts and collagen of fish skin are purchased from Sigma biological reagent company (Chinese Shanghai).
Adopt experiment of single factor, respectively three enzymolysis factors are investigated, be respectively hydrolysis temperature (37 DEG C, 45 DEG C and 50 DEG C), enzymolysis time (10,20,30,40 and 60 minutes) and enzyme-substrate proportioning (1:100,1:50,1:20,1:15 and 1:10 w/w).Take 1.65 grams of collagen of fish skin to be dissolved in 6ml Mili-Q water, then use 2mol/L NaOH by its pH regulator to 9.0.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 1000rpm again after cooling.After supernatant collection, respectively its Activity of Antifreeze is measured, to determine optimum enzymolysis condition.The enzymatic hydrolysis condition obtaining the enzymolysis solution with maximum Activity of Antifreeze is: enzymolysis pH is pH7-10, temperature 30-55 DEG C, enzymolysis time is 20-60 minute, enzyme-substrate proportioning is 1:15-1:20(w/w).Be preferably that pH is 9.0, temperature 45 C, enzymolysis time be 30 minutes, enzyme-substrate proportioning is 1:20(w/w);
(2) separation of enzymolysis product, purifying
First under optimum enzymolysis condition, carry out enzymolysis, the enzymolysis product obtained first is separated through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), and elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collect the peak with best Activity of Antifreeze, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) be separated, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5 M, flow velocity is 0.5mL/min.Collect the peak with best Activity of Antifreeze, RP-HPLC RPLC is utilized further to be separated again, the separation condition of reversed-phase HPLC is that flow velocity is 1mL/min, obtains highly purified specificity antifreeze peptide with 10-90% acetonitrile solution as elutriant.
(3) test of Activity of Antifreeze
The test of Activity of Antifreeze can be the provide protection of antifreeze peptide to low temperature freeze thawing such as series foods, bacterium, cell, live bodies.The bulgaricus ccm of activation to be inoculated in liquid nutrient medium 37 DEG C by the present invention, and seed liquor, as seed liquor, is inoculated in new liquid nutrient medium with the ratio of 1:100 by 130r/min overnight incubation, and 37 DEG C, 130r/min shaking table is cultured to OD
600about=1.0.Get the sterilized centrifuge tube of some 1.5mL, adding concentration degerming is after filtration 250 μ g/mL testing sample 900 μ L, by bacterium liquid dilution 10
4doubly, draw 100 μ L and be added in testing sample, mixing, coating, be inverted for 37 DEG C and cultivate 18h, meter colony number.Remaining sample-bacterium liquid mixed solution is placed in-20 DEG C of subzero treatment 24h, take out melt and be coated with, 37 DEG C be inverted cultivate 18h, count colony number, often group do two parallel.Then according to the survival rate of following formulae discovery bulgaricus ccm.
(4) determined amino acid sequence
The overall amino acid sequence utilizing protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) to measure specificity antifreeze peptide is: adgqtgqrge kgpagvkgda.
The present invention is only present in special peptide chain structure territory instead of overall protein theoretical basis in action based on the Activity of Antifreeze segment of antifreeze protein, to come from the collagen protein of fish-skin for starting material, be conceived to be controlled by the cutting condition of Sumizyme MP, be cut into the active polypeptide with specific peptide chain length and structural domain composition, and Activity of Antifreeze is realized efficiently.The present invention is that exploitation is based on the antifreeze peptide of food source and explore its widespread use based theoretical in food, medicine.
Accompanying drawing explanation
The CLC-HPLC-C18 color atlas of Fig. 1 purifying collagen of fish skin antifreeze peptide;
The specificity antifreeze peptide of Fig. 2 purifying is to the low-temperature protection effect of bulgaricus ccm; Wherein, A, B in figure represent blank bulgaricus ccm respectively, add 0.5%(w/w) the bulgaricus growth situation map of specificity antifreeze peptide sample.
Embodiment
Antifreeze peptide of the present invention is the polypeptide be made up of 20 amino acid.The overall amino acid sequence of described polypeptide is: adgqtgqrgekgpagvkgda.
Preparation method is as follows:
Take collagen of fish skin as raw material, Sumizyme MP is used to carry out enzymolysis to it, and according to the standard of international antifreeze protein Activity determination, set up Activity of Antifreeze detection system, optimize its enzymatic hydrolysis condition, the enzymatic hydrolysis condition obtaining the enzymolysis solution with maximum Activity of Antifreeze is: enzymolysis pH is 9.0, temperature is 45 DEG C, enzymolysis time is 30 minutes, enzyme-substrate proportioning is 1:20; Under optimum enzymolysis condition, carry out enzymolysis, the enzymolysis product obtained first is separated through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), and elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collect the peak with best Activity of Antifreeze, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) be separated, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5M, flow velocity is 0.5mL/min.Collect the peak with best Activity of Antifreeze, utilize RP-HPLC-C18 RPLC (long 15cm, diameter 0.8cm) be 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 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 10% acetonitrile and 90% water (v/v) place, obtain highly purified specificity antifreeze peptide.
Lyophilize obtains antifreeze peptide of the present invention, is the polypeptide be made up of 20 amino acid.The overall amino acid sequence of described polypeptide is: Ala-Asp-Gly-Gln-Thr-Gly-Gln-Arg-Gly-Glu-Lys-Gly-Pro-Ala-Gly-Val-Lys-Gly-Asp-Ala.
The instrument that the present invention adopts, detection means are as follows:
The Activity of Antifreeze detection system preparing antifreeze peptide of the present invention, adopts low temperature freeze thawing bacterium, detects and adds antifreeze peptide to the growth protecting effect after the freeze thawing of bacterium low temperature.The bulgaricus ccm of activation to be inoculated in liquid nutrient medium 37 DEG C, seed liquor, as seed liquor, is inoculated in new liquid nutrient medium with the ratio of 1:100 by 130r/min shaking table overnight incubation, and 37 DEG C, 130r/min shaking table is cultured to OD
600about=1.0.Get the sterilized centrifuge tube of some 1.5mL, adding degerming is after filtration 250 μ g/mL testing sample 900 μ L containing antifreeze peptide concentration, by bacterium liquid dilution 10
4doubly, draw 100 μ L and be added in testing sample, mixing, coating, be inverted in incubator for 37 DEG C and cultivate 18h, meter colony number.Remaining sample-bacterium liquid mixed solution is placed in-20 DEG C of subzero treatment 24h, take out melt and be coated with, in incubator 37 DEG C be inverted cultivate 18h, count colony number, often group do two parallel.Then the survival rate of bulgaricus ccm is calculated.Do blank test in contrast simultaneously.
The multiple separation and purification means such as application Sephadex G-50 molecular sieve, Sepharose SP C-25 ion-exchange chromatography, RP-HPLC RPLC, dialysis, realize the high efficiency separation purifying of the special antifreeze 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 specificity antifreeze peptide.
In order to understand content of the present invention, Characteristic further, hereby exemplify following examples:
embodiment 1
Take 1.65 grams of collagen of fish skin to be dissolved in 6ml Mili-Q water, then use 2mol/L NaOH by its pH regulator to 9.0.First this solution water-bath is heated to 45 DEG C, the ratio being then 1:20 according to enzyme-substrate proportioning again adds the Sumizyme MP of respective amount, and enzymolysis time is 30 minutes.Then go out enzyme 10 minutes in boiling water bath, and centrifugal 10 minutes of 14000rpm again after cooling, collects supernatant liquor for subsequent use.
Be separated by supernatant liquor Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm, collects the elution peak with best Activity of Antifreeze.
The elution peak with best Activity of Antifreeze of Sephadex G-50 gel chromatography separation is carried out again to next step separation, with Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm, diameter 2.0cm) be separated, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5M, flow velocity is 0.5mL/min.Collect each peak sample and measure Activity of Antifreeze, the sample obtaining best Activity of Antifreeze utilizes RP-HPLC-C18 RPLC to carry out further separation and purification again.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, and carries out gradient elution, collects the elution peak at 10% acetonitrile and 90% water (v/v) place, obtain highly purified specificity antifreeze peptide of the present invention, as shown in Figure 1.YP1 peak is the chromatographic peak of this specificity antifreeze peptide.
The specificity antifreeze peptide of purifying has the ability of very strong bacterium low-temperature protection, as seen from Figure 2, compared with blank (Fig. 2 A), with the addition of the specificity antifreeze peptide of 0.5% (w/w), (Fig. 2 B).The survival rate calculating bulgaricus ccm is 91.2%.
Protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) is utilized to measure the aminoacid sequence of specificity antifreeze peptide to the specificity antifreeze peptide of purifying.The overall amino acid sequence of described polypeptide is: Ala-Asp-Gly-Gln-Thr-Gly-Gln-Arg-Gly-Glu-Lys-Gly-Pro-Ala-Gly-Val-Lys-Gly-Asp-Ala.
SEQUENCE LISTING
<110> University of Fuzhou
<120> mono-kind utilizes Sumizyme MP enzymolysis collagen of fish skin to prepare antifreeze peptide
<160> 1
<170> BiSSAP 1.2
<210> 1
<211> 20
<212> PRT
<213> University of Fuzhou
<400> 1
Ala Asp Gly Gln Thr Gly Gln Arg Gly Glu Lys Gly Pro Ala Gly Val
1 5 10 15
Lys Gly Asp Ala
20
Claims (1)
1. an antifreeze peptide, is characterized in that the aminoacid sequence of described polypeptide is: Ala-Asp-Gly-Gln-Thr-Gly-Gln-Arg-Gly-Glu-Lys-Gly-Pro-Ala-Gly-Val-Lys-Gly-Asp-Ala.
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| CN103965495B (en) * | 2014-05-09 | 2017-03-08 | 江南大学 | A kind of preparation method of freeze proof collagen protein of fish skin COL base tertiary blending emulsion film |
| CN104109705B (en) * | 2014-06-19 | 2017-10-31 | 广东省农业科学院蚕业与农产品加工研究所 | A kind of enzyme process prepares the method with the marine collagen protein peptides for promoting Wound healing activity |
| CN106399436B (en) * | 2016-09-08 | 2019-12-20 | 福建农林大学 | Process for preparing fish skin antifreeze peptide by one-step enzymolysis |
| CN108118077A (en) * | 2016-11-30 | 2018-06-05 | 中南大学 | The technique that a kind of enzymatic hydrolysis salmon collagen prepares anti-oxidation peptide and antifreeze peptide |
| CN107759663B (en) * | 2017-11-02 | 2023-06-30 | 中国海洋大学 | Method for identifying nile tilapia skin collagen |
| CN108812771A (en) * | 2018-07-30 | 2018-11-16 | 武汉轻工大学 | A kind of anti-aging prebake bread and preparation method thereof containing composite modifying agent |
| CN108967473A (en) * | 2018-07-30 | 2018-12-11 | 武汉轻工大学 | A kind of prebake bread of improvement and preparation method thereof |
| CN108967474A (en) * | 2018-07-30 | 2018-12-11 | 武汉轻工大学 | A kind of anti-aging prebake bread and preparation method thereof containing hydrophilic colloid and collagen antifreeze peptide |
| CN108812770A (en) * | 2018-07-30 | 2018-11-16 | 武汉轻工大学 | A kind of antifreeze peptide containing composite collagen and the anti-aging prebake bread of plant hydrophilic colloid and preparation method thereof |
| CN108684786A (en) * | 2018-07-30 | 2018-10-23 | 武汉轻工大学 | A kind of anti-aging prebake bread and preparation method thereof |
| CN112641084A (en) * | 2021-01-15 | 2021-04-13 | 平顶山天晶植物蛋白有限责任公司 | Preparation method of antifreeze soybean protein isolate |
| CN115043909B (en) * | 2022-06-20 | 2024-07-12 | 福州大学 | Globefish skin antifreeze peptide and preparation method and application thereof |
| CN114990095B (en) * | 2022-06-28 | 2024-05-24 | 天津天隆江大生物科技有限公司 | Complex enzyme preparation for producing antifreeze protein polypeptide and application thereof |
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