CN101921328B - Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease - Google Patents
Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease Download PDFInfo
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- CN101921328B CN101921328B CN2010102775694A CN201010277569A CN101921328B CN 101921328 B CN101921328 B CN 101921328B CN 2010102775694 A CN2010102775694 A CN 2010102775694A CN 201010277569 A CN201010277569 A CN 201010277569A CN 101921328 B CN101921328 B CN 101921328B
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Abstract
The invention provides an antifreeze polypeptide and preparation method thereof. The method takes cow leather collagen as raw material and obtains the specific antifreeze polypeptide by screening and optimizing the enzymolysis condition of alkali protease, separating and purifying. The antifreeze polypeptide has the molecular weight of 2107Da and the amino acid full sequence of Gly-Glu-Arg-Gly-Phe-Pro-Gly-Glu-Arg-Gly-Ser-Pro-Gly-Ala-Gln-Gly-Leu-Gln-Gly-Pro-Arg. The invention breaks through the research thinking and method of antifreeze protein at home and abroad, overcomes the limitation of the number of purified antifreeze protein in natural organism and the safety worry of international FDA organization for transgene antifreeze protein in the food application, obtains the food-source high-efficiency antifreeze polypeptide, and lays the theoretical foundation for developing the antifreeze polypeptide based on the food source and exploring the wide application of the antifreeze polypeptide in the aspects of food and medical science.
Description
Technical field
The invention provides a kind of antifreeze peptide and preparation method thereof, related more specifically to a kind of antifreeze peptide that utilizes the preparation of Sumizyme MP enzymolysis of cow leather collagen, belong to biological technical field.
Background technology
The food and medicine product in low-temperature storage and transportation since the fluctuation of envrionment temperature change and suffer the problem of ice-crystal growth and recrystallization more and more to be subjected to people's attention repeatedly.The lifting repeatedly of temperature makes constantly growth of ice crystal, freeze thawing and recrystallization, havoc cell and weave construction and lose the due quality of product.Worldwide scientist is faced with serious challenge: how controlling ice-crystal growth and recrystallization, realize the ice-crystal growth control on the low temperature cold chain, is the key point of the numerous food and medicine product quality of restriction.
Antifreeze protein (antifreeze proteins, AFPs) or claim " ice structural protein " (ice structuring proteins), be that a class suppresses the growth of ice crystal and the active protein of recrystallization attached to ice crystal (ice crystals) surface.Antifreeze protein is owing to it has the control ice-crystal growth, reduces cell injury and keeps the original weave construction of product, quality and quality characteristics and outstanding meaning to become the hot research theme, very active to freeze proof proteic research both at home and abroad in recent years, " Nature " and " Science " follow up on closely latest Progress of antifreeze protein aspect.Present research mainly concentrates on from organisms such as polar region fish, land insect, plant and bacterium and is separated to multiple antifreeze protein, and seeks them in food, medical science and other industrial application.Be accompanied by the discovery of multiple antifreeze protein and going deep into of research, the research of its restriction organism antifreeze protein and two big key issues of application also show especially day by day: 1. the resulting quantity pettiness of natural separation and purification, very limited quantity limitation it in food and big rule application prospect medically; 2. be devoted to transgenic technology when enlarging the antifreeze protein output in organism source as scientist, the safety concerns of transgenosis antifreeze protein in food applications becomes the focal issue that consumers in general, European Union's tissue and international FDA tissue institute worry jointly again.
Same research also shows, the freeze proof active part of antifreeze protein only be present in partial specific polypeptides chain structure territory (domain) and be not whole protein in action, even sublimed antifreeze protein, freeze proof activity is often not high, still needs to probe into its active territory domain and further improves freeze proof efficient.So, how to obtain the high reactivity antifreeze peptide of the compact construction in food source, just become the urgent research direction of antifreeze protein.
Summary of the invention
In order to address the above problem, the invention provides a kind of antifreeze peptide and preparation method thereof, freeze proof activity is realized efficiently.
A kind of antifreeze peptide of the present invention, aminoacid sequence are Gly-Glu-Arg-Gly-Phe-Pro-Gly-Glu-Arg-Gly-Ser-Pro-Gly-Ala-Gln-Gly-Leu-Gln-Gly-Pro-Arg.The molecular weight of described antifreeze peptide is 2107 Da.
The preparation method of a kind of antifreeze peptide of the present invention is a raw material with the cow leather collagen, adopts Sumizyme MP that it is carried out enzymolysis, and separation and purification, lyophilize obtain antifreeze peptide; Described enzymatic hydrolysis condition is: enzymolysis pH is that pH7-10, temperature 30-55 ℃, enzymolysis time are that 20-60 minute, enzyme-substrate proportioning are 1:15-1:20(w/w); Described enzyme is a Sumizyme MP.Preferred enzymatic hydrolysis condition is: enzymolysis pH is 9.0,45 ℃ of temperature, enzymolysis time be that 30 minutes, enzyme-substrate proportioning are 1:15(w/w).The means of described separation and purification comprise Sephadex G-50 molecular sieve, Sepharose SP C-25 ion-exchange chromatography and RP-HPLC RPLC.
The concrete steps of described separation and purification are: enzymolysis product at first utilizes Sephadex G-50 gel chromatography to separate, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm; Collection has best freeze proof active peak, separates with Sulfopropyl-Sepadex C-25 cation-exchange chromatography again, and elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5M for the NaCl concentration gradient, and flow velocity is 0.5mL/min; Collection has best freeze proof active peak, utilize the RP-HPLC RPLC further to separate again, the separation condition of reversed-phase HPLC is to use the 10-90% acetonitrile solution as elutriant, and flow velocity is 1mL/min, obtains highly purified specificity antifreeze peptide; The molecular weight of described highly purified specificity antifreeze peptide is 2107 Da.
In order to realize such scheme, the concrete steps that the present invention takes are as follows:
(1) optimization of gelatin enzymatic hydrolysis condition
Enzyme that present technique adopted and cow leather collagen are available from Sigma biological reagent company (Chinese Shanghai).
Adopt experiment of single factor, respectively three enzymolysis factors are investigated, be respectively hydrolysis temperature (37 ℃, 45 ℃ and 50 ℃), 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 by weighing 1.65 gram cow leather collagens and be dissolved in the 6ml Mili-Q water, use 2mol/L NaOH then its pH regulator to 9.0.Earlier this solution water-bath being heated to needs temperature, then adds the enzyme of respective amount again by different enzyme-substrate proportionings, begins to react according to the predetermined reaction times.Then in boiling water bath, go out again enzyme 10 minutes, centrifugal 10 minutes of 1000rpm again after the cooling.Supernatant liquor is measured its freeze proof activity respectively after collecting, to determine optimum enzymolysis condition.The enzymatic hydrolysis condition that obtains having maximum freeze proof active enzymolysis solution is: enzymolysis pH is that pH7-10, temperature 30-55 ℃, enzymolysis time are that 20-60 minute, enzyme-substrate proportioning are 1:15-1:20(w/w).
(2) separation of enzymolysis enzymolysis product, purifying
Carry out earlier enzymolysis under optimum enzymolysis condition, the enzymolysis product that obtains separates through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm) earlier, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collection has best freeze proof active peak, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) separate, elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5M for the NaCl concentration gradient, and flow velocity is 0.5mL/min.Collection has best freeze proof active peak, utilize the RP-HPLC RPLC further to separate again, the separation condition of reversed-phase HPLC is to use the 10-90% acetonitrile solution as elutriant, and flow velocity is 1mL/min, obtains highly purified specificity antifreeze peptide.The molecular weight that utilizes substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) to measure the specificity antifreeze peptide of this purifying is 2107 Da.
(3) freeze proof active test
Utilize ice-creams matrix (Ice Cream Base Mix) to be antifreeze peptide research carrier.Get the ice-creams of 5uL, utilize the cold-heat-stage system, make its with the speed fast cooling of 40 ℃/min to-40 ℃, under this temperature, keep 5min and take pictures.Speed with 1 ℃/min is warming up to-14 ℃ more then, and then with every 3min frequency once reciprocation cycle 7 times between-14 ℃ and-12 ℃, with the variation of temperature of simulation ice-creams in two months storage transportations, and takes pictures and changes to observe ice crystal.
(4) mensuration of antifreeze peptide molecular weight
Utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) to measure the molecular weight of specificity antifreeze peptide.
(5) determined amino acid sequence
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) the amino acid complete sequence of mensuration specificity antifreeze peptide.
The present invention sees through the quantity limitation of present organism antifreeze protein purified research and the edible safety misgivings of transgenosis antifreeze protein, the freeze proof active part that is based on antifreeze protein only is present in special peptide chain structure territory (domain) rather than whole protein theoretical basis in action, with the collagen protein that comes from ox-hide is starting point, be conceived to cutting condition control by Sumizyme MP, be cut into active polypeptide, and freeze proof activity is realized efficiently with specific peptide chain length and structural domain composition.
The present invention has broken through the research ideas and methods of domestic and international existing antifreeze protein (polypeptide), overcome from the natural biological body quantity limitation of antifreeze protein purified and international FDA tissue to the safety concerns of transgenosis antifreeze protein in food applications, acquisition is based on the efficient antifreeze peptide in food source, for exploitation based on the antifreeze peptide in food source and explore its widespread use based theoretical in food, medicine.
Description of drawings
Fig. 1 is a collagen protein enzymolysis thing Sephadex G-50 elution profile;
Fig. 2 is that three parts of Sephadex G-50 elution peak suppress design sketch to Ice Crystal in Ice Cream growth and ice crystal thereof;
Fig. 3 is the Sulfopropyl-Sepadex C-25 elution profile of Fraction2;
Fig. 4 is for adding 1%(w/w) SP1, SP2 elution peak are to the Ice Crystal in Ice Cream growth and suppress the design sketch of ice-crystal growth;
Fig. 5 is the antifreeze peptide MALDI-TOF figure of purifying;
Fig. 6 is the effect of the specificity antifreeze peptide of purifying to Ice Crystal in Ice Cream growth and inhibition ice-crystal growth; Wherein, A, B represent blank ice-creams respectively, add 0.5%(w/w) the ice-crystal growth situation map of specificity antifreeze peptide sample.
Embodiment
The aminoacid sequence of antifreeze peptide of the present invention is Gly-Glu-Arg-Gly-Phe-Pro-Gly-Glu-Arg-Gly-Ser-Pro-Gly-Ala-Gln-Gly-Leu-Gln-Gly-Pro-Arg.
The preparation method is as follows:
With the cow leather collagen is raw material, use Sumizyme MP that it is carried out enzymolysis, and according to the active standard that detects of international antifreeze protein, set up freeze proof activity monitor system, optimize its enzymatic hydrolysis condition, the enzymatic hydrolysis condition that obtains having maximum freeze proof active enzymolysis solution is: enzymolysis pH is 9.0, temperature is that 45 ℃, enzymolysis time are that 30 minutes, enzyme-substrate proportioning are 1:15; Carry out enzymolysis under optimum enzymolysis condition, the enzymolysis product that obtains separates through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm) earlier, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collection has best freeze proof active peak, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) separate, elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5M for the NaCl concentration gradient, and flow velocity is 0.5mL/min.Collection has best freeze proof active peak, utilize the RP-HPLC RPLC further to separate again, the separation condition of reversed-phase HPLC is to use the 10-90% acetonitrile solution as elutriant, and flow velocity is 1mL/min, obtains highly purified specificity antifreeze peptide.Utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) to measure the molecular weight of the specificity antifreeze peptide of this purifying.
Lyophilize obtains antifreeze peptide of the present invention, carries out molecular weight determination and shows, the molecular weight of the specificity antifreeze peptide that obtains with the Sumizyme MP enzymolysis is 2107 Da.
Instrument, detection means that the present invention adopts are as follows:
The freeze proof activity monitor system of preparation antifreeze peptide of the present invention adopts in conjunction with cold cycling (cold-heat-stage) system, many power microscopes and automatic photography system, the freeze proof active inhibition ice-crystal growth detection system platform of group structure.Keep the low temperature environment of operating process with liquid nitrogen.The temperature fluctuation of analog equipment in the low-temperature storage process.By the system program setting, the temperature fluctuation that sample is set changes and 7～25 circulations repeatedly continuously, connects many power microscopes and photographic recording system in real time, and the record sample is in the cold-hot real-time ice-crystal growth situation in the working cycle repeatedly.
Use multiple separation and purification means such as 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.
Utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) to measure the molecular weight of specificity antifreeze peptide.
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) the amino acid complete sequence of mensuration specificity antifreeze peptide.
In order further to understand content of the present invention, characteristics and effect, exemplify following examples now:
Take by weighing 1.65 gram cow leather collagens and be dissolved in the 6ml Mili-Q water, use 2mol/L NaOH then its pH regulator to 9.0.Earlier this solution water-bath being heated to 45 ℃, is the enzyme of the ratio adding respective amount of 1:15 according to the enzyme-substrate proportioning more then, and enzymolysis time is 30 minutes.Then in boiling water bath, go out enzyme 10 minutes, centrifugal 10 minutes of 14000rpm again after the cooling, it is standby to collect supernatant liquor.
Supernatant liquor is separated with Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm, sees Fig. 1.Collect each peak and measure freeze proof activity (as shown in Figure 2).As can be seen from Figure 1, Fraction2 has best freeze proof activity, and statistical analysis shows that the ice crystal mean diameter is 5.02um, is significantly less than Fraction1(17.96um) and Fraction3(8.39um), have significant difference (P ﹤ 0.05).
Separate Fraction2 and carry out next step separation again, with Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm, diameter 2.0cm) separates, elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5M for the NaCl concentration gradient, flow velocity is 0.5mL/min, sees Fig. 3.Collect each peak and measure freeze proof activity (as shown in Figure 4).Clearly, the ice crystal diameter of SP2 is significantly less than SP1, and statistical analysis shows that its ice crystal size is respectively 3.35um and 9.16um, has significant difference (P ﹤ 0.05).
Utilize the RP-HPLC RPLC further to separate again to the sample of separating the SP2 part, obtain highly purified specificity antifreeze peptide.Utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) to measure the molecular weight of the specificity antifreeze peptide of this purifying.As can be seen from Figure 5, the molecular weight of specificity antifreeze peptide of the present invention is 2107 Da.
The specificity antifreeze peptide of purifying has the ability of very strong inhibition ice-crystal growth, and as seen from Figure 6, (Fig. 6 A) compares with blank, has added the specificity antifreeze peptide of 0.5% (w/w), ice crystal grow hardly (Fig. 6 B).
Specificity antifreeze peptide to purifying utilizes protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) aminoacid sequence of mensuration specificity antifreeze peptide.Obtaining its amino acid total order classifies as: Gly-Glu-Arg-Gly-Phe-Pro-Gly-Glu-Arg-Gly-Ser-Pro-Gly-Ala-Gln-Gly-Leu-Gln-Gly-Pro-Arg.
＜110〉University of Fuzhou
＜120〉utilize the Sumizyme MP enzymolysis of cow leather collagen to prepare antifreeze peptide
<160>?1
<210>?1
<211>?21
<212>?PRT
＜213〉artificial sequence
<220>
<400>?1
Gly?Glu?Arg?Gly?Phe?Pro?Gly?Glu?Arg?Gly?Ser?Pro?Gly?Ala?Gln?Gly
1 5 10 15
Leu?Gln?Gly?Pro?Arg
20
Claims (1)
1. antifreeze peptide, it is characterized in that: described amino acid sequence of polypeptide is Gly-Glu-Arg-Gly-Phe-Pro-Gly-Glu-Arg-Gly-Ser-Pro-Gly-Ala-Gln-Gly-Leu-Gln-Gly-Pro-Arg.
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| CN2010102775694A CN101921328B (en) | 2010-09-10 | 2010-09-10 | Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease |
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| CN103467586B (en) * | 2013-09-25 | 2015-04-15 | 福州大学 | Antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage and preparation method thereof |
| CN103555803B (en) * | 2013-11-05 | 2015-05-06 | 中国农业大学 | Fish skin antifreeze protein as well as preparation method and application thereof |
| CN104798868A (en) * | 2015-05-20 | 2015-07-29 | 福州大学 | Compound antifreeze agent and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101400695A (en) * | 2006-03-13 | 2009-04-01 | 日本水产株式会社 | Protein having ice nucleating activity |
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