CN103965294A - Antifreeze polypeptide, bionic antifreeze surface related to antifreeze polypeptide, and screening method and application of antifreeze polypeptide - Google Patents
Antifreeze polypeptide, bionic antifreeze surface related to antifreeze polypeptide, and screening method and application of antifreeze polypeptide Download PDFInfo
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- CN103965294A CN103965294A CN201310045867.4A CN201310045867A CN103965294A CN 103965294 A CN103965294 A CN 103965294A CN 201310045867 A CN201310045867 A CN 201310045867A CN 103965294 A CN103965294 A CN 103965294A
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Abstract
The invention relates to a method for screening a polypeptide fragment with antifreeze activity. The method comprises a step of screening amino acid sequences of natural antifreeze protein, antifreeze protein or antifreeze glycoprotein or properly improving amino acid sequences of natural antifreeze protein, antifreeze protein or antifreeze glycoprotein. The invention further relates to antifreeze polypeptide, a bionic antifreeze surface related to the antifreeze polypeptide, a coating containing the antifreeze polypeptide and a preparation method and application of antifreeze polypeptide. The antifreeze polypeptide inherits antifreeze characteristics of the antifreeze protein, has a simple structure, is easy to synthesize on large scale and to produce in batches and exerts better effects compared with the antifreeze protein when used for preparation of the antifreeze surface.
Description
Technical field
The present invention relates to biological chemistry, Materials science, surface and interface scientific domain, relates to the freeze proof surface that utilizes albumen and the polypeptide preparation with thermal hysteresis effect, and the method for preparing the freeze proof surface of Novel bionic.
Background technology
Thermal hysteresis effect is for to reduce the freezing point of solution in non-colligative mode, and do not change its fusing point, thereby the freezing point of the solution making and fusing point produce difference, and the difference of solution fusing point and freezing point is called as thermal hysteresis value.
Antifreeze protein (AFP) is the albumen that a class has thermal hysteresis effect.Absorption-inhibition the mechanism proposing according to people such as Raymond and Devries, antifreeze protein can be adsorbed on ice crystal surface, changes ice-crystal growth characteristic, suppresses ice-crystal growth.Conventionally, the thermal hysteresis value of the antifreeze protein aqueous solution is larger, represents that its Activity of Antifreeze is stronger.Compare with other salts solutions, antifreeze protein can play in lower concentration obvious reduction freezing point and delay icing effect.Antifreeze protein is extensively present in animals and plants and microbe.Antifreeze protein found so far from 1969, fish antifreeze protein be studied the most clearly.Specifically be divided into antifreeze glycoprotein (AFGP), I type AFP, II type AFP, III type AFP, IV type AFP.Antifreeze glycoprotein (AFGP) is the polymkeric substance consisting of Gly-Gly-Threonine tripeptides repeating unit, wherein on Threonine, is connected with a disaccharide group.I type AFP is rich in L-Ala (more than 60%), and has regular ɑ-spirane structure.The halfcystine (8%) that II type AFP contains high level, contains ɑ-spirane structure in its structure, beta sheet structure and a large amount of random structures.III type AFP is little globular protein (62 ~ 66 amino acid), has a flat ice bonding surface.4 antiparallel arrangements of ɑ-spirane structure in IV type AFP.Except fish antifreeze protein, people are also tenebrio molitor, and choristoneura fumigerana has been found to have the albumen of Activity of Antifreeze in the plants such as the first-class insect of red wing and winter wheat.Most insects and plant antifreeze protein, all have β chip architecture, and this structure can well be combined with base plane and the faceted pebble of ice, makes the antifreeze protein of this class have higher activity.All antifreeze proteins have non-colligative property to reduce freezing point and suppress the characteristic of ice-crystal growth.These features make antifreeze protein in fields such as industrial or agricultural and medical science, all have broad application prospects.For example, in industry, be used as frostproofer, in agricultural, anti-freeze gene can be accessed in the DNA sequence dna of crop, extend it and produce season, or medically for the cryopreservation of organ etc.
On the structure and composition of all kinds of antifreeze proteins, make a big difference, thereby cause the difference of its Activity of Antifreeze.In the extended familys of antifreeze protein, the Activity of Antifreeze of Properties of Insect Antifreeze Proteins will be far above most of fish antifreeze protein, the antifreeze protein THP26/27 for example separating from tenebrio molitor, and its Activity of Antifreeze exceeds 100 times than fish antifreeze protein.This proteinoid is called as " high reactivity antifreeze protein " (hyperactive AFP).Compare with common antifreeze protein, high reactivity antifreeze protein not only can be adsorbed on the faceted pebble of ice crystal, also can be adsorbed on the base plane of ice crystal.In fish protein, only the I type AFP in winter flounder has high reactivity.
High reactivity antifreeze protein structurally has salient feature.For example, in some Properties of Insect Antifreeze Proteins, aminoacid sequence Thr-Cys-Thr repeats in each β lamella, Thr lines up two row at two-dimensional space, distance between the Sauerstoffatom on its hydroxyl, with the lattice match of ice, Properties of Insect Antifreeze Proteins can be combined closely with ice crystal, thereby hinder ice-crystal growth.I type antifreeze protein in winter flounder body, the well-regulated αhelix of tool, its Thr and Asx residue of giving prominence to outside spiral can combine with the faceted pebble of ice crystal.Antifreeze protein has caused people's broad interest in the broad prospect of application in the fields such as food, agricultural, medical science and even material.But so far, the difficulty of antifreeze protein purification and preparation has limited its application greatly.Protein is with peptide bond, to be linked together and the compound that forms by 50 above amino acid, and the hydrolysate of protein is polypeptide.Polypeptide is comprised of 10 ~ 50 amino acid, and peptide and protein is the same, can be life active compound.Peptide synthesis technology is ripe, as solid-phase synthesis, and liquid phase synthesizing method, acid hydrolyzation, alkaline hydrolysis, enzyme process etc.Therefore, design and the synthetic polypeptide with Activity of Antifreeze, make to have the effect similar with antifreeze protein, is conducive to expand it in the application of every field and realizes large-scale industrial production.
Up to now; the preparation major limitation of anti-freeze material is being utilized hydrophobic or super hydrophobic surface, also has minority scientist that antifreeze protein is combined with polymer and modifies material surface, has all obtained good anti-freeze effect (P.Guo; Y.Zheng; M.Wei, C.Song, Y.Lin; L.Jiang; Adv.Mater.2012,24,2642; A.P.Esser-Kahn, V.Trang and M.B.Francis.J.Am.Chem.Soc.2010,132,13264.).But the anti-freeze effect of hydrophobic material is unsatisfactory, and super hydrophobic material preparation method is more loaded down with trivial details, and antifreeze protein is combined with polymer, need to, to protein modification reactive group, easily cause the inactivation of albumen.
The present invention according to the structure of several natural highly active proteins, form and with the binding mechanism of ice, the method that screening has the short-chain peptide of Activity of Antifreeze has been proposed.By the method, screen the polypeptide that has obtained having Activity of Antifreeze.Proposed a kind of method of preparing the freeze proof surface of Novel bionic simultaneously, be about to antifreeze protein/polypeptide and be combined in the surface that is modified with poly-Dopamine HCL layer.Freeze proof surface effect prepared by antifreeze peptide is remarkable, has the surface of freeze proof high-molecular polyivnyl alcohol to compare with finishing, and the freezing temperature that can reduce water of condensation reaches 5.5 ℃ most.This preparation method is simple, without special reaction conditions, and applied widely.
Summary of the invention
The present invention utilizes the mechanism of natural antifreeze protein and ice crystal effect, analyze the structure of natural high reactivity antifreeze protein, as I type fish antifreeze protein and Properties of Insect Antifreeze Proteins, find out wherein effectively fragment feature, according to its feature, screen and design the polypeptide fragment with Activity of Antifreeze.
One of object of the present invention is to provide a kind of screening to have the method for the polypeptide fragment of Activity of Antifreeze.
Two of object of the present invention is to provide a kind of polypeptide fragment of choosing from the aminoacid sequence of natural antifreeze protein, and the inventor has found the Activity of Antifreeze of this fragment first.
Three of the object of the invention is to provide a kind of bionical freeze proof material.
Four of the object of the invention is to provide a kind of antifreeze protein and antifreeze peptide is attached to material surface, prepares the method for bionical freeze proof material.This material, in industrial production, has broad application prospects in national defense construction and people's daily life.
The present invention realizes by following concrete technical scheme:
Screening has a method for the polypeptide fragment of Activity of Antifreeze, it is characterized in that, comprises the steps:
1), from natural antifreeze protein, antifreeze protein, screens in the aminoacid sequence of antifreeze glycoprotein, or with natural antifreeze protein, antifreeze protein, antifreeze glycoprotein is foundation, and its aminoacid sequence is suitably improved;
2) aminoacid sequence to screening, utilizes the method for solid phase synthesis to synthesize;
3) the polypeptide Activity of Antifreeze of gained sign step 2), selection has the polypeptide of thermal hysteresis effect.
According to the present invention, described method also further comprises:
4) characterize the structure of the polypeptide described in step 3) in the aqueous solution, preferably there is the polypeptide of certain secondary structure, for example, there is αhelix or in solution, can be assembled into the polypeptide of β chip architecture.
According to the present invention, it is characterized in that, described aminoacid sequence is selected from natural antifreeze protein, antifreeze protein, the aminoacid sequence on antifreeze glycoprotein and ice bonding surface, also preferably repeats aminoacid sequence.
In the present invention, the screening of step (1) foundation is that screening has thermal hysteresis effect and the aminoacid sequence that can be combined with ice face.
In the present invention, antifreeze protein hinders icing mechanism and is: antifreeze protein can be adsorbed on ice surface, stops ice crystal to be grown up, thus the structure of antifreeze peptide mate with ice crystal, its effect of competence exertion.Can be exactly the part that antifreeze protein is combined with ice with the sequence of ice crystal coupling.As for tumor-necrosis factor glycoproteins, because the aminoacid sequence of albumen is long, and majority is the repetition of certain aminoacid sequence, in order to choose the polypeptide of short chain as far as possible, and preferred a section in tumor-necrosis factor glycoproteins.But tumor-necrosis factor glycoproteins is not necessarily, with the aminoacid sequence of ice bonding surface be necessary.
According to the present invention, it is characterized in that, described polypeptide is selected from polypeptide water-soluble or alcohol dissolubility.Preferred water miscible polypeptide.
According to the present invention, it is characterized in that the preferred high reactivity antifreeze protein of described natural antifreeze protein.
According to the present invention, described antifreeze protein is selected from all kinds of antifreeze proteins in nature biotechnology body, comprises I type, II type, III type, IV type fish antifreeze protein, Properties of Insect Antifreeze Proteins, plant antifreeze protein, bacterium fungi antifreeze protein etc.
According to the present invention, described antifreeze glycoprotein is selected from the antifreeze glycoprotein in the Fish of polar region.According to the present invention, it is characterized in that, described method screening amino acid number is not more than 25 polypeptide fragment, and for example 20 polypeptide fragment, is also for example not more than 15 polypeptide fragment; Preferably be not more than 12 polypeptide fragment, the polypeptide fragment that more preferably amino acid number is 11, the polypeptide fragment that also more preferably amino acid number is 10.
According to the present invention, preferably antifreeze peptide fragment is that repeated fragment Thr-Cys-Thr-X-Ser-X-X-Cys-X-X-Ala-X(X in insect protein refers to a kind of in natural amino acid); Or described antifreeze peptide is polypeptide fragment DTA and TAA in I type fish antifreeze protein, or polypeptide fragment TCT.
According to polypeptide fragment of the present invention, it is characterized in that, described polypeptide has certain secondary structure, and described polypeptide fragment, in the aqueous solution, has αhelix or can be assembled into β chip architecture in solution.
The present invention also provides a kind of polypeptide with Activity of Antifreeze, it is characterized in that, described polypeptide fragment is from natural antifreeze protein, the fragment intercepting in the aminoacid sequence of antifreeze protein or antifreeze glycoprotein, or with natural antifreeze protein, antifreeze protein, antifreeze glycoprotein is foundation, carries out suitable improved aminoacid sequence.
According to the present invention, the preferred natural antifreeze protein of described polypeptide fragment, antifreeze protein, the repetition aminoacid sequence on antifreeze glycoprotein and ice bonding surface.
According to the present invention, the preferred high reactivity antifreeze protein of described natural antifreeze protein.
According to the present invention, described antifreeze protein is selected from all kinds of antifreeze proteins in nature biotechnology body, comprises I type, II type, III type, IV type fish antifreeze protein, Properties of Insect Antifreeze Proteins, plant antifreeze protein, bacterium fungi antifreeze protein etc.
According to the present invention, described antifreeze glycoprotein is selected from the antifreeze glycoprotein in the Fish of polar region.
According to the present invention, the amino acid number of described polypeptide fragment is not more than 25, for example, be not more than 20, is also for example not more than 15, is preferably not more than 12, and more preferably amino acid number is 11, and also more preferably amino acid number is 10.
According to the present invention, described polypeptide fragment is polypeptide fragment water-soluble or alcohol dissolubility.
According to the present invention, described polypeptide has certain secondary structure, as αhelix or in solution, can be assembled into β chip architecture.
According to the present invention, preferably, described antifreeze peptide is polypeptide fragment DTA and the TAA in I type fish antifreeze protein.Wherein, described DTA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:1; Described TAA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:2.Above-mentioned two kinds of antifreeze peptide fragments, in the aqueous solution, have αhelix, can effectively reduce solution freezing temperature.
According to the present invention, more preferably antifreeze peptide fragment is that repeated fragment Thr-Cys-Thr-X-Ser-X-X-Cys-X-X-Ala-X(X in insect protein refers to natural amino acid).In different Properties of Insect Antifreeze Proteins and identical antifreeze protein repeated fragment, X can be identical or different, preferably different.
According to polypeptide fragment of the present invention, it is characterized in that, described polypeptide fragment, in the aqueous solution, has αhelix or β chip architecture.
Compare with protein, the advantage of polypeptide is: simple in structure, be convenient to study its mechanism of action; Synthetic technology is ripe, easily obtains in a large number; Cost is relatively low; Design properly, its stability all can improve relatively with activity.
According to polypeptide fragment of the present invention, it is characterized in that, described polypeptide fragment, in the aqueous solution, has αhelix.
The present invention also provides a kind of isolated polypeptide fragment TCT, it is characterized in that having the aminoacid sequence as shown in SEQ IDNO:3.
According to the present invention, described polypeptide fragment TCT is by natural antifreeze protein, and antifreeze protein is separated in antifreeze glycoprotein.
According to above-mentioned polypeptide fragment, it is αhelix or β chip architecture, preferably β chip architecture.
Peptide T CT is a fragment of choosing from the aminoacid sequence of tenebrio molitor antifreeze protein and little chest turtle shell antifreeze protein, through DSC(Differential Scanning Calorimetry, differential scanning calorimeter) and the sign of circular dichroism spectrum, confirm that this fragment has good Activity of Antifreeze, and Stability Analysis of Structures, water-soluble, can in the aqueous solution, assemble and form β chip architecture.In DSC, compare with pure water freezing temperature, the freezing temperature of this peptide sequence can reduce by 7 ℃.In circular dichroism spectrum characterizes, the aqueous solution of TCT presents negative unimodal, the existence of susceptible of proof β chip architecture.
TCT amino acid list (Thr-Cys-Thr-Asp-Ser-Thr-Asn-Cys-Tyr-Lys-Ala-Thr)
The present invention also provides a kind of bionical freeze proof material, it is characterized in that, described material comprises natural antifreeze protein, antifreeze protein, antifreeze glycoprotein or antifreeze peptide of the present invention.
According to the present invention, described antifreeze peptide as described above.Described antifreeze protein is various mixture, mixture and the polymers that have the native protein of Activity of Antifreeze and include natural antifreeze protein active part.
According to the present invention, described antifreeze peptide is selected from the mixture that has the polypeptide fragment of Activity of Antifreeze or include the polypeptide fragment of Activity of Antifreeze, mixture and polymer.
According to the present invention, described antifreeze peptide is selected from polypeptide fragment DTA, polypeptide fragment TCT or the polypeptide fragment TCT in I type fish antifreeze protein.
According to the present invention, the preferred natural antifreeze protein of described polypeptide fragment, antifreeze protein, the repetition aminoacid sequence on antifreeze glycoprotein and ice bonding surface.
According to the present invention, the preferred high reactivity antifreeze protein of described natural antifreeze protein.
According to the present invention, described antifreeze protein is selected from all kinds of antifreeze proteins in nature biotechnology body, comprises I type, II type, III type, IV type fish antifreeze protein, Properties of Insect Antifreeze Proteins, plant antifreeze protein, bacterium fungi antifreeze protein etc.
According to the present invention, described antifreeze glycoprotein is selected from the antifreeze glycoprotein in the Fish of polar region.
According to the present invention, the amino acid number of described polypeptide fragment is not more than 25, is preferably not more than 12, and more preferably amino acid number is 11, and also more preferably amino acid number is 10.
According to the present invention, described polypeptide fragment is water-soluble polypeptide fragment.
According to the present invention, polypeptide has certain secondary structure, as αhelix or in solution, can be assembled into β chip architecture.
According to the present invention, described antifreeze peptide is polypeptide fragment DTA and the TAA in I type fish antifreeze protein, or peptide T CT.Wherein, described DTA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:1; Described TAA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:2, and described peptide T CT has the aminoacid sequence as shown in SEQ ID NO:3.
Preferably, described antifreeze peptide fragment, in the aqueous solution, has αhelix, can effectively reduce solution freezing temperature.More preferably antifreeze peptide fragment is the repeated fragment Thr-Cys-Thr-X-Ser-X-X-Cys-X-X-Ala-X in insect protein.
The bonding surface of some Properties of Insect Antifreeze Proteins and ice is very smooth β chip architecture, and β chip architecture refers to a kind of in natural amino acid by aminoacid sequence Thr-Cys-Thr-X-Ser-X-X-Cys-X-X-Ala-X(X) repeat to form.
The present invention also provides a kind of grafting method of bionical freeze proof material, it is characterized in that,
By Dopamine HCL or with the catechol derivatives of amido, modify bionical freeze proof material surface, at material surface, form a strata Dopamine HCL layer.
The method according to this invention, described antifreeze protein is the various mixtures that have the native protein of Activity of Antifreeze and include natural antifreeze protein active part, mixture and polymer etc., described in antifreeze peptide, be the various mixtures that there is the short-chain peptide of Activity of Antifreeze and include the short-chain peptide of Activity of Antifreeze, mixture and polymer etc.
According to the present invention, described antifreeze peptide preferably has as shown in SEQ ID NO:1, shown in SEQ IDNO:2, or the aminoacid sequence shown in SEQ ID NO:3.
According to the present invention, described Dopamine HCL is selected from small molecules Dopamine HCL or poly-Dopamine HCL.
In the present invention, in described Dopamine HCL, adjacent phenolic hydroxyl group and amino are the keys playing a role.The effect meeting of other segment affects the viscosity of poly-Dopamine HCL or similar structures to a certain extent.The effect of this quasi-molecule is exactly an instrumentality, under oxygen effect, forms the sticking poly-Dopamine HCL of tool.Poly-Dopamine HCL both can be combined in surface, again can be simultaneously react with amino etc., in conjunction with other molecules.
Small molecules Dopamine HCL, under alkaline environment, can spontaneous oxidation polymerization form the poly-Dopamine HCL (H.Lee, S.M.Dellatore, W.M.Miller, P.B.Messersmith, Science2007,318,426) with strongly adherent energy under the effect of oxygen.Poly-Dopamine HCL not only has strongly adherent, but also contain abundant functional group, can by Michael reaction or schiff base reaction grafting, have sulfydryl, the polymkeric substance of amino groups.
The material that is modified with Dopamine HCL layer is immersed in antifreeze protein/polypeptide solution, owing to all containing amino in protein and polypeptide.Poly-Dopamine HCL layer can react with the amino in antifreeze protein/polypeptide, thereby antifreeze protein/polypeptide has effectively been attached to material surface.
Aforesaid method of the present invention, utilizes poly-Dopamine HCL layer can adhere to each-characteristic of kind inorganic and organic materials, by material, is immersed in dopamine solution, forms the method realization with full-bodied Dopamine HCL layer polypeptide is attached to substrate surface.
The present invention also provides a kind of freeze proof material that contains antifreeze peptide of the present invention, it is characterized in that, described material comprises as surperficial base material.
According to the present invention, described antifreeze peptide as described above.Described antifreeze protein is various mixture, mixture and the polymers that have the native protein of Activity of Antifreeze and include natural antifreeze protein active part.
According to the present invention, described antifreeze peptide is selected from the mixture that has the polypeptide fragment of Activity of Antifreeze or include the polypeptide fragment of Activity of Antifreeze, mixture and polymer.
According to the present invention, described antifreeze peptide is selected from polypeptide fragment DTA and the TAA in I type fish antifreeze protein, or peptide T CT.Wherein, described DTA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:1; Described TAA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:2, and described peptide T CT has the aminoacid sequence as shown in SEQ ID NO:3.
According to the present invention, the preferred natural antifreeze protein of described polypeptide fragment, antifreeze protein, the repetition aminoacid sequence on antifreeze glycoprotein and ice bonding surface.
According to the present invention, the preferred high reactivity antifreeze protein of described natural antifreeze protein.
According to the present invention, described antifreeze protein is selected from all kinds of antifreeze proteins in nature biotechnology body, comprises I type, II type, III type, IV type fish antifreeze protein, Properties of Insect Antifreeze Proteins, plant antifreeze protein, bacterium fungi antifreeze protein etc.
According to the present invention, described antifreeze glycoprotein is selected from the antifreeze glycoprotein in the Fish of polar region.
According to the present invention, the amino acid number of described polypeptide fragment is not more than 25, is preferably not more than 12, and more preferably amino acid number is 11, and also more preferably amino acid number is 10.
According to the present invention, described polypeptide fragment is water-soluble polypeptide fragment.
According to the present invention, polypeptide has certain secondary structure, as αhelix or in solution, can be assembled into β chip architecture.
According to the present invention, described antifreeze peptide has as shown in SEQ ID NO:1, shown in SEQ ID NO:2, or the aminoacid sequence shown in SEQ ID NO:3.
According to the present invention, described substrate comprises the surface of various types of materials, glass for example, plastics, metal, polysilicon etc., and the uneven surface that has structure.
The present invention also provides a kind of substrate surface of the antifreeze peptide that contains any one of the present invention, it is characterized in that, described substrate comprises metal (for example aluminium/copper), plastics, glass, polysilicon, rubber, painted surface, and the uneven surface that has structure.
The present invention also provides a kind of coating, it is characterized in that institute, states coating and comprises antifreeze peptide of the present invention and substrate.
According to the present invention, described antifreeze peptide as previously mentioned.Described antifreeze protein is various mixture, mixture and the polymers that have the native protein of Activity of Antifreeze and include natural antifreeze protein active part.
According to the present invention, described antifreeze peptide is selected from the mixture that has the polypeptide fragment of Activity of Antifreeze or include the polypeptide fragment of Activity of Antifreeze, mixture and polymer.
According to the present invention, described antifreeze peptide is selected from polypeptide fragment DTA and the TAA in I type fish antifreeze protein, or peptide T CT.Wherein, described DTA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:1; Described TAA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:2, and described peptide T CT has the aminoacid sequence as shown in SEQ ID NO:3.
According to the present invention, described substrate comprises the surface of various types of materials, glass for example, plastics, metal (for example aluminium/copper), polysilicon, plastics, rubber, painted surface, and the uneven surface that has structure.
The present invention also provides a kind of aforesaid antifreeze peptide is grafted to surperficial method, it is characterized in that, comprises the antifreeze peptide described in any one of the present invention is grafted to substrate surface.
According to the present invention, described substrate comprises for example glass, plastics, metal (for example aluminium/copper), polysilicon, plastics, rubber, painted surface, and the uneven surface that has structure.
According to the present invention, described antifreeze peptide as previously mentioned.Described antifreeze protein is various mixture, mixture and the polymers that have the native protein of Activity of Antifreeze and include natural antifreeze protein active part.
According to the present invention, described antifreeze peptide is selected from the mixture that has the polypeptide fragment of Activity of Antifreeze or include the polypeptide fragment of Activity of Antifreeze, mixture and polymer.
According to the present invention, described antifreeze peptide is selected from polypeptide fragment DTA and the TAA in I type fish antifreeze protein, or peptide T CT.Wherein, described DTA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:1; Described TAA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:2, and described peptide T CT has the aminoacid sequence as shown in SEQ ID NO:3.
The present invention also provides a kind of method of preparing freeze proof surface, it is characterized in that, utilizes Dopamine HCL for medium, and the antifreeze peptide described in any one of the present invention is attached to substrate material surface.
The present invention also provides the antifreeze peptide of any one of the present invention in the application for anti-freeze or freeze proof material.
The present invention also provides the freeze proof surface of any one of the present invention in the application for anti-freeze or freeze proof material.
According to the present invention, described application comprises cable surface, refrigerator and interchanger coating, the surface that aircraft surface etc. are all kinds of has the frosting that delays to freeze to need.
Salt can reduce the freezing point of solution, and antifreeze protein and antifreeze peptide can be under lower concentration, and performance more obviously reduces the effect of freezing point of solution than high salt concentration solution.By each class methods at the freeze proof molecule of surface bonding, the concentration of freeze proof molecule is conventionally very low, and this makes antifreeze protein be applied in the freeze proof surperficial aspect of preparation, has great advantage, can more effectively reduce water of condensation at the freezing temperature of substrate surface, delay the freezing time of water of condensation.Antifreeze peptide possesses the freeze proof characteristic of antifreeze protein, simple in structure, and easily a large amount of synthetic and realization batch productions, are used for the freeze proof surface of preparation, are more better than antifreeze protein.
With existing, antifreeze protein end group is modified, be grafted on macromolecular chain, macromolecular chain being received to the method for preparing freeze proof surface in surface compares again, the inventive method is simple, step is few, only need to twice immersion in solution, without special reaction condition, avoid complicated separation and purification process simultaneously, can in reaction, not cause the inactivation of active substance.
Accompanying drawing explanation:
The column diagram of the average freezing temperature in surface that Fig. 1 surface that to be condensing drip modify at polypeptide DTA and PVA modify.
The column diagram of the average freezing temperature in surface that Fig. 2 surface that to be condensing drip modify at peptide T CT and PVA modify.
Embodiment
The present invention is elaborated by following embodiment, but those skilled in the art's understanding, described embodiment is not limitation of the present invention, any improvement of making on basis of the present invention and variation, all within protection scope of the present invention.
Embodiment 1:
The screening of antifreeze peptide:
All kinds of highly active proteins, because Properties of Insect Antifreeze Proteins has high heat stagnation value, and repeat aminoacid sequence Thr-Cys-Thr-X-Ser-X-X-Cys-X-X-Ala-X and can form smooth β chip architecture in Properties of Insect Antifreeze Proteins, can with the good combination of ice face.Choosing identical repetition aminoacid sequence Thr-Cys-Thr-Asp-Ser-Thr-Asn-Cys-Tyr-Lys-Ala-Thr in tenebrio molitor antifreeze protein and little chest turtle shell antifreeze protein synthesizes.In circular dichroism spectrum, show bear unimodal, confirmed the existence of its β-pleated sheet structure structure.Dsc is measured, and finds this peptide species, in the aqueous solution of 1mg/ml, has the thermal hysteresis value of 0.4 ℃.This peptide species, in lower concentration, has high thermal hysteresis value, is preferred antifreeze peptide.
Embodiment 2:
Antifreeze peptide (albumen) is for the preparation on freeze proof surface:
Compound concentration is the alkaline buffer solution Bicine(N of 10mM/ml, N-bicine N-), pH value is 8.3.By alkaline buffer solution Bicine dilution Dopamine HCL, the dopamine solution of preparation 2mg/ml.The silicon chip cleaning up is immersed in dopamine solution, in having the environment of oxygen, soaks.Water rinses the silicon chip that gained Dopamine HCL is modified, by silicon wafer blow-drying.Preparation antifreeze peptide (albumen) solution.Antifreeze peptide (albumen) strength of solution 0.2mg/ml.The silicon chip that is modified with poly-Dopamine HCL layer is immersed in antifreeze peptide solution and is soaked.Be soaked in water and be modified with the silicon chip of antifreeze peptide (albumen), soak, repeat this process three times.By silicon wafer blow-drying, or dry.The freeze proof surface that obtains having freeze proof characteristic.
Embodiment 3:
There is freeze proof surface and characteristic thereof prepared by the antifreeze peptide of αhelix:
The antifreeze peptide DTA with αhelix is attached to silicon base surface by the method for embodiment 2.0.1 ℃ of the thermal hysteresis value that DTA has at 1mg/ml solution.The silicon base of modification is placed in to airtight chamber, the water droplet of 0.5uL is dropped on the surface of modification, closed chamber is placed on cold platform.Speed with 10 ℃/min is warmed up to 50 ℃ by surface, and constant temperature 10min at 50 ℃ evaporates water droplet in closed chamber.Speed with 5 ℃/min is down to subzero 40 ℃ from normal temperature, makes saturated water vapour at surface condensation, freezes.Record the freezing temperature of each water droplet.Statistics is condensate in the water droplet freezing temperature of substrate surface, calculates the average freezing temperature of condensing drip.Compare with the substrate surface freezing temperature that the high-molecular polyivnyl alcohol (PVA) with Activity of Antifreeze is modified, can be observed, the surface that antifreeze peptide is modified has the effect of obvious reduction water droplet freezing temperature, can reduce about 2.5 degrees Celsius of freezing temperature, sees accompanying drawing 1.
Embodiment 4:
Can assemble and form freeze proof surface and characteristic thereof prepared by the antifreeze peptide of β chip architecture:
The antifreeze peptide TCT that can assemble formation β chip architecture is attached to silicon base surface by the method for embodiment 2.0.4 ℃ of the thermal hysteresis value that this peptide species has at 1mg/ml solution.The silicon base of modification is placed in to airtight chamber, the water droplet of 0.5uL is dropped on the surface of modification, closed chamber is placed on cold platform.Speed with 10 ℃/min is warmed up to 50 ℃ by surface, and constant temperature 10min at 50 ℃ evaporates water droplet in closed chamber.Speed with 5 ℃/min is down to subzero 40 ℃ from normal temperature, makes saturated water vapour at surface condensation, freezes.Record the freezing temperature of each water droplet.Statistics is condensate in the water droplet freezing temperature of substrate surface, calculates the average freezing temperature of condensing drip.Compare with the substrate surface freezing temperature that the high-molecular polyivnyl alcohol (PVA) with Activity of Antifreeze is modified, can be observed, the surface that antifreeze peptide is modified has the effect of obvious reduction water droplet freezing temperature, can reduce freezing temperature and reach 5 degrees Celsius, sees accompanying drawing 2.
Embodiment 5:
The surface of Dopamine HCL grafting antifreeze glycoprotein:
The antifreeze glycoprotein having in the Fish of polar region is attached to silicon base surface by the method for embodiment 2.The silicon base of modification is placed in to airtight chamber, the water droplet of 0.5uL is dropped on the surface of modification, closed chamber is placed on cold platform.Speed with 10 ℃/min is warmed up to 50 ℃ by surface, and constant temperature 10min at 50 ℃ evaporates water droplet in closed chamber.Speed with 5 ℃/min is down to subzero 40 ℃ from normal temperature, makes saturated water vapour at surface condensation, freezes.Record the freezing temperature of each water droplet.Statistics is condensate in the water droplet freezing temperature of substrate surface, calculates the average freezing temperature of condensing drip.Compare with the substrate surface freezing temperature that the high-molecular polyivnyl alcohol (PVA) with Activity of Antifreeze is modified, can be observed, the surface that antifreeze peptide is modified has the effect that reduces water droplet freezing temperature, can lower surface condensation water freezing temperature.
Embodiment 6:
The surface of Dopamine HCL grafting antifreeze protein:
By thering is the little chest turtle shell of Zhunger Basin antifreeze protein, by the method for embodiment 2, be attached to silicon base surface.The silicon base of modification is placed in to airtight chamber, the water droplet of 0.5uL is dropped on the surface of modification, closed chamber is placed on cold platform.Speed with 10 ℃/min is warmed up to 50 ℃ by surface, and constant temperature 10min at 50 ℃ evaporates water droplet in closed chamber.Speed with 5 ℃/min is down to subzero 40 ℃ from normal temperature, makes saturated water vapour at surface condensation, freezes.Record the freezing temperature of each water droplet.Statistics is condensate in the water droplet freezing temperature of substrate surface, calculates the average freezing temperature of condensing drip.Compare with the substrate surface freezing temperature that the high-molecular polyivnyl alcohol (PVA) with Activity of Antifreeze is modified, can be observed, the effect that the surface that antifreeze protein is modified has obvious reduction water droplet freezing temperature, can lower surface condensation water freezing temperature.
Embodiment 7:
The surface of Dopamine HCL grafting antifreeze protein:
By thering is I type fish antifreeze protein HPLC6, by the method for embodiment 2, be attached to silicon base surface.The silicon base of modification is placed in to airtight chamber, the water droplet of 0.5uL is dropped on the surface of modification, closed chamber is placed on cold platform.Speed with 10 ℃/min is warmed up to 50 ℃ by surface, and constant temperature 10min at 50 ℃ evaporates water droplet in closed chamber.Speed with 5 ℃/min is down to subzero 40 ℃ from normal temperature, makes saturated water vapour at surface condensation, freezes.Record the freezing temperature of each water droplet.Statistics is condensate in the water droplet freezing temperature of substrate surface, calculates the average freezing temperature of condensing drip.Compare with the substrate surface freezing temperature that the high-molecular polyivnyl alcohol (PVA) with Activity of Antifreeze is modified, can be observed, the effect that the surface that antifreeze protein is modified has obvious reduction water droplet freezing temperature, can lower surface condensation water freezing temperature.
Embodiment 8:
Macromolecular grafted antifreeze peptide:
Smooth turtle shell antifreeze protein is diluted in PBS damping fluid, slowly flows through the glass surface that is modified with glutaraldehyde.Amino in albumen reacts with surperficial aldehyde radical, smooth antifreeze protein can be connected in to glass surface.With NaCNBH3, reduce carbon-to-nitrogen double bon again.Obtain being connected with the surface of antifreeze protein.The freeze proof surface making is placed in to airtight chamber, the water droplet of 0.5uL is dripped from the teeth outwards, closed chamber is placed on cold platform.Speed with 10 ℃/min is warmed up to 50 ℃ by surface, and constant temperature 10min at 50 ℃ evaporates water droplet in closed chamber.Speed with 5 ℃/min is down to subzero 40 ℃ from normal temperature, makes saturated water vapour at surface condensation, freezes.Record the freezing temperature of each water droplet.Statistics is condensate in the water droplet freezing temperature of substrate surface, calculates the average freezing temperature of condensing drip.Compare with the substrate surface freezing temperature that the high-molecular polyivnyl alcohol (PVA) with Activity of Antifreeze is modified, can be observed, the surface that antifreeze protein is modified has the effect that reduces water droplet freezing temperature, can lower surface condensation water freezing temperature.
Embodiment 9:
For the icing situation of cold surface water of condensation in Reality simulation environment, surface prepared in embodiment 3 is placed in to (25 ℃) under room temperature, humidity 40 ~ 50%.On cold platform, with the speed cooling of 2 ℃/min.Guarantee water droplet abundant condensation on surface, surface temperature is in quasi-balanced state.Record the freezing temperature of water of condensation.Compare with the surface that is modified with the high-molecular polyivnyl alcohol (PVA) of Activity of Antifreeze, the surface that peptide T CT modifies can reduce and shows 10.7 ℃ of condensing drip freezing temperatures.Compare closed environment, peptide modified surface, delays the icing effect of condensing drip more obvious.
Claims (10)
1. screening has a method for the polypeptide fragment of Activity of Antifreeze, it is characterized in that, comprises the steps:
1), from natural antifreeze protein, antifreeze protein, screens in the aminoacid sequence of antifreeze glycoprotein, or with natural antifreeze protein, antifreeze protein, antifreeze glycoprotein is foundation, and its aminoacid sequence is suitably improved;
2) aminoacid sequence to screening, utilizes the method for solid phase synthesis to synthesize;
3) the polypeptide Activity of Antifreeze of gained sign step 2), selection has the polypeptide of thermal hysteresis effect.
2. according to the screening method of claim 1, it is characterized in that, described method also further comprises: 4) characterize the structure of the polypeptide described in step 3) in the aqueous solution, the polypeptide preferably with certain secondary structure, for example, have αhelix or in solution, can be assembled into the polypeptide of β chip architecture.
Preferably, described aminoacid sequence is preferably natural antifreeze protein, antifreeze protein, and the aminoacid sequence on antifreeze glycoprotein and ice bonding surface, also preferably repeats aminoacid sequence.More preferably, the preferred high reactivity antifreeze protein of described natural antifreeze protein.Also more preferably, described method screening amino acid number is not more than 25 polypeptide fragment, and for example 20 polypeptide fragment, is also for example not more than 15 polypeptide fragment; Preferably be not more than 12 polypeptide fragment, the polypeptide fragment that more preferably amino acid number is 11, the polypeptide fragment that also more preferably amino acid number is 10.
3. a polypeptide with Activity of Antifreeze, is characterized in that, described polypeptide fragment is from natural antifreeze protein, the fragment intercepting in the aminoacid sequence of antifreeze protein or antifreeze glycoprotein, or with natural antifreeze protein, antifreeze protein, antifreeze glycoprotein is foundation, carries out suitable improved aminoacid sequence.
Preferably, described antifreeze peptide is polypeptide fragment DTA and the TAA in I type fish antifreeze protein.Wherein, described DTA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:1; Described TAA polypeptide fragment has the aminoacid sequence as shown in SEQ ID NO:2.Above-mentioned two kinds of antifreeze peptide fragments, in the aqueous solution, have αhelix, can effectively reduce solution freezing temperature.
More preferably antifreeze peptide fragment is that repeated fragment Thr-Cys-Thr-X-Ser-X-X-Cys-X-X-Ala-X(X in insect protein refers to a kind of in natural amino acid).In different Properties of Insect Antifreeze Proteins and identical antifreeze protein repeated fragment, X is different.
4. a polypeptide fragment TCT for isolated natural antifreeze protein, is characterized in that, has the aminoacid sequence as shown in SEQID NO:3.Preferably, described TCT polypeptide fragment is β chip architecture.
5. a bionical freeze proof material, is characterized in that, described material comprises that claim 1-3 any one is from natural antifreeze protein, antifreeze protein, the polypeptide fragment TCT of the antifreeze peptide that antifreeze glycoprotein filters out or claim 4.
6. according to the freeze proof material of claim 5, it is characterized in that, described material comprises as surperficial base material.
7. a grafting method for claim 5 or 6 bionical freeze proof material, is characterized in that,
By Dopamine HCL or modify the surface of described bionical freeze proof material with the catechol derivatives of amido, at material surface, form a strata Dopamine HCL layer.
8. a substrate surface that contains the antifreeze peptide of, is characterized in that, described substrate comprises metal (for example aluminium/copper), plastics, glass, polysilicon, rubber, painted surface, and the uneven surface that has structure.
9. a coating, is characterized in that described coating comprises and comprises that claim 1-3 any one is from natural antifreeze protein, antifreeze protein, the antifreeze peptide that antifreeze glycoprotein filters out or the antifreeze peptide of claim 4 and substrate.
10. claim 1-3 any one is from a natural antifreeze protein, antifreeze protein, and the antifreeze peptide that antifreeze glycoprotein filters out, the polypeptide fragment TCT of claim 4, or the freeze proof substrate surface of claim 8 is in the application for anti-freeze and freeze proof material.
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