Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease

An antifreeze polypeptide and protease technology, applied in the biological field, can solve the problems of small quantity and limited application prospects of large laws.

Inactive Publication Date: 2010-12-22
FUZHOU UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

With the discovery and research of various antifreeze proteins, two key issues restricting the research and application of biological antifreeze proteins have become increasingly prominent: 1. The amount obtained by natural isolation and purification is very small and very limited 2. When scientists

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  • Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease
  • Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease
  • Antifreeze polypeptide prepared by enzymolysis of cow leather collagen by alkali protease

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preparation example Construction

[0034] The antifreeze activity detection system for preparing antifreeze polypeptide of the present invention adopts a combined cold-heat-stage system, multi-fold microscope and automatic camera system to construct an antifreeze activity inhibition ice crystal growth detection system platform. The cryogenic environment during operation was maintained with liquid nitrogen. Simulate temperature fluctuations of products during cryogenic storage. Through the system program setting, set the temperature fluctuation of the sample and repeat it continuously for 7 to 25 cycles, connect a multi-fold microscope and a real-time photographic recording system, and record the real-time ice crystal growth of the sample during the cold-hot cycle.

[0035] Sephadex G-50 molecular sieves, Sepharose SP C-25 ion exchange chromatography, RP-HPLC reversed-phase high-performance liquid chromatography, dialysis and other separation and purification methods are used to achieve efficient separation and ...

Embodiment 1

[0040] Weigh 1.65 g bovine hide collagen and dissolve it in 6 ml Mili-Q water, then adjust its pH to 9.0 with 2 mol / L NaOH. First heat the solution to 45°C in a water bath, then add the corresponding amount of enzyme according to the enzyme-substrate ratio of 1:15, and the enzymatic hydrolysis time is 30 minutes. Then inactivate the enzyme in a boiling water bath for 10 minutes, then centrifuge at 14000 rpm for 10 minutes after cooling, and collect the supernatant for later use.

[0041] The supernatant was separated by Sephadex G-50 gel chromatography (length 100cm, diameter 2.6cm), the eluent was deionized water, the flow rate was 2mL / min, and the elution peak was measured at 225nm, see figure 1 . Peaks were collected and assayed for antifreeze activity (eg figure 2 shown). From figure 1 It can be seen that Fraction2 has the best antifreeze activity. Statistical analysis shows that the average diameter of ice crystals is 5.02um, which is significantly smaller than F...

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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 [0001] The invention provides an antifreeze polypeptide and a preparation method thereof, and more specifically relates to an antifreeze polypeptide prepared by enzymatically hydrolyzing cowhide collagen with alkaline protease, which belongs to the field of biotechnology. Background technique [0002] The problem that food and pharmaceutical products repeatedly suffer from ice crystal growth and recrystallization due to fluctuations in ambient temperature during cryogenic storage and transportation has attracted increasing attention. The repeated rise and fall of temperature causes ice crystals to grow continuously, freeze-thaw and recrystallize, which seriously damages the cell and tissue structure and loses the quality of the product. Scientists around the world are facing serious challenges: how to control the growth and recrystallization of ice crystals, and realize the control of ice crystal growth on the low-temperature cold chain is the key to restrict...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/20C07K1/18C07K1/16C12P21/06
Inventor 汪少芸郑邦晓饶平凡
Owner FUZHOU UNIV
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