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Method for separating and purifying intestinal tract cathepsin B of sea cucumber

A cathepsin, separation and purification technology, applied in the field of freeze-drying, can solve the problems that there is no extraction method of sea cucumber intestinal cathepsin B, no literature and information reports, etc., and the effect of optimizing processing conditions and prolonging storage period can be achieved.

Active Publication Date: 2009-10-28
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no relevant extraction method for sea cucumber intestinal cathepsin B, and there are no relevant literature and information reports

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0033] Take fresh live sea cucumbers, remove the body wall and clean the intestines of sea cucumbers to remove sand and impurities. Homogenize it with 5 mmol / L sodium acetate buffer solution of pH 3.0 containing 5mmol / L L-Cys (L-cysteine) at 0°C with 5 times the volume of sea cucumber intestine; extract at 4°C for 4h , refrigerated and centrifuged at 6000×g (4° C.) for 60 min, and the supernatant was taken. Select the saturation of ammonium sulfate to be 65%, carry out fractional precipitation of ammonium sulfate, after refrigerating and centrifuging at 20000×g (4°C) for 60 minutes, take the precipitate, dissolve the precipitate with 50ml of the above-mentioned sodium acetate buffer solution, dialyze to desalinate, and concentrate by ultrafiltration , to obtain cathepsin B crude enzyme. Pass the obtained cathepsin B crude enzyme through DEAESepharose CL-6B ion-exchange chromatographic column, and use 50mmol / LL-Cys pH6.5 sodium phosphate buffer containing 0.5-2.0mmol / L NaCl fo...

example 2

[0035] Take fresh live sea cucumbers, remove the body wall and clean the intestines of sea cucumbers to remove sand and impurities. Homogenize it with 0°C 5mmol / L pH6.0 sodium acetate buffer solution containing 10mmol / L L-Cys (L-cysteine) containing 3 times the volume of sea cucumber intestine; extract at 4°C for 6h , using 15000×g (4°C) refrigerated centrifugation for 30 minutes, and the supernatant was taken. Select the saturation of ammonium sulfate to be 40%, carry out fractional precipitation of ammonium sulfate, refrigerate and centrifuge at 15000×g (4°C) for 60 min, take the precipitate, dissolve the precipitate with 50 ml of the above-mentioned sodium acetate buffer solution, dialyze to remove salt, and use polyethylene glycol After alcohol concentration, the cathepsin B crude enzyme is obtained. Pass the obtained cathepsin B crude enzyme through a DEAE Sepharose CL-6B ion-exchange chromatographic column, and use 20mmol / L L-Cys sodium phosphate buffer containing 0.01-...

example 3

[0037]Take fresh live sea cucumbers, remove the body wall and clean the intestines of sea cucumbers to remove sand and impurities. Homogenize it with a 0°C 150mmol / L pH6.8 sodium acetate buffer solution containing 20mmol / L L-Cys (L-cysteine) containing 20 times the volume of sea cucumber intestine; extract at 4°C for 15h , using 18000×g (4°C) refrigerated centrifugation for 10 minutes, and the supernatant was taken. Select the ammonium sulfate saturation to be 90%, carry out ammonium sulfate fractional precipitation, after refrigerated centrifugation at 18000×g (4°C) for 30 min, take the precipitate, dissolve the precipitate with 50 ml of the above-mentioned sodium acetate buffer solution, dialyze to remove salt, and use polyethylene Concentrate by diol method to obtain crude cathepsin B enzyme. Pass the obtained cathepsin B crude enzyme through a DEAE Sepharose CL-6B ion-exchange chromatographic column, and use 20mmol / L L-Cys sodium phosphate buffer containing 0.5-8.0mmol / L ...

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PUM

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Abstract

The invention relates to a method for separating and purifying intestinal tract cathepsin B of a sea cucumber, which comprises the following steps: using sodium acetate buffer solution containing L-Cys to lixiviate a sea cucumber intestine homogenate breaking buffer solution at a temperature of 4 DEG C; adopting ammonia sulfate to perform fractional precipitation; using the sodium acetate buffer solution to dissolve the precipitate; and concentrating the dissolved precipitate after dialysis and demineralization to obtain crude enzyme of the cathepsin B. The crude enzyme of the cathepsin B is processed by 2 to 3 methods of ion exchange chromatography chromatogram, gel chromatogram and high-efficiency liquid chromatogram to achieve the aim of purification.

Description

technical field [0001] The invention belongs to extracting protease from marine animals, and relates to the fields of ion exchange chromatography technology, gel chromatography technology, high performance liquid chromatography technology, freeze-drying technology and the like. Background technique [0002] There are many kinds of cathepsins in lysosomes. At present, 10 cathepsins have been purified and identified from the muscles and viscera of fish and shellfish, namely cathepsins A, B, C, D, E, H, L, L-like, X and S. [0003] Research on cathepsin B has mainly focused on fish. Currently, cathepsin B has been purified and identified from gray mullet, tilapiamossambica, mackerel, common Mackerel, chun salmon, carp muscle. [0004] Cathepsin B (CB) is a lysosomal cysteine ​​proteolytic enzyme with endopeptidase and carboxypeptidase activities, which can degrade muscle protein extensively. Studies have shown that at pH 5.0, CB is able to hydrolyze myosin, troponin, tropomy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
Inventor 朱蓓薇周大勇董秀萍于蕾孙黎明吴海涛宫国君刘晶
Owner DALIAN POLYTECHNIC UNIVERSITY
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