Method for preparing phenoloxidase active protein

A technology of active protein and phenol oxidase, applied in biochemical equipment and methods, oxidoreductase, enzyme, etc., can solve the problems of complex and expensive equipment, achieve the effect of improving product purity, simplifying production process, and low cost

Active Publication Date: 2018-12-14
湛江博康海洋生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chromatography and spectroscopy have become the main detection methods for phenolic substances due to their advantages of high accuracy and high sensitivity, but the equipment is complex and expensive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 adopts the following steps to prepare:

[0033] (1) Take 40ml of Limulus plasma and place it in a water bath at 10°C, add dropwise 57.7ml of 100% saturated ammonium sulfate solution, stir while adding, after the dropwise addition, put it in a refrigerator at 4°C for 1 hour;

[0034] (2) After standing for 1 hour, take it out, and use a high-speed refrigerated centrifuge to centrifuge for 30 minutes at 4°C and 11,000r / min to obtain Limulus plasma hemocyanin, which is stored at 4°C for later use;

[0035] (3) Take Limulus plasma hemocyanin and dissolve it in 0.01mol / L, pH=7 PBS buffer (protein concentration is 10mg / mL), put it into a MW3500Da dialysis bag, dialyze overnight, collect the solution, and store it at 4°C. spare;

[0036] (4) Purify the solution collected in step (3) through a SephacrylS-100 propylene dextran gel column, elute with 50mmol / L Tris-HCl at pH=7.0, collect the solution that absorbs at 280nm, and obtain blood Blue protein solution is p...

Embodiment 2

[0038] Embodiment 2 adopts the following steps to prepare:

[0039] (1) Take 40ml of Limulus plasma and place it in a water bath at 10°C, add dropwise 57.7ml of 100% saturated ammonium sulfate solution, stir while adding, after the dropwise addition, put it in a refrigerator at 4°C for 1 hour;

[0040] (2) After standing for 1 hour, take it out, and use a high-speed refrigerated centrifuge to centrifuge for 30 minutes at 4°C and 11,000r / min to obtain Limulus plasma hemocyanin, which is stored at 4°C for later use;

[0041] (3) Take Limulus plasma hemocyanin and dissolve it in 0.01mol / L, pH=7 PBS buffer (protein concentration is 10 mg / mL), put it into a MW3500Da dialysis bag, dialyze overnight, collect the solution, and store it at 4°C. spare;

[0042] (4) Purify the solution collected in step (3) through a SephacrylS-100 propylene dextran gel column, elute with 50mmol / L Tris-HCl at pH=7.0, collect the solution that absorbs at 280nm, and obtain blood Blue protein solution is ...

Embodiment 3

[0044] Embodiment 3 adopts the following steps to prepare:

[0045] (1) Take 40ml of Limulus plasma and place it in a water bath at 10°C, add dropwise 57.7ml of 100% saturated ammonium sulfate solution, stir while adding, after the dropwise addition, put it in a refrigerator at 4°C for 1 hour;

[0046] (2) After standing for 1 hour, take it out, and use a high-speed refrigerated centrifuge to centrifuge for 30 minutes at 4°C and 11,000r / min to obtain Limulus plasma hemocyanin, which is stored at 4°C for later use;

[0047] (3) Take Limulus plasma hemocyanin and dissolve it in 0.01mol / L, pH=7 PBS buffer (protein concentration is 10 mg / mL), put it into a MW3500Da dialysis bag, dialyze overnight, collect the solution, and store it at 4°C. spare;

[0048] (4) Purify the solution collected in step (3) through a SephacrylS-100 propylene dextran gel column, elute with 50mmol / L Tris-HCl at pH=7.0, collect the solution that absorbs at 280nm, and obtain blood Blue protein solution is ...

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Abstract

The invention discloses a preparation method of a phenoloxidase active protein. The preparation method comprises the following steps: salting out proteins from a raw material waste blood plasma residual on Tachypleus Amebocyte Lysate by a saturated ammonium sulfate solution, performing refrigerating centrifugation to obtain limulus hemocyanin, performing dialysis desalting on a PBS solution of thehemocyanin through an MW 3500 Da dialysis bag, and carrying out Sephacryl S-100 allyl dextran gel column purification and concentrating treatment to obtain high-efficiency phenoloxidase active hemocyanin, wherein the initial reaction rate Vi of the high-efficiency phenoloxidase active hemocyanin is 74.52 nmol.(min.mg). The product obtained in the invention has a high purity and a high activity, and can be used for preparing a phenolic pollutant micro-detector with the advantages of high efficiency, simplicity and low cost; and the preparation method is simple, and simultaneously achieves desalting, separating, purifying and concentrating effects by cooperating the MW 3500 Da dialysis bag with the Sephacryl S-100 allyl dextran gel column.

Description

technical field [0001] The invention relates to a method for preparing an active protein, in particular to a method for preparing an active protein of phenoloxidase, and belongs to the technical field of extraction of natural active substances. Background technique [0002] Phenols mainly include phenol, cresol, aminophenol, dinitro-o-cresol, phenolic acid, and pentachlorophenol. Phenols are mainly derived from coking, oil refining, gas production, phenol, insulating materials, medicine, and papermaking. Exhaust gas and waste water. Phenol is a medium-strength chemical poison and a foul-smelling substance. It can invade the human body through the digestive tract, respiratory tract and skin and mucous membranes, and combine with the protein in the protoplasm of cells to make the cells lose their vitality. It has toxic effects, low concentration can cause cumulative chronic poisoning, high concentration can cause acute poisoning and even coma death. Phenol is listed as one o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02
CPCC12N9/0059C12Y110/03001
Inventor 邓春梅洪鹏志康信煌何兰珍张国光
Owner 湛江博康海洋生物有限公司
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