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A kind of preparation method of phenoloxidase active protein

A technology of active protein and phenol oxidase, applied in biochemical equipment and methods, redox enzymes, enzymes, etc., can solve the problems of complex and expensive equipment

Active Publication Date: 2021-07-06
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, and place it in a refrigerator at 4°C for 1 hour after the addition;

[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 dialysis bag of MW3500Da, 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 ph...

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 phenol oxidase active protein. The present invention uses the residual waste plasma of Limulus reagent as raw material, salts out the protein through saturated ammonium sulfate solution, freezes and centrifuges to obtain Limulus hemocyanin, and then desalts the PBS solution of hemocyanin through MW3500Da dialysis bag, and passes through SephacrylS‑ 100 propylene dextran gel column purification, concentrated treatment to obtain high-efficiency phenoloxidase activity of hemocyanin, the reaction initial velocity Vi = 74.52 nmol · (min · mg). The product obtained by the present invention has high purity and high activity, and can be used to prepare efficient, convenient, and low-cost micro-detectors for phenolic pollutants; the preparation method of the present invention is simple, and adopts MW3500Da dialysis bags and SephacrylS-100 acryl-dextran gel The combination of columns can achieve the effects of desalination, separation, purification and concentration at the same time.

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 Patents(China)
IPC IPC(8): C12N9/02
CPCC12N9/0059C12Y110/03001
Inventor 康信煌邓春梅洪鹏志何兰珍张国光
Owner 湛江博康海洋生物有限公司
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