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Method for conducting double aqueous phase extraction, separation and purification on papain through modified polyethylene glycol-sodium tartrate

A papain, polyethylene glycol technology, applied in the directions of hydrolase, biochemical equipment and methods, enzymes, etc., can solve the problems of increasing the distribution coefficient and yield of the target product, increasing the difficulty of operation and economic cost, and accelerating the equivalence of the system , to achieve the effect of fast system phase separation, easy operation and high separation efficiency

Inactive Publication Date: 2016-11-23
瑞安市智造科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing two-phase extraction technology needs to be supplemented by centrifugation and other operations after the phase separation of the system, thereby accelerating the phase separation of the system, and adding neutral salt KCl to the system to increase the distribution coefficient and yield of the target product increases the operational difficulty and economic cost

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] (1) Preparation of modified polyethylene glycol

[0022] 10g octanoic acid, 60g PEG1000 and 0.1g SO 4 2- / ZrO 2 Put the solid superacid catalyst in a rotary evaporator, mix it evenly, react at 130°C, and divide the water under reduced pressure. After the pH test paper detects that the reaction solution is neutral, stop the reaction, cool to room temperature, filter, and remove the filtrate by distillation. After adding moisture, 65.3 g of modified polyethylene glycol was obtained.

[0023] (2) Preparation of papain crude extract

[0024] Add 10g of papaya milk to 500g of deionized water and soak for 6 hours, freeze at -20°C for 3 hours, then thaw at 25°C, repeat the freeze-thaw process 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant Obtained 18g of crude extract containing papain, the purity was 51.4%, and the yield was 92.5%.

[0025] (3) Aqueous two-phase extraction

[0026] Take 13g of the crude extract containing p...

Embodiment 2

[0028] (1) Preparation of modified polyethylene glycol

[0029] 10g octanoic acid, 80g PEG6000 and 0.12g SO 4 2- / ZrO 2 Place the solid superacid catalyst in a rotary evaporator, mix it evenly, react at 140°C, and divide the water under reduced pressure. After the pH test paper detects that the reaction solution is neutral, stop the reaction, cool to room temperature, filter, and remove the filtrate by distillation. After adding moisture, 82.9 g of modified polyethylene glycol was obtained.

[0030] (2) Preparation of papain crude extract

[0031] Add 10g of papaya milk to 600g of deionized water and soak for 4 hours, freeze at -20°C for 5 hours, then thaw at 25°C, repeat the freeze-thaw process 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant 17.4 g of crude extract containing papain was obtained, the purity was 53.8%, and the yield was 93.4%.

[0032] (3) Aqueous two-phase extraction

[0033] Take 12.8g of crude extract cont...

Embodiment 3

[0035] (1) Preparation of modified polyethylene glycol

[0036] 10g octanoic acid, 100g PEG2000 and 0.15g SO 4 2- / ZrO 2 Place the solid superacid catalyst in a rotary evaporator, mix it evenly, react at 150°C, and divide the water under reduced pressure. After the pH test paper detects that the reaction liquid is neutral, stop the reaction, cool to room temperature, filter, and remove the filtrate by distillation. After removing moisture, 104.9 g of modified polyethylene glycol was obtained.

[0037] (2) Preparation of papain crude extract

[0038] Add 10g of papaya milk to 700g of deionized water and soak for 5 hours, freeze at -20°C for 8 hours, and then thaw at 25°C, repeat the freeze-thaw process for 3 times to break the wall, then centrifuge the obtained solution, and collect the supernatant 16.6 g of crude extract containing papain was obtained, the purity was 55.0%, and the yield was 91.2%.

[0039] (3) Aqueous two-phase extraction

[0040] Take 13.6g of the crud...

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Abstract

The invention provides a method for conducting double aqueous phase extraction, separation and purification on papain through modified polyethylene glycol-sodium tartrate. The method comprises the steps that papaya juice is subjected to wall breaking through a repetitive freeze-thawing method; supernatant liquor is collected, and a papain crude extract is obtained; modified polyethylene glycol and sodium tartrate are added into the crude extract, double aqueous phase extraction and layering are conducted in sequence, an upper phase is taken and put into a dialysis membrane of which the interception relative molecular mass is 10,000 to be dialysed for 15 h to 20 h, and a papain aqueous solution is obtained; freeze drying is conducted at -15 DEG C to -10 DEG C for 8 h to 15 h, and papain powder is obtained, wherein the average molecular weight of PEG in modified macrogol ester is 600 or 1,000 or 2,000. According to the method for conducting double aqueous phase extraction, separation and purification on the papain through modified polyethylene glycol-sodium tartrate, the selected double aqueous phase system is high in selectivity to the papain and high in system phase separation speed, phase separation can be conducted rapidly without the help of equipment such as a centrifugal machine, operation is easy and convenient, the equipment is conventional, the method is suitable for industrial production, and the obtained papain product has high purity and yield.

Description

technical field [0001] The invention belongs to the technical field of biochemical separation, and relates to a method for separating and purifying papain, in particular to a method for extracting, separating and purifying papain with modified polyethylene glycol-sodium tartrate two-phase extraction. Background technique [0002] Papain is widely found in the milk of immature papaya fruits. It is a protein single chain composed of 212 amino acids. It has the characteristics of high temperature resistance, strong activity, good stability, and strong protein hydrolysis ability. It is a pure natural product that can act on various proteins. It is not sensitive to pH changes, metal ions and detergents. It has many uses and has been widely used in various industries such as food, chemical industry, light textile industry, medicine and health. In meat processing, papain is the best meat tenderizer because of its higher thermal stability compared with other proteases. Papain is u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50
CPCC12N9/63C12Y304/22002
Inventor 任新年
Owner 瑞安市智造科技有限公司
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