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Preparation method for high-activity lumbrukinase

A lumbrokinase and high-activity technology, which is applied in the field of preparation of high-activity lumbrokinase, can solve problems such as the blank of kitchen waste, and achieve the effects of safe use, low cost and easy operation.

Inactive Publication Date: 2011-11-16
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the management of food waste in most cities in our country is basically blank, and it is in a state of self-disposal by the emission unit.

Method used

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  • Preparation method for high-activity lumbrukinase

Examples

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Effect test

Embodiment 1

[0037] A preparation method for highly active lumbrokinase, comprising the following steps (such as figure 1 shown):

[0038] 1) Using dry kitchen waste powder as raw material to raise Eisenia chinensis;

[0039] 2) Wash fresh earthworms and add 100mmol / L PBS (pH7.8) pre-cooled at 4°C at 1:1 (w / v), mash them in a high-speed tissue masher, filter with four layers of gauze to obtain the filtrate, and obtain crude protein solution. Place for 10min, centrifuge at 6000rpm, 4°C for 10min, remove the precipitate, and keep the supernatant;

[0040] 3) The supernatant is pressurized by the pump and enters the STM-DGM multifunctional membrane separation system (which can intercept molecules with a molecular weight greater than 10kD). : 12L / (m 2 .h), concentration ratio: 5.0:1, obtain crude enzyme liquid after ultrafiltration;

[0041] 4) At 4°C, add solid ammonium sulfate to the crude enzyme solution to make its saturation reach 20%, let it stand for 3.5 hours, centrifuge at 6000r / ...

Embodiment 2

[0045] A preparation method for highly active lumbrokinase, comprising the following steps (such as figure 1 shown):

[0046] 1) Using dry kitchen waste powder as raw material to raise Eisenia chinensis;

[0047] 2) Wash fresh earthworms and add 100mmol / L PBS (pH7.8) pre-cooled at 4°C at 1:1 (w / v), mash them in a high-speed tissue masher, filter with four layers of gauze to obtain the filtrate, and obtain crude protein solution. Place for 10min, centrifuge at 6000rpm, 4°C for 10min, remove the precipitate, and keep the supernatant;

[0048] 3) The supernatant is pressurized by the pump and enters the STM-DGM multifunctional membrane separation system (which can intercept molecules with a molecular weight greater than 10kD). : 12L / (m 2 .h), concentration ratio: 5.2:1, obtain crude enzyme liquid after ultrafiltration;

[0049] 4) At 4°C, add solid ammonium sulfate to the crude enzyme solution to make its saturation reach 30%, let it stand for 3.7 hours, centrifuge at 6000r / ...

Embodiment 3

[0053] A preparation method for highly active lumbrokinase, comprising the following steps (such as figure 1 shown):

[0054] 1) Using dry kitchen waste powder as raw material to raise Eisenia chinensis;

[0055] 2) Wash fresh earthworms and add 100mmol / L PBS (pH7.8) pre-cooled at 4°C at 1:1 (w / v), mash them in a high-speed tissue masher, filter with four layers of gauze to obtain the filtrate, and obtain crude protein solution. Place for 10min, centrifuge at 6000rpm, 4°C for 10min, remove the precipitate, and keep the supernatant;

[0056] 3) The supernatant is pressurized by the pump and enters the STM-DGM multifunctional membrane separation system (which can intercept molecules with a molecular weight greater than 10kD), working temperature: 28°C, feed liquid pH: 7.8, pressure: 0.7MPa, membrane flux : 9L / (m 2 .h), concentration ratio: 5.2:1, obtain crude enzyme liquid after ultrafiltration;

[0057] 4) At 4°C, add solid ammonium sulfate to the crude enzyme solution to ...

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Abstract

The invention discloses a preparation method for high-activity lumbrukinase. The method comprises the following steps: (1) cleaning earthworms, adding a phosphate buffer solution (PBS), carrying out triturating and filtering, taking the filtrate for centrifugation so as to obtain a supernatant; (2) carrying out ultrafiltration to obtain a crude enzyme solution; (3) carrying out fractional precipitation with ammonium sulfate and dissolving the precipitates with PBS to obtain an enzyme solution; (4) purifying the enzyme solution with Sephadex G-25 column; (5) purifying the enzyme solution with DEAE-cellulose column so as to obtain lumbrukinase. According to the invention, earthworms are fed by dry powder of kitchen waste and then lumbrukinase is extracted from the earthworms, thereby realizing resource utilization and transfer of added value of kitchen waste; conventional separating and purifying methods for enzyme are improved and optimized, and enzyme specific activity of lumbrukinasethat is extracted and purified by the method provided in the invention reaches 42000 U / mg, meeting requirements for enzyme for pharmaceutical purposes. The method provided in the invention has the characteristics of simple operation and low cost, and is easy for expanded industrial production.

Description

technical field [0001] The invention belongs to the field of biochemical pharmacy, and in particular relates to a preparation method of highly active lumbrokinase. Background technique [0002] How to deal with kitchen waste is a difficult problem, and it is also a problem that restricts urban development. It is imminent to solve the problem of messy kitchen waste disposal. According to statistics, in 2000, the amount of food waste produced in my country was 45 million tons, and it increased at an annual rate of about 10%, and the annual increase in food waste reached 5 million tons. At present, the management of kitchen waste in most cities in my country is basically blank, and it is in a state of self-disposal by the emission unit. This directly leads to three main flow directions of food waste: first, most of the food waste is collected without treatment and is directly fed to chickens and pigs; The dining table; thirdly, part of the kitchen waste has not been sorted, a...

Claims

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Application Information

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IPC IPC(8): C12N9/48
Inventor 马广智王高锋梁学世黄儒强陈孔亮潘淦林盛张璋虞为彭乐武佳韵陈世文张涛郭文康陈纯纯芦双高艳明赵盼盼黄陈玲邓金鹏
Owner SOUTH CHINA NORMAL UNIVERSITY
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