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Enzymology modification method for improving vitality of lipase

A modification method and lipase technology are applied in the field of modification of lipase, which can solve the problems of complex molecular structure operation and enzyme inactivation, and achieve the effects of simple steps, mild enzymatic modification conditions and less equipment.

Inactive Publication Date: 2013-07-10
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The molecular structure of chemically modified enzymes is complex to operate, and the enzymes are easily inactivated during the modification process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Free lipase and trypsin were added according to the protein mass ratio of 1:1. In this experiment, 50 μL of lipase and 200 μL of 1 mg / mL trypsin were added. In the control group, trypsin solution was replaced by 200 μL 0.001 mol / L HCl;

[0030] (2) The treatment system is 4ml disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH7.0, 0.05mol / L);

[0031] (3) The treatment time is 20 minutes, and the temperature of the water bath is 37 degrees;

[0032] (4) After the treatment, put the Erlenmeyer flask in a 37-degree constant temperature water bath shaker to preheat for 10 minutes, add 4ml of substrate, shake at 180rpm for 20min, add 15mL of 95% ethanol to stop the reaction;

[0033] (5) After the reaction is terminated, titrate the fatty acid produced by the reaction with 0.05mol / L sodium hydroxide standard solution, using phenolphthalein as the indicator. Calculate the activity of lipase after modification and the lipase of control group acco...

Embodiment 2

[0035] (1) Free lipase and trypsin were added at a protein mass ratio of 1:5. In this experiment, 50 μL of lipase and 200 μL of 5 mg / mL trypsin were added. In the control group, trypsin solution was replaced by 200 μL 0.001 mol / L HCl;

[0036] (2) The treatment system is 4ml disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH7.0, 0.05mol / L);

[0037] (3) The treatment time is 30 minutes, and the temperature of the water bath is 30 degrees;

[0038] (4) After the treatment, put the Erlenmeyer flask in a 37-degree constant temperature water bath shaker to preheat for 10 minutes, add 4ml of substrate, shake at 180rpm for 20min, add 15mL of 95% ethanol to stop the reaction;

[0039] (5) After the reaction is terminated, titrate the fatty acid produced by the reaction with 0.05mol / L sodium hydroxide standard solution, using phenolphthalein as the indicator. Calculate the activity of lipase after modification and the lipase of control group according to for...

Embodiment 3

[0041] (1) Free lipase and trypsin were added at a protein ratio of 1:5. In this experiment, 50 μL of lipase and 200 μL of 5 mg / mL trypsin were added. As a control, trypsin solution was replaced by 200 μL 0.001 mol / L HCl;

[0042] (2) The treatment system is 1ml disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH8.0, 0.05mol / L);

[0043] (3) The treatment time is 40 minutes, and the temperature of the water bath is 25 degrees;

[0044] (4) After the treatment, add 4 mL of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution (pH7.0, 0.05mol / L), place the Erlenmeyer flask in a 37-degree constant temperature water bath shaker to preheat for 10 minutes, and then add 4ml of substrate, shake at 180rpm for 20min, add 15mL of 95% ethanol to stop the reaction;

[0045] (5) After the reaction is terminated, titrate the fatty acid produced by the reaction with 0.05mol / L sodium hydroxide standard solution, using phenolphthalein as the indicator. ...

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Abstract

The invention provides an enzymology modification method for improving the vitality of a lipase. The method comprises the following steps: an enzyme solution with a certain concentration is prepared from trypsin and an HCl solution; a free lipase or an immobilized lipase and a protease which are added to a buffer solution with a certain pH value according to a certain proportion and well mixed are incubated for a period of time in a thermostatic water bath; the vitality of the lipase is determined by an olive oil emulsification process at a temperature of 37 DEG C; and the vitality improvement rate of a modified lipase is obtained through comparing with the activity of the lipase which is not processed by the protease. The method of the present invention has the advantages of simple step, easy operation, substantial improvement of the enzyme activity of the free lipase and the immobilized lipase (the vitality improvement rate of the free lipase reaches 32%, and the vitality improvement rate of the immobilized lipase reaches 38%), and mild reaction condition, and does not cause the denaturalization and the deactivation of the lipase because of extreme conditions.

Description

technical field [0001] The invention relates to the technical field of lipase modification, in particular to an enzymatic modification method for improving lipase activity. Background technique [0002] Lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) are a special class of acylhydrolases that catalyze ester hydrolysis or alcoholysis, ester synthesis, transesterification, lactone synthesis, peptide synthesis, high It is one of the most widely used enzymes in organic synthesis reactions such as polymer synthesis and chiral drugs. It is mainly used in industries such as food, chemistry, oleochemicals, agricultural chemistry, papermaking, washing and biosurfactant synthesis and biodiesel production. [0003] There are two main obstacles to the industrial application of lipase, one is high cost, and the other is the stability and activity of lipase. In terms of solving these problems, many researchers have studied the stability and activity improvement of lipase, mainly fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/20
Inventor 黄惠华刘自琴刘艳丰王娟
Owner SOUTH CHINA UNIV OF TECH
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