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Method for separating and purifying Salsolinol synthetase

A technology for separation, purification and enzyme synthesis, which is applied in the field of protein purification and can solve problems such as non-enzymatic activity detection, enzymatic properties, and lack of separation and purification.

Inactive Publication Date: 2017-12-12
BEIJING UNIV OF TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is speculated that Sal synthetase may be a new enzyme that can catalyze the production of endogenous neurotoxins, but it has not been isolated and purified, and there is no systematic study on enzyme activity detection and enzymatic properties

Method used

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  • Method for separating and purifying Salsolinol synthetase
  • Method for separating and purifying Salsolinol synthetase
  • Method for separating and purifying Salsolinol synthetase

Examples

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Embodiment 1

[0020] 1. Acid precipitation

[0021] 1.1 Prepare 1M perchloric acid (PCA) solution (1M perchloric acid, 10mM sodium metabisulfite and 10mM EDTA) and 2M potassium carbonate solution;

[0022] 1.2 Mix the crude enzyme solution with 1M PCA at a volume ratio of 5:1, mix well, centrifuge at 17,000g / min, 4°C for 10min, and take the supernatant;

[0023] 1.3 Neutralization: add a certain proportion of K to the supernatant 2 CO 3 Solution (the molar mass of perchloric acid is K 2 CO 3 2 times of that), put it at -80°C for 15min, take it out, centrifuge at 17,000g / min, 4°C for 10min, take the supernatant, and remove the perchloric acid in the solution through this step.

[0024] 2. Ultrafiltration

[0025] The ultrafiltration method uses the pressure difference as the driving force to selectively separate the target substance through the microporous structure on the membrane surface. It is a membrane separation technology. Under a certain external force, when the original soluti...

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Abstract

The invention discloses a method for separating and purifying Salsolinol synthetase, and belongs to the field of protein purification. The method includes pretreating crude enzyme liquid of the Salsolinol synthetase according to a perchloric acid precipitation method; adding K2CO3 into the crude enzyme liquid, subjected to perchloric acid treatment, of the Salsolinol synthetase, putting the crude enzyme liquid at the temperature of 80 DEG C for a while, such as 15 minutes, taking out the crude enzyme liquid for centrifugation, and taking supernate; performing ultra-filtration on the obtained supernate, at least further separating and purifying Sal synthetase of the ultra-filtrated filtrate according to a hydrophilic interaction chromatography (HILIC) method by an ultra-filtration tube with the molecular cut off of 3kD. By the method, the Salsolinol synthetase can be purified completely.

Description

technical field [0001] The invention belongs to the field of protein purification, in particular, the invention relates to a method for separating and purifying Salsolinol synthetase. Background technique [0002] Sal synthase (Salsolinol synthase) is the first key enzyme in the synthesis and metabolism of catechol isoquinolines. It is an enzyme that catalyzes dopamine (DA) and acetaldehyde (AcH) to generate dopaminergic neurotoxic substance Salsolinol. , which directly affects the metabolic process of Sal and its derivatives in the body, is an important endogenous pathogenic factor of Parkinson's disease and is closely related to its pathogenesis. So far, there are few research reports on Sal synthase, and only a few researchers have done some research on its properties, but its specific properties are still unclear. At present, it is speculated that Sal synthetase may be a new enzyme that can catalyze the production of endogenous neurotoxins, but it has not been isolated ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88
CPCC12N9/88
Inventor 陈薛钗邓玉林
Owner BEIJING UNIV OF TECH