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A large-scale, enzyme-induced technology, applied in the field of protein extraction, can solve the problems of PH450 enzyme activity and quality reduction, inability to achieve high-quality application, etc., to achieve high efficiency, easy induction and separation, and good quality.
Active Publication Date: 2018-10-30
无斑时代国际生物科技(深圳)有限公司
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However, in the process of preparation in this way, since the expressed genetically engineered bacteria obtain the PH450 enzyme protein product according to the expression of the gene sequence, since there is no functional modification step after expression in the metabolic process in the body, the prepared in this way The activity and quality of PH450 enzymes are greatly reduced, far from meeting the requirements of high-quality applications
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[0017] Example of the present invention proposes a large amount of preparation method of PH450 enzyme, comprises the steps:
[0018] S10, carrying out mass culture of cyanobacteria, and inducing with PH450 enzyme inducer during the culture process;
[0019] S20, after the mass culture in step S10 is completed, the total protein of cyanobacteria cells is extracted;
[0020] S30, using human PH450 enzyme as the antigen for immune reaction to prepare polyclonal antibody;
[0021] S40, using polyclonal antibody as an affinity medium to prepare an affinity chromatography column;
[0022] S50, pass the total protein of cyanobacteria cells extracted in step S20 on the affinity chromatography column in step S40.
[0023] In the above-mentioned method of the present invention, the process of its preparation is induced and cultivated by a large number of cyanobacteria, and a large amount of cell-modified PH450 enzymes are produced in the process of cyanobacteria metabolism, which has ...
Embodiment 1
[0043] S11, configure BG-11 medium (configure according to the mother liquor composition culture recorded in the microbiological experiment manual), and obtain cyanobacteria species at the same time (the present invention preferably chooses the crystal cyanobacteria of Lake Neuchâtel and "Shuisheng No. 1042" cyanobacteria, and selects Its purpose is to be more active than other cyanobacteria in the pigment metabolism of the usual domestic "Shuisheng No. 1042", and the amount of enzyme production is higher, which is suitable for the mass production of the present invention); S12, inoculate the cyanobacteria species to BG Cultivation in -11 medium: first transfer a small amount of algae to fresh medium, the inoculation ratio is 300,000 to 2 million cells / ml medium (generally inoculate as many as possible), and use a synchronous incubator placed in a cool white fluorescent tube (200-400footcandle) for 1-2 days to observe the initial growth status, after the growth is better, move ...
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Abstract
The invention provides a large-scale preparation method of PH450 enzyme. The large-scale preparation method includes: acquiring blue algae, cultivating the blue algae on a large scale, and using PH450 enzyme inducer for induction in the process of cultivation; after large-scale cultivation is finished, collecting blue algae cells, and extracting total protein of the blue algae cells; using human PH450 enzyme as antigen for immunoreaction to prepare polyclonal antibody; taking the polyclonal antibody as an affinity matrix to prepare an affinity chromatographic column; subjecting the extracted total protein of the blue algae cells to column passing in the affinity chromatographic column. On the basis that the blue algae and the human PH450 enzyme are almost completely identical in three-dimensional structure and activity function of protein and the blue algae is simple in metabolism and easy to induce and separate, a blue algae induced cultivation product is subjected to affinity chromatography by using specific antibody of the PH450 enzyme; compared with existing preparation methods through genetic engineering bacterium fermentation, the large-scale preparation method has the advantages that the PH450 enzyme prepared on a large scale is active enzyme generated during blue algae cell metabolism, thereby being high in quality and having activity; the purifying and separating process is simple, so that the large-scale preparation method is higher in benefit.
Description
technical field [0001] The invention belongs to the technical field of protein extraction, and in particular relates to a large-scale preparation method of PH450 enzyme. Background technique [0002] PH450 pigment metabolism enzyme system, or also called CYPH450 enzyme, P450 enzyme, is a kind of monooxygenase that mainly exists in the liver and intestinal tract, mostly located in the endoplasmic reticulum of cells, and catalyzes a variety of endogenous and exogenous substances (including macrophages). Most clinical drugs) metabolism, because the absorption peak measured by spectrophotometry is around 450nm, hence the name. It is mainly distributed in the SER (endoplasmic reticulum membrane), but also exists in the membranes of plasma membranes, mitochondria, Golgi bodies, peroxisomes, nuclear membranes and other organelles. , metabolized into water-soluble substances, so that toxic substances excreted. As a terminal oxygenase, it participates in the process of detoxificati...
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