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Method for preparing C1,2-position dehydrogenated steroid compound

A steroidal compound and dehydrogenation technology, applied in the field of biochemistry, can solve the problem of difficult solubility of membrane proteins, etc., and achieve the effect of single product and high-efficiency conversion

Active Publication Date: 2020-07-31
武汉艾默佳华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there are many examples of heterologous expression, due to the poor solubility of the membrane protein itself, most of them exist in the form of inclusion bodies, which is a technical problem to be solved urgently in its industrial application

Method used

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  • Method for preparing C1,2-position dehydrogenated steroid compound
  • Method for preparing C1,2-position dehydrogenated steroid compound
  • Method for preparing C1,2-position dehydrogenated steroid compound

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, construction and shake flask expression of recombinant KstD fusion enzyme plasmid:

[0034] Escherichia coli BL21(DE3) cells containing the recombinant plasmid were cultured in LB medium containing 35 μg / ml kanamycin and rotated at 37° C. at 200 rpm. When OD600nm reached 0.8, 0.4 mM isopropyl-β-D-thiogalactopyranoside (final concentration) was added, the culture temperature was lowered to 20° C., and culture was continued for 18-20 hours. It was found by SDS-PAGE that the expression of the soluble target enzyme protein was significantly increased ( figure 2 ).

Embodiment 2

[0035] Embodiment 2, high-density fermentation prepares KstD fusion enzyme:

[0036] In order to obtain a high-density cell culture with a large amount of enzymes, the high-density medium was optimized, and the cells were cultured in a 15L fermenter with some modifications, such as replacing glucose by glycerol, yeast powder as a nitrogen source, induced by lactose, etc. At the beginning of the fermentation, the temperature of the fermentor was controlled at 37 °C to allow rapid cell growth. During the fed-batch phase, the dissolved oxygen was controlled between 10% and 30%, and the pH was controlled between 6.2 and 7.8. Before the induction phase, the cell culture temperature was lowered to 20°C and maintained for a period of time, then lactose mother solution was added until the final concentration reached 0.4mM, and the induction time was 18h-20h. Induce protein expression. After fermentation, collect E. coli cells by centrifugation at 4°C, resuspend in 50mM Tris-HCl buff...

Embodiment 3

[0038] Embodiment 3, KstD fusion enzyme activity

[0039] Take an appropriate amount of the centrifuged supernatant of 2.3, Mbp-KstD2 at 30 °C with a Nano Drop 2000 spectrophotometer (Thermo Scientific) at 600 nm (ξ600 nm = 18.7 × 103

[0040] cm-1M-1) using phenazine methyl sulfate (PMS) and 2,6-dichlorophenol-diphenol (DCPIP). The reaction mixture (1 ml) consisted of 50 mM Tris-HCl buffer (pH 7.0), 150 mM PMS, 8 mM DCPIP, appropriate concentration of supernatant or cell extract and 100 mM AD methanol. Activity is expressed as units per milligram of protein; 1 U is defined as a reduction of 1 μmolmin-1 DCPIP. No activity was detected in the reaction mixture without 4-androstenone-3,17-dione (AD). The remaining supernatant was subjected to SDS-PAGE electrophoresis analysis, and SDS-PAGE used 8% separating gel and 5% stacking gel to identify whether the protein was expressed correctly. Protein expression in the fermenter as Figure 4 shown.

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Abstract

The invention provides a method for preparing a C1,2-position dehydrogenated steroid compound. The method comprises the following steps: an expression vector for expressing recombinant KstD is constructed, wherein an amino acid sequence of the recombinant KstD is SEQ ID. 3; the recombinant KstD is expressed by using the expression vector in a cell culture manner to obtain a crude enzyme liquid; and a substrate androstane-4-alkene-3,17-dione (4-AD) to the crude enzyme liquid, and the C1,2-position dehydrogenated steroid compound is obtained through reaction. The soluble KstD fusion enzyme is obtained through modification of KstD211 of mycobacterium HGMS2; by use of the KstD fusion enzyme efficiently expressed by recombinant Escherichia coli, the KstD fusion enzyme is prepared through high-density fermentation, and the enzyme specific activity reaches 31.6 U / mg.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to a method for preparing C1,2-position dehydrosteroid compounds. Background technique [0002] Steroid hormone drugs are an important class of drugs in clinical practice, and their anti-inflammatory activity is particularly significant. Steroid drugs are widely used in the prevention and treatment of various diseases. In addition, changes in the structure of the steroidal core could lead to more potent steroid drugs. For example, when unsaturation was introduced into the 1,2-position of hydrocortisone acetate (HA) by Δ1-dehydrogenation, the anti-inflammatory activity of the product prednisolone acetate (PA) was enhanced three to fourfold. However, due to the complexity of the steroid structure, traditional chemical methods are difficult to specifically modify steroid intermediates, and the chemical methods have defects such as low conversion rate, many by-products, and environmental po...

Claims

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Application Information

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IPC IPC(8): C12P33/02C12N9/02
CPCC12P33/02C12N9/001C12Y103/99004
Inventor 苏正定成细瑶宋士奎周曦
Owner 武汉艾默佳华生物科技有限公司
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