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Method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by enzymatic method

A technology of enzymatic preparation and dihydroxy, which is applied in fermentation and other directions, can solve the problems of low substrate concentration, substrate conversion, low reaction efficiency, etc., and achieve high product yield and good quality

Inactive Publication Date: 2015-01-07
ENZYMEWORKS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of the above reactions catalyzed by the isolated enzyme can convert the R1 group in Compound 1 to a hydroxyl substrate, and even when 4AD is converted, there are problems such as low substrate concentration and low reaction efficiency.
Usually in the field of steroid conversion, enzymes cannot convert similar substrates due to strong substrate specificity (Current Organic Chemistry, 2012, 16, 2551-2582, Applied Microbiology Biotechnology, 2012, 96, 133-142, Journal of Biochemistry , 1995, 117, 1043-1049, Molecular Microbiology, 1995, 15, 895-905, CN 102168099A, etc.)

Method used

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  • Method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by enzymatic method
  • Method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by enzymatic method
  • Method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by enzymatic method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 (Preparation of KSDH):

[0032] The KSDH enzyme powder was prepared by conventional methods: The KSDH enzyme powder was derived from Arthrobacter simplex IFO12069 described in the literature J.Biochem.117,1043-1049 and the literature Lett.Appl.Microbiol.44 (2007)563-568. The KSDH gene fragment (synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.) was ligated with the pET30a plasmid digestion product, and then transformed into the competent E.coli BL21(DE3) strain. The positive clones were screened and inoculated into resistant ones. In liquid LB medium, culture at 37°C to OD600 to 0.8, add inducer IPTG, continue culture for 16 hours, centrifuge to collect the precipitate, add phosphate buffer to suspend, sonicate in an ice water bath for 10 minutes, centrifuge to take the supernatant, freeze Dry to obtain KSDH enzyme powder.

Embodiment 2

[0033] Example 2 (Verification of KSDH catalytic reaction):

[0034] 10mg of substrate and 40mg of KSDH enzyme powder were added to 2ml pH 7.0 phosphate buffer, stirred at 30°C for 20 hours, and the conversion rate detected by HPLC / MS was 16.5% (such as figure 1 Shown).

Embodiment 3

[0035] Example 3 (temperature optimization):

[0036] 20mg of substrate and 20mg of KSDH enzyme powder were added to 2ml pH 7.0 phosphate buffer, stirred at different temperatures for reaction, and the conversion rate detected by HPLC / MS was as follows figure 2 Shown.

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Abstract

The invention relates to a method for preparing 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione by an enzymatic method. The method is characterized by adding a substrate 11A, 17A-dihydroxy-pregna-1-ene-3,20-dione, 3-sterone-1-dehydrogenase, menadione and a cosolvent to an aqueous phase buffer solution with a pH value of 8-11 and stirring to react at a temperature of 30-50 DEG C to obtain 11A, 17A-dihydroxy-pregna-1,4-diene-3,20-dione, wherein the concentration of the substrate is 5-10g / l in an initial reaction system; the feeding amount of 3-sterone-1-dehydrogenase is 0.5-4 times the mass of the substrate; the cosolvent is selected from one of or a composite of more of dimethyl sulfoxide, polyethylene glycol-400 and Tween-60. The method has the advantages that the concentration of the reactive substrate can be 10g / L and the conversion rate within 3 hours can be 99% at most; the product has high yield and good quality; the method is suitable for industrial production.

Description

Technical field [0001] The invention belongs to the technical fields of biopharmaceuticals and biochemicals, and specifically relates to a method for preparing 11A,17A-dihydroxy-pregna-1,4-diene-3,20-dione by an enzymatic method. Background technique [0002] From compound 1:11A-hydroxypregnant-1,4-dien-3-one, its structural formula is Especially the 11A,17A-dihydroxy-pregna-1,4-diene-3,20-dione (R 1 Is hydroxyl, R 2 Is acetyl, R 3 As an intermediate, a variety of steroid compounds with good anti-inflammatory effects can be prepared, such as prednisolone (trade name Donison) and dexamethasone (Dexamethasone), etc. These drugs have a wide range of clinical applications. . [0003] Since plant sterols have gradually replaced diosgenin as the main raw material for steroid production, fermentation and chemical methods are used to prepare compound 1 from 4-androstenedione (4AD) derived from sterols as the starting material. 1 Is hydroxyl, R 2 Is acetyl, R 3 The route for hydroxy) has b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P33/02
Inventor 梁晓亮李国庆唐园园
Owner ENZYMEWORKS
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