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Mycobacterium fortuitum capable of effectively producing 9alpha-OH-AD and application thereof

A technology of Mycobacterium fortuitousus, -OH-AD, which is applied in the direction of bacteria, fermentation, and microbial-based methods, can solve the problems of up to 336 hours of transformation cycle and low conversion rate of substrates, and achieve good industrial application prospects, The effect of reduced conversion time

Active Publication Date: 2019-04-19
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

British Patent GB1530730 discloses the mutant strain Mycobacterium fortuitum NRRL B-8119 with 9α-hydroxylase by treating Mycobacterium fortuitum ATCC6842 with nitrosoguanidine mutagenesis, and this mutant strain can convert sitosterol, cholesterol or Stigmasterol produces 9α-OH-AD, but the conversion period is as long as 336 hours, and the conversion rate of the substrate is extremely low, and only a small amount of product 9α-OH-AD can be obtained

Method used

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  • Mycobacterium fortuitum capable of effectively producing 9alpha-OH-AD and application thereof
  • Mycobacterium fortuitum capable of effectively producing 9alpha-OH-AD and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Mutagenesis screening of ARL-91

[0023] ARTP and LiCl complex mutagenesis of Mycobacterium fortuitum:

[0024] (1) wash the slant seeds of starting bacterial strain Mycobacterium fortuitum (Mycobacterium fortuitum) (this bacterial strain is preserved by applicant's laboratory) with 0.5% Tween 80 sterile aqueous solution, make thalline OD 600 The value is controlled at 1.0±0.2. Inoculate into 50mL seed culture medium (same as Example 2) with 3% inoculum size, cultivate at 30°C and 200r / min until logarithmic growth phase (22-24h);

[0025] (2) ARTP mutagenesis

[0026] Adjusting step (1) to prepare the starting strain cultivated into OD 600 = 0.6~0.8 bacteria suspension, use a pipette gun to draw 10 μL on the mutagenesis slide, carry out ARTP mutagenesis, the mutagenesis time is 60s, wash the bacteria on the mutagenesis slide to get the bacteria suspension, for the mutagenesis The bacterial suspension was serially diluted, and 100 μL was spread on the agar...

Embodiment 2

[0033] Example 2: Fermentative production of 9α-OH-AD by Mycobacterium fortuitousus

[0034] Experimental strains: ARL-91 and starting strains;

[0035] (1) Seed cultivation

[0036] Take the fresh ARL-91 strain and one slant of the starting strain that have been cultivated for 2 to 3 days, and wash the slant seeds with 0.5% Tween80 sterile aqueous solution to make the bacteria OD 600 The value is controlled at 1.0±0.2. Inoculate into 50mL seed medium with 3% inoculum amount, cultivate at 30°C and 200r / min until logarithmic growth phase (22-24h).

[0037] The seed medium components are as follows: diammonium hydrogen phosphate 1.5g / L, yeast powder 9g / L, glycerol 10g / L, sodium dihydrogen phosphate 0.5g / L, disodium hydrogen phosphate 0.5g / L, Tween 80 0.5g / L, adjust the pH to 7.3;

[0038] (2) Fermentation culture

[0039] The seed solution was inserted into the fermentation medium with an inoculation amount of 8%, and phytosterol with a final concentration of 10g / L was adde...

Embodiment 3

[0047] Example 3 Fermentation of ARL-91 to produce 9α-OH-AD

[0048] (1) The production strain is ARL-91, and the seed culture is the same as in Example 1;

[0049] (2) Fermentation culture: insert the seed liquid into the fermentation medium according to the inoculation amount of 10%, add phytosterols with a final concentration of 5g / L in the fermentation medium, and ferment at 28~30°C and 160r / min After 48h, the production rate of 9α-OH-AD reached 96.2% after the fermentation.

[0050] The composition of the fermentation medium is as follows: ammonium sulfate 2.0g / L, urea 0.2g / L, sodium dihydrogen phosphate 1.0g / L, disodium hydrogen phosphate 0.5g / L, Tween 80 0.3g / L, glycerin 8g / L L. Phytosterol 5g / L, methyl-β-cyclodextrin 15.8g / L, pH 7.3, sterilized at 120-122°C for 20min under a pressure of 0.1-0.15mpa.

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Abstract

The invention belongs to the technical field of steroid biotransformation, and particularly relates to a mycobacterium fortuitum capable of effectively degrading phytosterol side chains and producing9alpha-OH-AD and application thereof. The mycobacterium fortuitum is a mutagenic strain obtained by physico-chemical combination mutagenesis, and the yield of 9alpha-OH-AD is up to 97%; when the concentration of a phytosterol substrate is up to 95.7% and is 1.21 times of that of original strains, and the biotransformation time is 72 hours and is 24 hours shorter than the original strains. Comparedwith the prior art, mycobacterium fortuitum using phytosterol as the substrate for producing 9alpha-OH-AD is highest in conversion rate and shortest in conversion time, and the mycobacterium fortuitum has a good industrial application prospect.

Description

Technical field: [0001] The invention belongs to the technical field of steroid biotransformation, and in particular relates to a mycobacterium fortuitum capable of effectively degrading side chains of phytosterols and high-yielding 9α-OH-AD and its application. technical background: [0002] 9α-hydroxyandrost-4-ene-3,17-dione (9α-hydroxyandrost-4-ene-3,17-dione, 9α-OH-AD) is an important precursor for the production of corticosteroids with halogens at the 9α position The body plays a very important role in regulating the body. Many steroid hormone drugs are produced with 9α-OH-AD as the starting material; 9α-OH-AD can be used to synthesize hydrocortisone, 17α-hydroxy Various important steroid drugs such as progesterone, eplerenone, dexamethasone, betamethasone, cortisone, fluocinolone acetate, fludroxone, etc. have important commercial value. [0003] Many microorganisms such as Mycobacterium, Arthrobacter and Corynebacterium can use phytosterols as carbon sources to produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P33/00C12R1/33
CPCC12P33/00C12N1/205C12R2001/33
Inventor 王敏申雁冰周海杰龙科艺骆健美夏梦雷屠琳娜马赛
Owner TIANJIN UNIV OF SCI & TECH
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