A kind of bacterial strain producing 4-hp and the preparation method of high-yield 4-hp

A technology for strains and inoculants, applied in the field of efficient production of 4-HP strains and high-yield 4-HP preparation, can solve problems such as low conversion rate, poor stability, and inability to continuously produce 4-HP, and achieve high conversion efficiency. , the effect of improving quantity and quality, and improving production efficiency

Active Publication Date: 2020-01-21
广东本科生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Therefore, the technical problem to be solved in the present invention is to overcome the low conversion rate of microbial conversion production 4-HP in the prior art, 4-HP can only be produced in the form of trace by-products and the fermentation process has poor stability, and cannot continue to produce 4-HP. The problem of HP, thereby providing a stable and efficient production of 4-HP Mycobacterium parafortuitous, and the method of using the strain to prepare high-yield 4-HP

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Mutation screening of Mycobacterium parafortuitous strain BK12

[0036] 1. Screening of Mycobacterium parafortuitous strain BK11

[0037] 1.1 Preparation of medium

[0038] (1) Enrichment medium: KH 2 PO 4 1.0g / L, NaNO 4 3.0g / L, NH 4 Cl 4.0g / L, MgSO 4 ·7H 2 0 6.5g / L, FeS0 4 ·7H 2 0 0.01g / L, Tween-800.5g / L, phytosterol 10.0g / L. Adjust the pH of the medium to 7.0-7.2 before sterilization.

[0039] (2) Plate separation medium: Add 20 g of agar powder to every 1 L of enrichment medium to obtain a liquid plate separation medium; pour the liquid plate separation medium into a petri dish, and obtain a separation plate after the medium solidifies.

[0040] (3) Basic plate medium: yeast extract 5.0g / L, glycerol 10.0g / L, MgSO 4 ·7H 2 O 3.0g / L, K 2 HPO 4 0.5g / L, NH 4 CI 1.0g / L, agar powder 20.0g / L, adjust the pH of the medium to 7.0-7.2, and then sterilize at high temperature to obtain a liquid basic plate medium; pour the liquid basic plate medium into...

Embodiment 2

[0054] This embodiment provides a method for transforming and producing 4-HP using Mycobacterium parafortuitum (Mycobacterium parafortuitum) bacterial strain BK12, specifically comprising the following steps:

[0055] 1. Expanded culture of strains

[0056] (1) Preparation of seed medium: yeast extract 1.2kg, glucose 1.5kg, water 97.3kg, control pH to 7.0;

[0057] (2) Inoculate the preserved Mycobacterium parafortuitous strain BK12 from an eggplant bottle in 8L seed culture medium, cultivate at 30°C for 55 hours at 250rpm to obtain an expanded culture solution suitable for inoculation, and the OD600 of the expanded culture solution for 5.

[0058] 2. Microbial transformation into 4-HP

[0059] Preparation of fermentation medium: glucose 0.5kg, yeast extract 0.3kg, peptone 0.2kg, corn steep liquor 3.5kg, potassium dihydrogen phosphate 0.04kg, sodium nitrate 0.3kg, soybean oil 8kg, substrate phytosterol 1.5kg, water 85.3kg, Control the pH to 7.0; the volume of the fermenter ...

Embodiment 3

[0062] This embodiment provides a method for transforming and producing 4-HP using the Mycobacterium parafortuitum (Mycobacterium parafortuitum) strain BK12, which specifically includes the following steps:

[0063] 1. Expanded culture of strains

[0064] (1) Preparation of seed medium: peptone 1.5kg, glycerol 2.0kg, water 96.5kg, control pH to 7.5;

[0065] (2) Inoculate the Mycobacterium parafortuitous strain BK12 from an eggplant bottle in 8L seed culture medium, and cultivate it at 32°C for 48 hours at 200rpm to obtain an expanded culture solution suitable for inoculation. The OD600 of the expanded culture solution is 7 .

[0066] 2. Microbial transformation into 4-HP

[0067] Preparation of fermentation medium: glucose 1.2kg, yeast extract 0.2kg, peptone 0.5kg, corn steep liquor 2.5kg, potassium dihydrogen phosphate 0.06kg, sodium nitrate 0.5kg, soybean oil 10kg, substrate phytosterol 2.0kg, water 83kg, Control the pH to 7.6; the volume of the fermenter is 100L;

[00...

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Abstract

The invention discloses a Mycobacterium parafortuitum strain producing 4-HP, which is classified as Mycobacterium parafortuitum BK12 and has been preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO.M2017084; The new strain obtained after variable screening can efficiently transform sterols to produce 4-HP, produce a fermentation broth with 4-HP as the main product, simplify subsequent separation and extraction steps, and have high production stability. The present invention also discloses a preparation method for obtaining high-yield 4-HP by using said Mycobacterium parafortuitously; the preparation method of high-yield 4-HP of the present invention can make up to 3 parts of sterol in the fermentation medium When the species is converted into 4-HP, and the sterol input is 3 parts, the conversion rate reaches 90%, thereby increasing the single production yield on the basis of ensuring the single conversion rate.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, and in particular relates to a high-efficiency 4-HP production strain and a high-yield 4-HP preparation method. Background technique [0002] Steroids are the second class of drugs next to antibiotics and are widely used in the clinical field. For example, adrenal cortex hormones have anti-inflammatory, anti-allergic, anti-shock and anti-allergic effects, and are widely used in the treatment of rheumatoid Arthritis, bronchial asthma, eczema and other diseases; glucocorticoids can treat and alleviate collagen diseases, anaphylactic shock and other refractory or dangerous diseases, and are also indispensable drugs for the treatment of endocrine diseases such as Addison's; various Sex hormones are the main drugs for the treatment of male organ decline and some gynecological diseases, and are the main components of oral contraceptives. In addition, steroid hormone drugs also have the effects of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P33/00C12R1/32
CPCC12P33/00C12N1/205C12R2001/32
Inventor 林钦鹏林汉标林智德辜丹锋黄滨谢婉玲陈斯涛
Owner 广东本科生物工程股份有限公司
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