Bacterial strain for microbial transformation phytosterin as yield per unit androstane diene diketone

A technology for androstadiene dione and phytosterol is applied in the field of fermentation in bioengineering, which can solve the problems of high cost, low yield, difficult industrial production and the like, and achieves that the conversion rate does not decrease, the product purity is high, and the conversion rate is high. Effect

Inactive Publication Date: 2009-04-08
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, people used chemical methods to cut off the side chains of these sterols to make them intermediates for the preparation of steroid drugs, but it was difficult to apply to industrial production due to low yield and high cost.

Method used

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  • Bacterial strain for microbial transformation phytosterin as yield per unit androstane diene diketone
  • Bacterial strain for microbial transformation phytosterin as yield per unit androstane diene diketone
  • Bacterial strain for microbial transformation phytosterin as yield per unit androstane diene diketone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Effective transformation of phytosterols into androstenedione and androstenedione strain screening

[0036] Soil samples collected near an oil refinery in Bengbu, weighing 10g soil samples and 100mL triangular flask with glass beads, shake well and break up, let stand for 2h, take supernatant and filter, absorb a small amount of clear liquid and phytosterol as the only carbon Phytosterol-converting bacteria were enriched in the source liquid culture medium (plus Istatin). Take a little enrichment solution and spread it properly on the solid medium with phytosterol as the only carbon source for culture. Pick 200 well-growing single colonies and save them on a slant.

[0037] Medium composition: CH 3 COONH 4 1.5g, MgSO 4 ·7H 2 O 0.2g, K 2 HPO 4 0.4g, KH 2 PO 4 0.8g, FeSO 4 ·7H 2 O 5mg, ZnSO 4 ·7H 2 O 2 mg, MnCl 2 4H 2 O 0.5 mg.

[0038] Substrates: stigmasterol 28.70%, β-sitosterol 42.26%, campesterol 24.48%, other sterols 4.56%.

[0039] The...

Embodiment 2

[0040] Embodiment 2: Ultraviolet mutagenesis screening of a single-producing androstadienedione strain

[0041] Using the strain obtained in Example 1 as the starting strain, formulate its growth curve and ultraviolet lethality curve, after mutagenesis, dilute it in an appropriate gradient and spread it in a solid medium with phytosterol as the only carbon source in a dark room for 48 hours. Pick a single colony clone from a plate with a large difference in the growth status of the colony and inoculate it into three solid media of SM1 (SM+phytosterol), SM2 (SM+AD), and SM3 (SM+ADD), and culture it at 30°C for 48h. initial screening. Select the colonies that grow well on SM1 and SM2, and do not grow or grow slowly on SM3 for re-screening in shake flask fermentation, and select the strains with improved conversion rate and increased proportion of ADD products. The colony was identified as Bacillus sp ST06-95.

Embodiment 3

[0042] Embodiment 3: the research of the transformation ability of mutant strain

[0043] The bacterial strain ST06-95 obtained by screening in Example 2 was cultivated for 48h on solid medium (beef extract 0.3, peptone 0.5, NaCl 1.5, agar 2.0, pH 7.0), and then transferred to seed medium (beef extract 0.3, peptone 1.0 , NaCl 1.5, glycerol 1.5, yeast extract 0.3, pH 7.0) for 45 hours, and inoculated in the fermentation medium (phytosterol 0.8, glucose 1, citric acid 0.2, glycerin 0.25, ferrous ammonium citrate 0.05, K 2 HPO 4 0.05, MgSO 4 0.05, (NH 4 ) 2 HPO 4 1.0, Tween80 0.3, pH 7.0) and fermented for 7 days. The fermentation broth was extracted with ethyl acetate, and the content of AD and ADD in the solution was analyzed by HPLC. Such as image 3 As shown, the peak time corresponding to the standard sample AD is 9.7 min, and the peak time corresponding to the standard sample ADD is 11.1 min. After the strains after mutagenesis are fermented, the AD content in the...

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Abstract

The invention discloses phytosterol of microbial transformation, which is a strain which only produces androstene dione, pertaining to the fermentation technology field in biological engineering. The screened strains are classified and named as starch degrading bacillus (Bacillus a myloiquefaciens.) ST 06-95 and preserved in the China Center for Type Culture Collection with a preservation number of CCTCC No. M208135. Samples are collected from the nature separated for primary screening and put in slant preservation; wild strains ST 06 are taken as starting strains and screened by ultraviolet mutagenesis to obtain strains ST06-95 which mainly produce androstenone; and the growth of strains enters a logarithmic growth phase at the temperature of 30 DEG C, the pH value of 7.0 and a rotation speed of 220r / min and after culture lasting for 60h. Thallus in the growth phase are inoculated into a fermentation culture medium according to inoculation amount of 12 percent (V / V) and fermented for 6 days to 7 days at the temperature of 30 DEG C, the pH value of 7.0 and a rotation speed of 220r / min. When quantitative detection is carried out on the fermented products, the product content of ADD exceeds 1500mg / l, and ADD accounts for 98 percent of the total amount of AD and ADD. The microbial transformation provides a basis for the industrialization of the microbial transformation of steroidal medicaments.

Description

technical field [0001] The invention relates to the production of androstadienedione, a key intermediate of steroid medicine, by transforming phytosterols with microorganisms, and belongs to the technical field of fermentation in bioengineering. Background technique [0002] In 1949, Hench et al. found that cortisone had outstanding curative effect on rheumatoid arthritis, and confirmed that both cortisone and hydrocortisone belonged to adrenal cortex hormones, making steroid corticosteroids a drug with high curative effect and high economic value. Because the content of hormones in animals is extremely low, it cannot be prepared in large quantities by directly extracting from animal raw materials. At present, semi-synthetic methods are mostly used, that is, raw materials with steroidal nucleus cyclopentane polyhydrophenanthrene are obtained from animals and plants, and then prepared by enzymatic transformation and chemical transformation. Sterols, the starting raw material...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/01C12P33/00C12R1/07
Inventor 饶志明许正宏赵有玺周楠迪夏海峰钟传圣
Owner JIANGNAN UNIV
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