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Nocardia sp. capable of converting quininone into (R)-3-quinuclidinol and conversion method

The technology of Nocardia and quinine alcohol is applied in the field of bioengineering and can solve the problems of derivative reaction and low optical purity of 3-quinine alcohol, and achieves simple reaction process, strong adaptability to catalytic environment and less by-products. Effect

Active Publication Date: 2014-01-15
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The optical purity of (R)-3-quinine alcohol obtained by this method is not high, and there are derivatization reactions

Method used

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  • Nocardia sp. capable of converting quininone into (R)-3-quinuclidinol and conversion method
  • Nocardia sp. capable of converting quininone into (R)-3-quinuclidinol and conversion method
  • Nocardia sp. capable of converting quininone into (R)-3-quinuclidinol and conversion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Screening and identification of strains

[0051] 1.1 Medium

[0052] Inorganic salt medium: K 2 HPO 4 ·3H 2 O 1.0g, Na 2 HPO 4 ·3H 2 O 1.0g, (NH 4 ) 2 HPO 4 2.0g, NaNO 3 2.0g, MgSO 4 ·7H 2 O 0.2g, CaCl 2 2H 2 O 10mg, FeSO 4 ·7H 2 O 1.0mg, ZnSO 4 0.1mg, distilled water 1000mL.

[0053] A certain concentration of quinine hydrochloride was added to the inorganic salt medium to prepare the corresponding enrichment medium.

[0054] 1.2 Isolation and purification of strains

[0055] Put 5g of the collected sample into a 250mL shaker flask containing 50mL of sterile water, add glass beads, shake on a shaker at 250r / min for 2h, and let it stand for 3-5min. After the soil particles settle, take 2mL of the supernatant and add Into 50mL liquid enrichment medium (containing quinine hydrochloride 5g / L). After 3-4 days of culture at 30° C. on a shaker at 200 r / min, transfer to the next batch of enrichment medium (containing quinine hydrochloride 7...

Embodiment 2

[0059] Example 2: Catalytic properties of Nocardia sp.WY1202

[0060] The growth curve and enzyme production curve of Nocardia sp.WY1202 cultured at 30°C in the fermentation medium in embodiment 1 are as follows: figure 2 shown. Depend on figure 2 It can be seen that in the first 40h of bacterial cell culture, the conversion rate increases with the growth of the bacterial cell, and the conversion rate is the highest in the middle and late logarithmic growth phase (40h), reaching 92.7%. In the later stage of cell culture, the conversion rate of quinine ketone began to decline, but the decline was not obvious.

Embodiment 3

[0061] Embodiment 3: Preliminary optimization of Nocardia sp.WY1202 fermentation conditions

[0062] 1 Single factor experiment of carbon source

[0063] The addition amount of different carbon sources in the fermentation medium was 15g / L.

[0064] Table 1, the reaction of different carbon source fermentation bacteria transforming quinine into (R)-3-quinine alcohol

[0065]

[0066] From image 3 It can be seen from the results that glycerol is the most suitable carbon source for cell growth, followed by glucose, but for the conversion of quinine, glycerol is not a particularly ideal carbon source. When glucose was used as the carbon source, the conversion rate of quinine ketone was the highest. Considering the conversion rate and dry cell weight comprehensively, glucose was the optimal carbon source.

[0067] 2 Single factor experiment of nitrogen source

[0068] The addition amount of different nitrogen sources in the fermentation medium was 10g / L.

[0069] Table 2, ...

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Abstract

The invention discloses a Nocardia sp. WY1202 and a production method for (R)-3-quinuclidinol through fermentation of quininone by using Nocardia sp. WY1202. According to the invention, quininone hydrochloride is used as a sole carbon source for the strain of Nocardia sp. WY1202 which is screened from soil in a farm orchard in Xiqing District, Tianjin City, China, and is preserved in China General Microbiological Collection Center with an accession number of CGMCC No. 5095. The production method comprises the following steps: with the Nocardia sp.WY1202 as a strain, carrying out primary seed culture and secondary fermentation amplification culture; taking a fermentation culture solution, collecting thalli through centrifugation, washing the thalli and re-suspending the thalli in a buffer solution; adding the substrate quininone hydrochloride, adding glucose and carrying out centrifugation after a reaction for 24 to 48 h; adjusting the pH value of a supernatant until the supernatant is alkaline; drying the supernatant through pressure reduced spinning; subjecting an obtained residue to solid-liquid extraction with an organic solvent; and drying an obtained filtrate through spinning so as to obtain (R)-(-)-3-quinuclidinol. According to the invention, the product (R)-(-)-3-quinuclidinol has a yield of 95%, a purity of 96% and an e.e value of more than 95%; the production method is simple to operate, has low energy consumption, accords with requirements for green chemistry and is applicable to large-scale bioconversion production for (R)-(-)-3-quinuclidinol.

Description

Technical field: [0001] The invention relates to the field of bioengineering, in particular to a bacterial strain for fermenting and synthesizing chiral quinine alcohol and a production method thereof. Background technique: [0002] [0003] (R)-(-)-3-Quinuclidinol, English name: (R)-(-)-3-Quinuclidinol, chemical name: (R)-(-)-1-azabicyclo[2.2.2] Octan-3-ol, molecular formula C 7 h 13 NO, the molecular weight is 127.18, the CAS number is 25333-42-0, the EINECS number is 246-857-6, the pure product of R-3-quinine alcohol is white crystal, the melting point is 217-224 ° C, the boiling point is 120 ° C, the specific The optical rotation is -44.5° (c=3g / 100ml, 1N HCl), and the solubility in water is 100g / 100mL. (R)-3-quinine alcohol is the important intermediate of synthetic multiple anticholinergic drugs, such as solifenacin (Bossard P.[P].DE19715465,1997) and ravatorate (You Qidong, Lin Guoqiang, hand Sexual drugs: research and application Beijing: Chemical Industry Pre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P17/18C12R1/365
Inventor 李键煚王玉吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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