Method for preparing optical activity chirality secondary alcohol with rhodotorula reductase formulation

An optically active, reductase technology, applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems of limited large-scale industrial application, high price, etc., achieve good industrial application development prospects, easy to prepare, The effect of high substrate concentration

Inactive Publication Date: 2008-12-03
江苏华荣生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used coenzyme regeneration enzymes include glucose dehydrogenase, glucose 6-phosphate dehydrogenase, formate dehydrogenase, etc., but the price of these enzymes is relatively expensive, which limits their large-scale industrial application

Method used

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  • Method for preparing optical activity chirality secondary alcohol with rhodotorula reductase formulation
  • Method for preparing optical activity chirality secondary alcohol with rhodotorula reductase formulation
  • Method for preparing optical activity chirality secondary alcohol with rhodotorula reductase formulation

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Experimental program
Comparison scheme
Effect test

Embodiment 1-8

[0023] Take 5mg of Rhodotorula reductase crude enzyme preparation, add to 1ml NaHC 3 -Na 2 CO 3 In the buffer (100mM, pH 9.0), various substrates of different concentrations were added, such as acetylpyridine, benzene ring or methyl-substituted acetophenone derivatives, and 20 μl of NADPH solution with a concentration of 10 mM and 72 mg of mannitol were added, Shake the reaction on a 30 °C thermostatic shaker. After reacting for 24 hours, extract with an equal volume of ethyl acetate, add anhydrous sodium sulfate to the extract and dry it, and analyze the reaction conversion rate and the optical purity of the product by gas chromatography. The results are shown in Table 1.

[0024] Table 1 The crude enzyme preparation of Rhodotorula catalyzes the asymmetric reduction of acetylpyridine and aromatic ketones

[0025]

[0026]

Embodiment 9

[0028] Take 50mg of Rhodotorula reductase crude enzyme preparation, add to 10ml NaHCO 3 -Na2 CO 3 Buffer (100mM, pH 9.0), add 0.12g 2-acetylpyridine (final concentration is 100mM), add 0.1ml concentration of 10mM NADP + 0.9 g of mannitol was added to the solution, and the reaction was shaken on a constant temperature shaker at 30° C., and samples were taken intermittently to measure the conversion rate of the reaction. After 1 hour of reaction, the conversion rate was 17.1%. After 24 hours, the substrate was completely converted, and 0.098 g of the product (S)-2-pyridine-1-ethanol was isolated with an optical purity of >99%.

Embodiment 10

[0030] Take 10mg of partially purified Rhodotorula reductase preparation, add to 50ml NaHCO 3 -Na 2 CO 3 Buffer (100mM, pH9.0), add 0.605g 2-acetylpyridine (final concentration is 100mM), add 1ml concentration of 10mM NADP + 3.6 g of mannitol was added to the solution, and the reaction was shaken on a constant temperature shaker at 30° C., and samples were taken intermittently to measure the conversion rate of the reaction. After 1 hour of reaction, the conversion rate was 41.5%. By 5 hours, the substrate had been completely reduced and transformed. Add 50ml of ethyl acetate to extract the product (S)-2-pyridine-1-ethanol in the reaction solution, let stand to separate layers, take out the solid organic phase of ethyl acetate, and add 50ml of ethyl acetate to the water phase to extract again, so Repeat 4 times. Combine the ethyl acetate organic phases, add an appropriate amount of anhydrous sodium sulfate to dry overnight, filter, and remove the ethyl acetate in the filtra...

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Abstract

The invention discloses a method for preparing optical activity chiral secondary alcohol by using a rhodotorula reducing enzyme preparation agent. The rhodotorula reducing enzyme preparation agent is extracted from a rhodotorula cell, and comprises aldehyde reductase and mannitol dehydrogenase. Through the coupled chemical reaction of the aldehyde reductase and the mannitol dehydrogenase, mannite which is low in price can be used as cosubstrate, so that the in-situ regeneration and the cyclic utilization of coenzyme NADPH are realized during the reducing reaction process of a carbonyl group. The complete and highly-selective reduction of the high-concentration carbonyl substrate can be realized only through adding the coenzyme with catalytic amount in a synthesized reaction system. The production process is simple, the product is easy to extract and purify, and the rhodotorula reducing enzyme preparation agent can be used for preparing the chiral secondary alcohol with various high optical purity, in particular to (S)-aromatic secondary alcohol.

Description

Technical field: [0001] The invention belongs to the field of biochemical industry, and relates to the preparation of a reductase preparation containing carbonyl reductase and mannitol dehydrogenase extracted from rhodotorula, and a method for preparing corresponding optically active chiral secondary alcohols by reducing carbonyl compounds. Background technique: [0002] Optically active chiral secondary alcohols are important intermediates in the synthesis of many pharmaceuticals, pesticides, spices, and other fine chemicals. Split or asymmetric synthesis. The synthesis of optically active chiral secondary alcohols by chemically catalyzing the asymmetric reduction of ketones usually requires transition metal complexes with complex structures as catalysts. The catalysts are expensive, difficult to prepare and recover, and cause serious pollution to the environment. Their production and application are limited. limit. Biological synthesis of optically active chiral secondar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P41/00C12P7/22C12P7/02C12R1/645
Inventor 许建和杨巍朱月珍陆帮荣叶双庚潘江
Owner 江苏华荣生物科技有限公司
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