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6- cyano-(3r, 5r)-dyhydroxyl hexanoic acid tert-butyl ester prepared by biological catalysis, and bacterial strain thereof

A technology of tert-butyl hydroxycaproate and strains, which is applied in the field of biocatalytic preparation of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate and strains, and can solve the problems of insufficient diastereomeric induction, diastereomeric Problems such as low de value of tert-butyl hydroxycaproate

Active Publication Date: 2012-08-22
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many defects in the chemical synthesis method of tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate. The synthesis requires the use of highly toxic triethylboron and cryogenic conditions at -85°C, and the diastereoinduction is not sufficient , the product 6-cyano-(3R,5R)-dihydroxyhexanoic acid tert-butyl ester has a low de value, and the boride waste treatment formed by this reaction needs to go through tedious repeated methanol quenching, vacuum distillation, etc.
At present, there is no report that Pichia guilliermondii has diastereoselective carbonyl reductase activity of (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester

Method used

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  • 6- cyano-(3r, 5r)-dyhydroxyl hexanoic acid tert-butyl ester prepared by biological catalysis, and bacterial strain thereof
  • 6- cyano-(3r, 5r)-dyhydroxyl hexanoic acid tert-butyl ester prepared by biological catalysis, and bacterial strain thereof
  • 6- cyano-(3r, 5r)-dyhydroxyl hexanoic acid tert-butyl ester prepared by biological catalysis, and bacterial strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Screening microbial strains with catalytic (R)-6-cyano-5-hydroxyl-3-oxoylhexanoic acid tert-butyl ester reduction activity

[0041] Collect soil samples from all over the country, take 1.0g soil samples and disperse them into 10.0mL, 0.85% normal saline solution, mix well; take 2.0mL bacterial suspension and inoculate to 28mL containing 100mM (R)-6-cyano-5- Hydroxy-3-oxohexanoic acid tert-butyl ester enrichment medium (preparation: 50.0g glucose, 100.0g soybean sprouts boiled for 30 minutes, added 22.8g (R)-6-cyano-5-hydroxy-3-oxohexanoic acid tert-butyl ester, add water to make up to 1.0L, pH is natural); at 28°C and 150rpm, culture on a shaking table until the culture medium becomes turbid; transfer to fresh sterile In the enrichment medium (the composition is the same as before), the shaking culture was continued for 4 days under the conditions of 30° C. and 150 rpm, and the second batch of enrichment culture was obtained.

[0042] The second batch of enr...

Embodiment 2

[0045] Example 2: Influence of (R)-6-cyano-5-hydroxy-3-oxoylhexanoic acid tert-butyl concentration on Pichia guilliermondii X25 carbonyl reductase volume enzyme activity and diastereoselectivity in fermentation medium

[0046] For the Pichia guilliermondii X25 bacterial classification that will be preserved on the test tube slant, pick an inoculation loop thallus and inoculate to contain 0g / L,0.5g / L,1.0g / L,2.0g / L,3.0g / L,4.0g / L In the sterile fermentation medium of L, 5.0g / L, 7.0g / L, 8.0g / L (R)-6-cyano-5-hydroxy-3-oxoylhexanoic acid tert-butyl ester, other fermentation medium The component formula is as follows: yeast extract 25.0g / L, glucose 25.0g / L, (NH 4 ) 2 HPO 4 2.5g / L, KH 2 PO 42.5g / L, MgSO 4 30mg / L, NaCl 1.0g / L, solvent is water, pH 6.0. Cultivate at 28°C, 150rpm for 2 days, and collect the fermentation broth.

[0047] Pipette 20 mL of fermentation broth, centrifuge at 12,000 rpm for 8 minutes, discard the supernatant, collect the thalli, wash the thalline three ...

Embodiment 3

[0048] Embodiment 3: The impact of culture temperature on volume enzyme activity and diastereoselectivity of Pichia guilliermondii X25 carbonyl reductase

[0049] To the Pichia guilliermondii X25 bacterial classification that will be preserved on the test tube slant, pick an inoculation loop thalline and inoculate to the non-butyl ester containing 4.0g / L (R)-6-cyano-5-hydroxyl-3-carbonylhexanoate In the bacterial culture medium, here (R)-6-cyano-5-hydroxyl-3-oxoylhexanoic acid tert-butyl plays the role of inducer, and the other component formulations of the fermentation medium are as follows: yeast extract 25.0g / L, Glucose 25.0g / L, (NH 4 ) 2 HPO 4 2.5g / L, KH 2 PO 4 2.5g / L, MgSO 4 30mg / L, NaCl 1.0g / L, solvent is water, pH7.0, cultured at 18°C, 21°C, 24°C, 28°C, 32°C, 34°C, 37°C, 150rpm for 2 days, Collect the broth.

[0050] Pipette 20 mL of fermentation broth, centrifuge at 12,000 rpm for 8 minutes, discard the supernatant, collect the thalli, wash the thalline three...

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Abstract

The invention provides a Pichia guilliermondii X25 which is enriched and screened from soil, has high diastereoselectivity and is a carbonyl reductase active microbial new strain, and application of the Pichia guilliermondii X25 in preparation of 6-cyano-(3R, 5R)-dyhydroxyl hexanoic acid tert-butyl ester by biological asymmetric reduction (R)-6-cyano-5-hydroxyl-3-carbonyl hexanoic acid tert-butylester. The strain is preserved in China Center for Type Culture Collection (CCTCC), the address is Wuhan university, Wuhan, China, the post code is 430072, the CCTCC No. is M 2011386, and the preservation date is November11th,2011. The optical pure 6-cyano-(3R, 5R)-dyhydroxyl hexanoic acid tert-butyl ester prepared by using carbonyl reductase generated by the Pichia guilliermondii X25 to convert (R)-6-cyano-5-hydroxyl-3-carbonyl hexanoic acid tert-butyl ester is high in stereoselectivity, mild in reaction condition and environment-friendly.

Description

(1) Technical field [0001] The present invention relates to a bacterial strain with R-carbonyl reductase activity—Pichia guilliermondii X25, and its preparation of optically pure 6-cyano-(3R,5R)-di Application of tert-butyl hydroxycaproate. (2) Background technology [0002] Chiral drugs are of great significance in the prevention and treatment of many diseases, and their synthesis research has been highly valued by academia, enterprises and the government. Chiral alcohols with specific functional groups are key chiral building blocks for the synthesis of many chiral drugs, and are widely used in fine chemicals such as pharmaceuticals, pesticides, and spices. [0003] Cardiovascular disease has become one of the most threatening diseases to human beings, and its morbidity and mortality have surpassed malignant tumors and ranked first. According to the statistics of the World Health Organization, more than 30 million people die of cardiovascular and cerebrovascular diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/16C12P13/00C12R1/84
Inventor 郑裕国王亚军毛芳芳魏积福
Owner ZHEJIANG UNIV OF TECH
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