Method for preparing 2,2-dimethyl cyclopropanecarboxamide and bacterial strain thereof by biological catalysis

A technology of culture and location, which is applied in the field of biocatalytic preparation of 2,2-dimethylcyclopropanecarboxamide and its strains, can solve the problems of excessive waste water, low product yield, harsh reaction conditions and the like

Active Publication Date: 2010-12-22
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical synthesis of 2,2-dimethylcyclopropylcarboxamide has the disadvantages of harsh reaction conditions, lengthy reaction steps, many by-products, low product yield and more waste water.

Method used

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  • Method for preparing 2,2-dimethyl cyclopropanecarboxamide and bacterial strain thereof by biological catalysis
  • Method for preparing 2,2-dimethyl cyclopropanecarboxamide and bacterial strain thereof by biological catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Screening microbial strains with catalytic 2,2-dimethylcyclopropanenitrile hydration activity

[0041] Collect soil samples around chemical plants in Zhejiang Province, take 1.0g of soil samples and disperse them into 10.0ml of 0.95% normal saline solution, mix thoroughly; take 2.0ml of bacterial suspension and inoculate to 50.0ml containing 1.0‰ (v / v) 2,2-Dimethylcyclopropanecarbonitrile rich medium (preparation: 10.0g glucose, 1.0g K 2 HPO 4 , 0.2gMgSO 4 , 1.0gKH 2 PO 4 , 0.002gFeSO 4 ·7H 2 O, 0.002g CoCl 2 ·6H 2 O, 0.015g CaCl 2 , the pH is natural, and the water is added to 1.0L; the culture medium is sterilized at 121°C for 20 minutes, and after cooling to room temperature, 1.0mL of 2,2-dimethylcyclopropanenitrile is added, here, racemic 2,2-dimethyl cyclopropanecarbonitrile as the only carbon source and nitrogen source of the enrichment medium), shake culture under the conditions of 30°C and 150rpm until the culture medium is turbid; ) was tra...

Embodiment 2

[0044] Example 2: Preparation of biocatalyst R.boritolerans CCTCC M 208108 cells

[0045] The new nitrile hydratase that obtains from the present invention's breeding produces bacterial classification R.boritolerans CCTCC M208108 on the test tube inclined surface and picks a ring thalline, inoculates to 20.0ml aseptic seed culture medium (preparation: glucose 10.0g, yeast extract 3.0g , NaCl 1.0g, K 2 HPO 4 0.3g, KH 2 PO 4 0.3g, MgSO 4 0.2g, pH natural, make up to 1.0L with water. The medium was sterilized at 121° C. for 20 minutes), and cultured with shaking at 30° C. and 150 rpm for 24 hours.

[0046] Investigate the induction effect of different inducers, design the fermentation medium of the control group, the composition is: 10.0g / l glucose, 8.0g / l yeast powder, 1.0g / l KH 2 PO 4 , 1.0g / l K 2 HPO 4 , 1.0g / l NaCl, 0.2g / l MgSO4, 0.01g / l FeSO4, 0.05g / l CaCl 2 , the solvent is water; the fermentation medium of the experimental group was added 1.0g / l sodium glutamate,...

Embodiment 3

[0049] Embodiment 3: 2,2-dimethylcyclopropanamide biotransformation in the phosphate buffer system

[0050] For the biosynthesis of 2,2-dimethylcyclopropanamide, the biocatalyst R. boritolerans CCTCC M 208108 bacterium cells prepared by the present invention are selected. Select 10.0ml pH7.0 phosphate buffer (50.0mM) for the transformation system, the final concentration of R.boritolerans CCTCC M 208108 cells is 0.1gDCW / L, the substrate is fed in 3 batches, and 5.0μL 2,2-dimethylcyclopropane is added to each batch For cyanonitrile, the conversion temperature is 30°C, each batch is converted for 5 minutes, and the total conversion time is 15 minutes. After the transformation, centrifuge at 10,000rpm for 10min, discard the precipitate, collect the supernatant, and then filter the transformation liquid through a 0.45μm microfiltration membrane. The permeate is analyzed by gas chromatography. The conversion rates of the three batches are 100%, 99.1% and 100% respectively. , the c...

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Abstract

The invention provides a Rhodococcus boritolerans FW815 enriched in soil and sewage and sieving high nitrile hydratase active mircoorganism strain, and a process that applies the strain to biological catalysis methods for the preparation of 2,2-dimethyl cyclopropane formamide. The 2,2-dimethyl cyclopropane formamide is prepared by converting 2,2-dimethyl cyclopropane formonitrile via the nitrile hydratase generated by the strain, wherein, the reaction medium is water, constituents of the reaction system are simple, the biological catalysis efficiency is high, the conversion time is short, theconversion rate for substrates is high, the yield of the 2,2-dimethyl cyclopropane formamide product is high and the extraction and purification processes are simple.

Description

(1) Technical field [0001] The invention relates to a bacterial strain with high nitrile hydratase activity and its application in biocatalytic preparation of 2,2-dimethylcyclopropanamide. (2) Background technology [0002] Nitriles are a class of compounds containing cyano functional groups, which play a very important role in organic synthesis. The cyano group is an important source of C1, which is usually introduced into organic matter as a "water-stable carbanion" to form amines, imines, amides, amidines, carboxylic acids, and carbonyl compounds, etc. However, the traditional chemical conversion of nitriles has the disadvantages of high energy consumption, harsh reaction conditions, strong acid or strong base, high temperature and metal catalysts, low reaction selectivity, and the formation of a large number of by-products. Nitrile compounds also exist in the natural environment and in bacteria, fungi, plants, and animals. In biological systems, known nitrile hydratases...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12R1/01C12P13/02C12N1/20
Inventor 郑裕国王亚军沈寅初
Owner ZHEJIANG UNIV OF TECH
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