Application of saccharomyces cerevisiae in catalyzing citral to generate dextrorotatory citronellal and method for preparing levorotatory isopulegol

A technology of isopulegol and Lithospora, which is applied to the application of Trichosporidium lubilis in selectively catalyzing citral to generate dextro-citronellal, and the field of preparing levo-isopulegol, which can solve the problem of neral aldehyde The problems of low stereoselectivity and low optical purity of products can achieve the effect of good industrial application prospects, good optical purity and high stereoselectivity.

Active Publication Date: 2022-07-29
SUZHOU BAIFUAN ENZYME TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The ene reductases reported so far have high stereoselectivity for geranial, but generally not high stereoselectivity for neral, resulting in the optical purity of the product when the mixture of cis / trans citral is used as the reaction substrate. not tall

Method used

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  • Application of saccharomyces cerevisiae in catalyzing citral to generate dextrorotatory citronellal and method for preparing levorotatory isopulegol
  • Application of saccharomyces cerevisiae in catalyzing citral to generate dextrorotatory citronellal and method for preparing levorotatory isopulegol
  • Application of saccharomyces cerevisiae in catalyzing citral to generate dextrorotatory citronellal and method for preparing levorotatory isopulegol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of T. lubilis CGMCC 1292 wet cells

[0044] Seed liquid medium formula: glucose 1%, peptone 0.5%, beef extract 0.8%, pH 7.0. High temperature sterilization at 121°C for 20min.

[0045] Fermentation medium formula: glucose 15g / L, beef extract 8g / L, peptone 5g / L, KH 2 PO 4 2.0g / L, Na 2 HPO 4 2.0g / L, NaCl 0.5g / L, MgSO 4 0.5g / L, pH 7.0. Sterilize at 121℃ for 20min.

[0046] Take the slant of the yeast CGMCC 1292 stored at 4°C, pick a loop and inoculate it into a 250 mL shake flask containing 50 mL of seed liquid medium. Incubate for 24h at 30°C, 200rpm shaker, transfer to a 1L shake flask with 300mL fermentation medium at 5% (v / v) inoculum, continue to culture at 30°C, 200rpm for 36h, and harvest wet cells by centrifugation .

Embodiment 2

[0047] Example 2 Preparation of the crude enzyme powder of Rhizopus bilis CGMCC 1292

[0048] 100 g of cells harvested in Example 1 were weighed, suspended in 1 L of Tris-HCl buffer (100 mM, pH 8.0), and cooled in an ice-water bath. The cell suspension was disrupted twice using a high pressure homogenizer at 1800 bar. The disrupted liquid was centrifuged at 4°C and 15000 rpm for 10 min to obtain a clear centrifuged supernatant, which was a cell-free extract. The cell-free extract was quickly frozen in a -80°C refrigerator, and then freeze-dried using a freeze dryer to obtain freeze-dried crude enzyme powder.

Embodiment 3

[0049] Example 3 Determination of Stereoselectivity Catalyzed by Trichosporidium licheniformis citral olefinic reductase

[0050] In a 2 mL Eppendorf tube, add 100 μL of the cell-free extract as described in Example 2, 10 μL of neral or geranial in methanol (100 mM), 20 μL of NADPH in water (100 mM), and 870 μL of Tris-HCl buffer (100 mM) , pH 8.0), placed on a shaking mixer, 30 ° C, 1000 rpm shaking reaction for 2 h. Extract three times with equal volume of ethyl acetate, add anhydrous sodium sulfate to the extract and dry, and detect the stereo configuration and optical purity of the product citronellal by gas chromatography with a chiral chromatographic column.

[0051] The gas chromatography column used is a BGB-174 chiral chromatography column, 30m×0.25mm×0.25μm, the temperature of the injector and the detector is 250°C, the initial column temperature is 40°C, and the temperature is increased to 120°C at a rate of 4°C / min , maintained for 1 min, and then heated to 180 °C...

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Abstract

The invention discloses an application of Trichosporon loubiereii in selective catalysis of citral to generate dextro-citronellal, and the preservation number of the Trichosporon loubiereii is CGMCC (China General Microbiological Culture Collection Center) 1292. The invention further discloses a preparation method of the Trichosporon loubiereii. The invention further discloses a method for preparing the L-isopulegol. According to the present invention, the citral olefinic bond reductase catalyst derived from the lebuliferae trichosporon CGMCC 1292 has high stereoselectivity on cis/trans-citral, and the optical purity of the product d-citronellal is superior to the optical purity of the product d-citronellal reported at present. On the basis, a citral olefinic bond reductase reaction step and a metal catalytic cyclization reaction step of the product dextro-citronellal are organically coupled in series, so that the consumption of an organic solvent is reduced, the process is simpler, the optical purity of the product is good, and the industrial application prospect is better.

Description

technical field [0001] The invention belongs to the technical field of biology and chemical engineering, and more particularly relates to the application of T. ruberiformis in selectively catalyzing citral to generate dextro-citronellal, and the coupled chemical cyclization using the T. lubilis yeast A method for preparing L-isopulegol with a catalyst. Background technique [0002] L-menthol is commonly known as menthol. As a natural source of essence and spice, it has remarkable cooling properties and is widely used in the fields of daily chemicals, tobacco, food, and medicine and health. The global annual demand is as high as 40,000 tons. However, naturally extracted L-menthol needs to occupy a lot of arable land, and its output fluctuates greatly due to the influence of the climate, which cannot meet the market demand. Therefore, about 30% of the menthol currently available in the market is produced by chemical synthesis. [0003] BASF, Takasago and Dezhixin have indepen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/26C12P41/00C07C29/00C07C35/18C12N1/16C12N9/02C12R1/645
CPCC12P41/002C12N9/001C07C29/00C12P7/26C12Y103/01C12N1/16C07B2200/07C07C35/18
Inventor 欧阳鹏飞钱小龙戴忆思张雄寅张傲南
Owner SUZHOU BAIFUAN ENZYME TECH
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