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A kind of method that utilizes single enzyme to produce various alkenal fragrances

A technology for alkenals and fragrances, which is applied in the field of producing a variety of alkenals fragrances by using a single enzyme, which can solve the problems of single product, unstable enzyme activity, and many types of enzymes, and achieve low reaction costs, mild and easy-to-control reaction conditions, The effect of high reaction efficiency

Active Publication Date: 2017-12-01
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with chemical synthesis, enzymatic synthesis of alkenals has high efficiency and high yield, and the enzymes needed for synthesis are extracted from natural plants, which has little environmental pollution, but it also has the following problems: 1. The substrate is specific, so a set of reaction enzymes can only produce one main alkenal substance, and cannot obtain multiple alkenal substances at the same time, so the product is single;
[0005] 2. During the reaction process, the reaction conditions of the two enzyme systems need to be controlled at the same time, so the production conditions become complicated and difficult to control, which is not suitable for large-scale production;
[0006] 3. The reaction enzyme extracted from plants takes C18 fatty acid as the reaction substrate, and the volatile substance formed is mainly C6 hexenal, so the volatile spices derived from C20 fatty acid such as C8 cannot be obtained by this enzymatic method;
[0007] 4. The reaction enzymes extracted from plants are crude enzyme liquids. There are many types of enzymes and unstable enzyme activities, resulting in unstable products and not suitable for production.

Method used

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  • A kind of method that utilizes single enzyme to produce various alkenal fragrances
  • A kind of method that utilizes single enzyme to produce various alkenal fragrances
  • A kind of method that utilizes single enzyme to produce various alkenal fragrances

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: the preparation of cloning enzyme PhLOX

[0024] 1. Extract the genomic DNA of the porphyra thallus with the CTAB method (cetyltrimethylammonium bromide method), and the steps are as follows:

[0025] 1) After preheating 2% CTAB extract in a water bath at 65°C, dispense 15mL of the extract into 50mL centrifuge tubes, add 0.1% mercaptoethanol, mix well and set aside;

[0026] 2) Take 500mg of fronds, quickly grind them into powder with liquid nitrogen in a sterile mortar, and immediately transfer them to CTAB extract;

[0027] 3) After oscillating evenly, heat in a 65°C water bath for 60 minutes, during which time the centrifuge tubes are repeatedly inverted several times every 10 minutes;

[0028] 4) Put it into a centrifuge for centrifugation, the centrifugation conditions are: 4°C, 8000rpm, 10min, after the centrifugation, suck the supernatant;

[0029] 5) Add an equal volume of phenol / chloroform / isoamyl alcohol mixture (ratio: 25 / 24 / 1) to the above su...

Embodiment 2

[0061] Add 100 μM linolenic acid to 0.5 mL of 25 mg / L clone enzyme PhLOX protein purification solution obtained in Example 1, mix well and incubate at 20° C. for 15 min to obtain an enzymatic hydrolysis reaction solution.

[0062] The product extraction and detection methods are as follows: add 1 mL of ethyl acetate (chromatographically pure) to the reaction solution, and shake at 4 °C for 1 h in the dark. 12000rpm, 4°C, centrifuge for 10min, collect the upper ethyl acetate phase, and add 0.5mL ethyl acetate to the lower phase to repeat extraction once, then collect the upper organic phase. Then combine the two collections, add 1 mL of pre-cooled deionized water, vortex and mix well, and ice-bath for 5 minutes to wash away water-soluble impurities. 12000rpm, 4°C, centrifuge for 10min, take the upper ethyl acetate phase, and use LC-MS to analyze the substrate consumption, such as figure 1 As shown, it was found that the substrate consumption reached 38.7%, of which figure 1 T...

Embodiment 3

[0064] Add 100 μM arachidonic acid to 5 mL of 25 mg / L concentration of cloning enzyme PhLOX protein purification solution obtained in Example 1, mix well, and incubate at 20° C. for 1 h to obtain an enzymatic hydrolysis reaction solution.

[0065] The product extraction and detection method is: add 10mL ethyl acetate (chromatographically pure) to the reaction solution, shake at 4°C in the dark for 1h, separate the upper ethyl acetate phase, and add 5mL ethyl acetate to the lower phase to repeat the extraction The upper organic phase was collected after one pass. Then combine the two collections, add 10mL of pre-cooled deionized water, vortex and mix thoroughly, and ice-bath for 5min to wash away the water-soluble impurities. The upper ethyl acetate phase was separated and analyzed for substrate consumption by LC-MS, e.g. figure 2 As shown, it was found that the substrate consumption reached 100%, where figure 2 The upper spectral line in the middle represents before the re...

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Abstract

The invention relates to a method for producing a variety of alkenal fragrances by using a single enzyme. The enzyme used in the method is a cloned enzyme, which has dual functions like lipoxygenase and hydroperoxide lyase, and can utilize a single enzyme in one step. Complete the enzymatic hydrolysis of linolenic acid, arachidonic acid, DHA, EPA and other polyunsaturated fatty acids to obtain short-chain natural alkenal fragrance compounds with different carbon numbers. Compared with the prior art, the method has mild and easy-to-control reaction conditions, high reaction efficiency, no harmful substances, low reaction cost, and can react with various substrates to obtain various pure natural fragrance compounds.

Description

technical field [0001] The invention relates to a production method of alkenal fragrances, in particular to a method for producing various alkenal fragrances by using a single enzyme. Background technique [0002] Alkenals are important constituents of natural spices and fragrances. They exist in trace amounts in the volatile substances of many plants, and can be detected in the fruits, leaves, stems and other tissues of plants as well as in marine algae. Such as: 1-octene-3-ol, also known as pine moldol, is a compound with a strong and pleasant aroma of soil, mushrooms and licorice. It mainly exists in mint, thyme and fresh Among mushrooms, it is a natural equivalent spice. It is widely used in beverages, candies, iced foods, baked foods, condiments and tobacco flavors. It is a valuable spice in the spice industry. 2-Nonenal is an edible spice that can give some foods a light woody aroma. It mainly exists in melons, peaches, grapes, tomatoes, carrots, soybeans, sesame and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P1/00C12P7/24C12P7/04C12P7/26C11B9/00
Inventor 陈海敏朱竹君陈娟娟严小军
Owner NINGBO UNIV
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