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Preparation method of (2S,3S)-2,3-butanediol

A technology of butanediol and diacetyl, which is applied in the field of biochemistry, can solve the problems of low concentration and optical purity, low chiral purity, and low efficiency, and achieve easy-to-obtain raw materials, simple synthetic routes, and high efficiency Effect

Pending Publication Date: 2020-09-29
ENZYMASTER NINGBO BIO ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low concentration and optical purity of (2S,3S)-2,3-butanediol in previous studies limited its application.
Although, in the prior art, whole cells of recombinant Escherichia coli are used to produce (2S, 3S)-2,3-butanediol from diacetyl, there are problems such as low efficiency and low chiral purity in this method.

Method used

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  • Preparation method of (2S,3S)-2,3-butanediol

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Embodiment 1

[0033] This embodiment provides a kind of preparation method that contains the solution of ketoreductase, it comprises the following steps:

[0034] (1) Insert the coding gene of ketoreductase between the Nco I and EcoR I restriction sites of the pET28a (+) carrier to obtain a recombinant vector; the nucleotide sequence of the coding gene of the ketoreductase is shown in the sequence listing Shown in SEQ ID NO: 1; the amino acid sequence of the ketoreductase is shown in SEQ ID NO: 2 in the sequence listing.

[0035] (2) Transform the above recombinant vector into Escherichia coli BL21 to obtain a recombinant strain.

[0036] (3) Inoculate the above-mentioned recombinant strain into LB solid medium containing chloramphenicol, and place it at 37°C for activating culture for 20 hours to obtain colonies; use an inoculation loop to pick a single colony and inoculate it into 50 mL of LB medium (with chloramphenicol added) in a 250mL Erlenmeyer flask, cover the bottle with a sealing...

Embodiment 2

[0039] This embodiment provides a kind of (2S,3S)-2, the preparation method of 3-butanediol, it comprises the following steps:

[0040] (1) Get 6.06g of the solution containing ketoreductase obtained in the above-mentioned embodiment 1 and 20mL concentration of 0.01mol / L phosphate buffered saline solution and place it in a clean 250mL single-necked flask with a magnet, and place the single-necked flask In a water bath at 20° C., the solution in the one-necked flask was stirred and mixed at a speed of 400 rpm to obtain a mixed solution.

[0041] (2) During the stirring process, keep the temperature of the water bath constant, and sequentially add 0.202g of nicotinamide adenine dinucleotide, 100mL of isopropanol and 20.2g of 2,3-butanedione to the above mixture A sealed reaction was carried out to obtain a reaction solution.

[0042] (3) The above reaction solution was filtered under reduced pressure to obtain a light yellow filtrate, and then the filtrate was subjected to rota...

Embodiment 3

[0045] This embodiment provides a kind of (2S,3S)-2, the preparation method of 3-butanediol, it comprises the following steps:

[0046] (1) Get 10.1g of the solution containing ketoreductase obtained in the above-mentioned embodiment 1 and 20mL concentration of 0.2mol / L phosphate buffered saline solution and place it in a clean 250mL single-necked flask with magnets, and place the single-necked flask In a water bath at 45° C., the solution in the one-necked flask was stirred and mixed at a speed of 400 rpm to obtain a mixed solution.

[0047] (2) During the stirring process, keep the temperature of the water bath constant, and sequentially add 0.606g of nicotinamide adenine dinucleotide, 180mL of isopropanol and 20.2g of 2,3-butanedione to the above mixture A sealed reaction was carried out to obtain a reaction solution.

[0048] (3) Suction filtration of the above reaction solution under reduced pressure to obtain a light yellow filtrate, and then place the filtrate at 55° C...

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Abstract

The invention discloses a preparation method of (2S,3S)-2,3-butanediol, and belongs to the technical field of biochemistry. The preparation method comprises the following steps of: stirring and mixingsolution containing ketoreductase with buffer solution to obtain mixed solution; in the stirring process, adding nicotinamide adenine dinucleotide, isopropanol and 2,3-butanedione into the mixed solution to perform closed reaction, so that reaction solution is obtained; carrying out reduced pressure suction filtration and rotary evaporation treatment on the reaction solution to obtain extractingsolution; and carrying out rectification separation on the extracting solution to obtain the (2S,3S)-2,3-butanediol. Meanwhile, the invention provides an amino acid sequence of the ketoreductase for catalyzing 2,3-butanedione to generate the (2S,3S)-2,3-butanediol. The invention further provides a nucleotide sequence for coding the ketoreductase. The preparation method is simple in synthetic routeand high in efficiency; 2,3-butanedione is reduced into (2S,3S)-2,3-butanediol by selecting the ketoreductase in an isopropanol system; the conversion rate of 2,3-butanedione can reach up to 95%; andin addition, the chiral purity of the prepared (2S,3S)-2,3-butanediol product can also reach 98%.

Description

technical field [0001] The invention relates to the technical field of biochemistry, in particular to a preparation method of (2S,3S)-2,3-butanediol. Background technique [0002] 2,3-butanediol (2,3-bd) is an important vicinal diol with 3 stereoisomers: meso-2,3-butanediol, (2R,3R)-2,3 - Butanediol and (2S,3S)-2,3-butanediol. As an important platform chemical, 2,3-bd can be used to produce valuable derivatives such as methyl ethyl ketone and 1,3-butadiene. Optically active isomers can act as antifreeze agents. Optically pure 2,3-bd also serves as a good building block for the asymmetric synthesis of chiral compounds containing two vicinal stereocenters. Therefore, it is necessary to develop a practical technique to produce optically pure 2,3-bd. [0003] Among them, (2S,3S)-2,3-butanediol is an important chiral compound, which acts as a good building block in the asymmetric synthesis of high-value chiral compounds. However, the low concentration and optical purity of (...

Claims

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Application Information

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IPC IPC(8): C12P7/18C12N9/04C12N15/53C12N15/70C07C29/76C07C29/80C07C31/20C12R1/19
CPCC12P7/18C12N9/0006C12N15/70C07C29/76C07C29/80C12Y101/01184C07B2200/07C07C31/207
Inventor 张城孝余梦娇朱莹托马斯·道斯曼蔡宝琴章兆琪洪瑞梅方茂海
Owner ENZYMASTER NINGBO BIO ENG CO LTD
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