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Method for preparing L-tertiary leucine based on biological brick tandem double enzymes

A technology of tert-leucine and bio-bricks, which is applied in the direction of introducing foreign genetic material using carriers, recombinant DNA technology, bacteria, etc., can solve the problems of low utilization efficiency, unfavorable production costs, and low theoretical yield, and achieve cost savings , low cost, good chiral selectivity

Inactive Publication Date: 2015-12-16
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the theoretical yield of the former ideal product L-tert-leucine is lower than 50%, and the latter, although the theoretical yield is high, requires the addition of a large amount of expensive coenzyme NADH
Krix et al. used formate dehydrogenase to couple leucine dehydrogenase to realize the regeneration of expensive coenzymes. However, the input of coenzymes in this method is as high as 2mM, and the economy is poor.
In the technical scheme disclosed by CN102978251.A, the input amount of the enzyme used accounts for 4% of the substrate, and the amount of the enzyme is too large, the utilization efficiency is not high, and it is not economical
Weiming Liu et al. used double plasmids to express formate dehydrogenase and leucine dehydrogenase to realize the regeneration of coenzymes. However, this method needs to use double antibiotics, which is not conducive to protecting the environment and saving production costs, and the economy is poor

Method used

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  • Method for preparing L-tertiary leucine based on biological brick tandem double enzymes
  • Method for preparing L-tertiary leucine based on biological brick tandem double enzymes
  • Method for preparing L-tertiary leucine based on biological brick tandem double enzymes

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Embodiment 1

[0037] A method for preparing L-tert-leucine based on biological bricks in series with double enzymes, comprising the steps of:

[0038] (1) Construction of tandem biological brick elements capable of tandem expression of leucine dehydrogenase and formate dehydrogenase (such as figure 1 shown), wherein the gene sequence of leucine dehydrogenase is shown in SEQID01, and the gene sequence of formate dehydrogenase is shown in SEQID02, specifically including:

[0039] 1) constructing the first biological brick element capable of expressing the leucine dehydrogenase alone, specifically:

[0040]a. Using the plasmid pUC18-leudh containing the leucine dehydrogenase gene as a template, PCR amplification was performed with primers LeuDH-F1 and LeuDH-R1 to obtain the leucine dehydrogenase gene sequence, wherein LeuDH-F1 and LeuDH- R1 is shown in SEQID3 and SEQID4 respectively;

[0041] b. Digest the above leucine dehydrogenase gene sequence and terminator B0015 with EcoRI and SpeI res...

Embodiment 2

[0053] Step (1) to step (3) are the same as embodiment 1, and step (4) is as follows:

[0054] The above-mentioned cell fluid, trimethylpyruvate, amino donor, coenzyme and auxiliary substrate for coenzyme regeneration are placed in a buffer system with pH = 7.0 for shaking reaction, and the product L-tert. Leucine, the specific reaction system is: 0.030mol / L trimethylpyruvate, 1mol / LNH 4 Cl-NH 3 Buffer system, 0.005mL 1mM NADH, the volume of the above reaction is 15mL, the reaction temperature is 37°C, the reaction time is 8h, and the shaking rate is 180rpm. The reacted material is centrifuged to remove the precipitate, the supernatant is added with an equal volume of methanol, mixed and oscillated evenly, the precipitate is discarded by centrifugation, and the supernatant is diluted for later use. Detected by high performance liquid chromatography, the calculated yield of the product L-tert-leucine is 90.82%, and the e.e. value is greater than 99.0%.

Embodiment 3

[0056] Step (1) to step (3) are the same as embodiment 1, and step (4) is as follows:

[0057] The above-mentioned cell fluid, trimethylpyruvate, amino donor, coenzyme and auxiliary substrate for coenzyme regeneration are placed in a buffer system with pH = 7.0 for shaking reaction, and the product L-tert. Leucine, the specific reaction system is: 5% ammonia water, 0.060mol / L trimethylpyruvate, 1mol / LNH 4 Cl-NH 3 Buffer system, 0.005mL 1mM NADH, the volume of the above reaction is 15mL, the reaction temperature is 37°C, the reaction time is 12h, and the shaking rate is 180rpm. The reacted material is centrifuged to remove the precipitate, the supernatant is added with an equal volume of methanol, mixed and oscillated evenly, the precipitate is discarded by centrifugation, and the supernatant is diluted for later use. Detected by high performance liquid chromatography, the calculated yield of the product L-tert-leucine is 85.39%, and the e.e. value is greater than 99.0%.

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Abstract

The invention discloses a method for preparing L-tertiary leucine based on biological brick tandem double enzymes. The method comprises the following steps: (1) constructing a tandem biological brick element capable of realizing tandem expression of leucine dehydrogenase and formate dehydrogenase; (2) introducing the tandem biological brick element into Escherichia coli (E.coli) so as to construct E.coli engineering bacteria based on tandem expression of leucine dehydrogenase and formate dehydrogenase; (3) inoculating the E.coli engineering bacteria into a liquid enlarged culture medium containing chloromycetin for carrying out culture and induced expression to obtain fermentation liquid, carrying out refrigerated centrifugation to obtain cells, and re-suspending and washing with a buffer solution so as to obtain cell sap; and (4) putting the cell sap, trimethylpyruvic acid, amino donors, coenzymes and cosubstrates for regeneration of the coenzymes in a buffer system for carrying out vibration reaction, and carrying out whole-cell catalysis asymmetric reductive amination to obtain the product, namely L-tertiary leucine. The method disclosed by the invention has the advantages that the product conversion rate is high, the chiral selectivity is better, the reaction conditions are mild, the operation is simple, the expensive coenzymes can be regenerated, and the cost is saved by expressing the double enzymes with single cells.

Description

technical field [0001] The invention belongs to the technical field of L-tert-leucine preparation, and in particular relates to a method for preparing L-tert-leucine based on a biological brick tandem double enzyme. Background technique [0002] As a non-natural chiral amino acid, L-tert-leucine has been widely used in the chemical, food and pharmaceutical industries because of its large steric hindrance due to its hydrophobic tert-butyl group, which makes it easier to control the molecular conformation in organic synthesis. , especially L-tert-leucine is used as a chiral drug intermediate for the synthesis of biological inhibitors, anti-virus, anti-cancer, etc. Therefore, L-tert-leucine has high commercial application value. For example, the anti-HIV drug Atazanavir (Atazanavir) synthesized by Bristol-Myers Squibb Company using L-tert-leucine as a drug intermediate has a sales volume of 1.4 billion US dollars in 2009 . [0003] L-tert-leucine can be obtained by chemical s...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12N15/70C12N1/21
Inventor 方柏山江伟
Owner XIAMEN UNIV
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