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Method for preparing highly pure L-tertiary leucine through biological process

A tertiary leucine biosynthesis technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of high price and high cost, and achieve the effect of low cost, low pollution and high selectivity

Active Publication Date: 2014-08-06
山东斯递尔化工科技有限公司
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  • Abstract
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Problems solved by technology

Because the reagents triethyloxonium tetrafluoroborate, N-chlorosuccinimide and tert-butyllithium are expensive, the cost is high; in addition, the industrial scale preparation is prone to produce harmful oxides

Method used

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  • Method for preparing highly pure L-tertiary leucine through biological process
  • Method for preparing highly pure L-tertiary leucine through biological process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the construction of two kinds of recombinant bacteria containing leucine dehydrogenase gene and formate dehydrogenase gene

[0027] Genomic DNA was extracted from Bacillus cereus and Candida boidinii respectively, and the target gene fragments of fdh (formate dehydrogenase) and LeuDH (leucine dehydrogenase) were obtained by PCR method respectively. Gene fragments; then, express the fdh target gene and LeuDH target gene through the plasmid pETDuet-1. For detailed operation methods, see Organic Process Research & Development, 2006, 10, 666-669.

Embodiment 2

[0028] Embodiment 2, self-induced fermentation culture of genetically engineered bacteria (two kinds of recombinant bacteria culture methods are the same). Add 100 μl of genetically engineered bacteria seed solution into 10 ml LB medium containing 100 μg / ml kanamycin, and cultivate overnight at 37° C. and 200 rpm. Take 400 μl of the bacterial solution in the logarithmic growth phase and insert it into 40 ml of the following medium containing 100 μg / ml kanamycin, and incubate at 37° C., 200 rpm for 12 hours. The medium composition is: 1% tryptone, 0.3% yeast extract, 25mM Na 2 HPO 4 , 25mM KH 2 PO 4 , 50mM NH 4 Cl, 5mM Na 2 SO 4 , 2mM MgSO 4 , 1.0% glycerol, 0.05% glucose, 0.25% lactose, 100 μM FeCl 3 .

Embodiment 3

[0029] Example 3, preparation of the recombinant cell suspension: the fermented broth was centrifuged (10000g, 5 minutes, 4° C.) to obtain crude bacteria, washed and suspended with pH6.0-7.0 phosphate buffer, used for the following experiments or stored in- 20°C for use.

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Abstract

The invention discloses a method for preparing highly pure L-tertiary leucine through a biosynthesis process. The method comprises the following steps: inoculating an Escherichia coli seed liquid expressing leucine dehydrogenase gene and formic dehydrogenase gene into in a fermenting culture medium of a self-induction system, carrying out fermenting culture, centrifuging to obtain crude thalli, and adding a buffer solution to obtain a cell suspension; adding substrates comprising trimethylpyruvic acid and ammonium formate, and carrying out a biological catalysis reaction to obtain an trimethylpyruvic acid conversion solution; and centrifuging the conversion solution, adjusting the pH value of the solution, carrying out column chromatography, precipitating, and concentrating to obtain highly pure L-tertiary leucine. The method fully uses the high effectiveness, the specificity and the mildness of an enzyme; and compared with traditional chemical synthesis methods, the method disclosed in the invention has the advantages of high selectivity, mild conditions, simple process, low cost, small pollution and the like.

Description

technical field [0001] The invention relates to a method for preparing L-tert-leucine by biosynthesis. Background technique [0002] L-tert-leucine is prepared to produce antiviral drugs such as Atazanavir (anti-HIV drug), Boceprevir (anti-HCV drug), and Telaprevir (anti-HCV drug). It is a drug intermediate. At the same time, due to the large steric hindrance of tert-butyl, tert-leucine is used as a chiral metal ligand for asymmetric synthesis. [0003] At present, the preparation methods of L-tert-leucine mainly include chemical synthesis and biosynthesis. Among them, chemical synthesis mainly includes three methods: chemical synthesis, chemical resolution and chiral synthesis. In 1993, U.Groth et al. used 2,5-diethylpyrazine as raw material (Liebigs Ann.Chem.1993, 715-719) to synthesize tert-leucine ethyl ester through tert-butylation and hydrolysis, with a yield of 70 %. Because the reagents triethyloxonium tetrafluoroborate, N-chlorosuccinimide and tert-butyllithium ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C12R1/19
Inventor 朱嘉震史峻嵩李海存王颖赵新远尹传祥王金才
Owner 山东斯递尔化工科技有限公司
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