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Aminoketone asymmetric reductase and nucleic acid thereof

An amino acid and amino ketone technology, applied in the directions of oxidoreductase, genetic engineering, plant genetic improvement, etc., can solve the problems of unfavorable economy, low yield, and many processes.

Inactive Publication Date: 2008-12-24
DAIICHI FINE CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] As mentioned above, in order to realize the production of pseudoephedrine with desired optical activity by 1-phenyl-2-methylamino-1-propanol, once the ephedrine of the optically active red complex is obtained, it needs to be reversed to Therefore, there are many and complicated procedures and the problems of low yield
[0008] Moreover, in the above-mentioned manufacture of pseudoephedrine, on the one hand, when the raw material ketone body is reduced, considerable diastereomer (stereo) isomer by-products are produced, on the other hand, as diastereomer (stereo) isomer It is difficult to recover the raw materials of the body, and it is also unfavorable economically.
[0009] In addition, although according to the method described in JP-A-8-98697, the optically active 2-amino-1-phenylethanol compound can be produced from a 2-amino-1-phenethyl alcohol compound having an asymmetric carbon atom in the molecule and a specific microorganism can be used to produce optically active 2-amino-1- Phenylalcohol derivatives, but for β-aminoalcohols with two asymmetric carbon atoms, there is currently no efficient way to make them

Method used

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  • Aminoketone asymmetric reductase and nucleic acid thereof
  • Aminoketone asymmetric reductase and nucleic acid thereof
  • Aminoketone asymmetric reductase and nucleic acid thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0222] (refinement of aminoketone asymmetric reductase)

[0223] Into a test tube containing 5 ml of medium with a composition (pH 7.0) of 1% glucose, 0.3% dipotassium hydrogen phosphate, 0.02% magnesium sulfate heptahydrate, 1.5% peptone, 0.2% sodium chloride, and 0.1% yeast extract Rhodococcus erythropolis MAK-34 was implanted, and shaking culture was carried out at 28°C for 3 days, and the obtained test sample was inoculated into 500 ml medium having the above composition, and shaking culture was carried out at 28°C for 3 days. This pre-culture solution was inoculated into a 501 medium having the above-mentioned composition supplemented with 0.1% of 1-amino-2-hydroxypropane, and cultured at 28° C. for 2 days.

[0224] The above-mentioned culture solution was processed with a centrifuge to obtain 3785 g of wet bacteria. In 1 kg of the thalli, add 1000 ml (pH 8.5) of 10 mM tris hydrochloric acid buffer solution containing 10% glycerin, 1 mM nickel chloride, 1 mM nicotinic ac...

Embodiment 2

[0238] (Cloning of Aminoketone Asymmetric Reductase Gene and Discovery in Escherichia coli)

[0239] (1) Determination of the amino acid partial sequence of the refined enzyme

[0240] Dissolve 1 nmol of lyophilized aminoketone asymmetric reductase (subunit molecular weight about 28,500) in 50 μl of 50 mM Tris hydrochloride buffer (pH 8.6) containing 8 M urea, and heat at 37 ° C 1 hour to get modified. Then 50 μl of 50 mM tris hydrochloride buffer solution (pH 8.6) was added to make the urea concentration 4M. Add 0.5 μl (0.006 nmol, E / S=1 / 167) of lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.), and digest at 30° C. for 6 hours. The obtained digested peptides were fractionated by a reverse phase column (Amersham Biosciences), and the amino acid sequence was analyzed with a 476A protein sequencer from ABI Company. The result is as follows.

[0241] ①MFNSIEGRSVVVTGGSK (N-terminal) (sequence number 3)

[0242] ②RLGEMTSEDMDSVFGVNVK (serial number 4)

[0243] ③AAQMGF...

Embodiment 3

[0329] (Formation of d-(1S,2S)-pseudoephedrine)

[0330] Each of the microorganisms shown in Table 1 was implanted in 5 ml of medium containing 1% glucose, 0.5% peptone, and 0.3% yeast extract, and cultured with shaking at 30° C. for 48 hours. After centrifuging the culture solution to obtain bacterial cells, the bacterial cells were placed in a test tube, and 1 ml of a 0.1 M sodium phosphate buffer solution (pH 7.0) was added thereto to suspend them. Furthermore, 1 mg of d 1-2-methylamino-propiophenone hydrochloride was added to the suspension, and the mixture was shaken at 30° C. for 24 hours to perform a reaction. After the reaction was finished, the reaction solution was centrifuged to remove the thalline, and the supernatant was added to HPLC to obtain optically active pseudoephedrine (made by μBondapakphenyl Waters, 4mm in diameter and 300mm long, and the eluent was 0.05M sodium phosphate buffer solution ( Contains acetonitrile 7%), pH 5.0, flow rate 0.8ml / min, detectio...

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Abstract

A protein, which is an aminoketone asymmetric reductase, having an effect of producing d-pseudoephedrine by acting on l-2-methylaminopropiophenone, and having the following physiochemical properties: substrate: l-2-methylaminopropiophenone optimum pH: pH 8.1 optimum temperature: 55 DEG C coenzyme: NADP molecular weight: about 28500 Da homotetramer and a nucleic acid coding the protein.

Description

technical field [0001] The present invention relates to an aminoketone asymmetric reductase, a nucleic acid encoding the enzyme, a microorganism producing the enzyme, and a method for producing optically active aminoalcohols using them. Background technique [0002] Ephedrine has been used since ancient times for sweating, antipyretic, antitussive and other purposes, and it is also known that d-pseudoephedrine has anti-inflammatory effects. In addition, it is known that 1-ephedrine has pharmacological effects such as vasoconstriction, blood pressure raising effect and sweating, so it is used medically as a sympathetic nerve stimulant. Moreover, 1-ephedrine can also be used for the treatment of bronchial asthma. That is, a method for producing optically active β-aminoalcohols containing optically active ephedrine is important in the production of pharmaceuticals or their intermediates, and thus an efficient production method is desired. [0003] At present, as a method for ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N1/15C12N1/19C12N1/21C12N5/10C12P7/02C12N9/04C12P13/00
CPCC12N9/0006C12P13/001C12P13/008C12N9/0004
Inventor 坂本惠司北伸二津崎和也森川忠则清水昌片冈道彦
Owner DAIICHI FINE CHEM CO LTD
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