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L-threonine aldolase mutant R318M and application thereof

A technology of threonine aldolase and mutant, which is applied in the field of L-threonine aldolase mutant R318M, can solve the problems of low diastereoselectivity, hindering application and the like

Active Publication Date: 2020-10-30
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The low diastereoselectivity greatly hinders the application of this enzyme in the synthesis of L-threo-DOPS

Method used

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  • L-threonine aldolase mutant R318M and application thereof
  • L-threonine aldolase mutant R318M and application thereof
  • L-threonine aldolase mutant R318M and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Preparation of L-threonine aldolase R318M mutant

[0043] 1. Design of L-threonine aldolase mutants

[0044] The wild-type L-threonine aldolase gene (L-TA gene, SEQ ID NO: 2) was isolated from feces samples of healthy black bears in the Sichuan Black Bear Protection and Incubation Base in our laboratory. The L-TA gene The full length of the open reading frame is 1032bp, and the encoded L-threonine aldolase consists of 343 amino acids. The isolation and cloning process of the gene is described in the patent application text with the application number 2019109657806, the publication number CN110592058A, and the invention title "threonine aldolase, its coding gene and its application in the biosynthesis of droxidopa" , which is incorporated herein by reference in its entirety.

[0045] The amino acid sequence (343aa) of the wild-type L-threonine aldolase is:

[0046] MYSFKNDYSEGAHPRILETLLRTNLEQCEGYGKDTYCEEAENLIKNKLNNESIEVHFISGGTQTNLIAISAFLRPHEGVISADTGHIFVNEAG...

Embodiment 2

[0093] Example 2. Utilize L-TA R318M mutant to synthesize droxidopa and detect de value

[0094] 1. Synthesis of droxidopa

[0095] The substrate solution was prepared in 10 mM PBS at pH 7.4, containing 1 M glycine, 60 mM 3,4-dihydroxybenzaldehyde and 50 μM pyridoxal 5-phosphate. Add 20 U of the L-TA R318M enzyme prepared in Example 1 to each ml of the substrate solution, mix well, place at 25° C. for 2 h, and then obtain a sample by ultrafiltration (10 kDa, 4000×g, 30 min).

[0096] 2. Detection of droxidopa and calculation of de value

[0097] The sample obtained after ultrafiltration in step 1 was diluted 10 times to achieve better HPLC separation effect. The amount of droxidopa (L-threo-DOPS) and its diastereoisomer (L-erythro-DOPS) in the sample was detected by HPLC. HPLC instrument model: Agilent 1260. Parameter settings: Chromatographic column: COSMOSIL 5C18-MS 4.6×150mm; wavelength 280nm, column temperature 30°C; mobile phase: 90% 0.1% (w / v, g / mL) 1-heptanesulfonic...

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Abstract

The invention relates to threonine aldolase, in particular to an L-threonine aldolase mutant R318M and application thereof. The amino acid sequence of the L-threonine aldolase mutant is shown as SEQ ID NO: 3, and the L-threonine aldolase mutant is obtained by changing the 318th amino acid of L-threonine aldolase with the amino acid sequence of SEQ ID NO: 1 from Arg to Met. The mutant has diastereoselectivity (de) of 77.1% in a reaction of catalyzing 3, 4-dihydroxy benzaldehyde and glycine to synthesize droxydopa [L-threonine-(3, 4-dihydroxy) phenylserine], the diastereoselectivity (de) is morethan 2.5 times that of a wild type, and the mutant has huge application potential in industrial production of biosynthesis of droxydopa.

Description

technical field [0001] The invention relates to threonine aldolase, in particular to an L-threonine aldolase mutant R318M and its application. Background technique [0002] Droxidopa (L-threo-DOPS, L-threo-3,4-dihydroxyphenylserine) has a β-hydroxy amino acid with two chiral centers, and is an anti-Parkinson's disease drug approved by the FDA in 2014 for improving Stiffness of gait and orthostatic dizziness caused by Parkinson's disease; improvement of orthostatic hypotension, orthostatic dizziness and fainting caused by Shy-Drager syndrome or familial amyloid neuropathy; improvement of orthostatic hypotension in hemodialysis patients dizziness and fatigue. [0003] At present, the synthesis methods of droxidopa mainly include chemical synthesis and enzyme catalysis. Among them, the chemical synthesis method mainly obtains chiral droxidopa through addition reaction, esterification reaction and chemical resolution. Although the operation is simple, a large amount of water, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12N15/70C12P13/04C12Y401/02005
Inventor 王伯初赵文艳潘银平杨碧玲祝连彩戚娜
Owner CHONGQING UNIV
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