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L-threonine aldolase mutant R318L and application thereof

A technology of threonine aldolase and mutants, which is applied to the L-threonine aldolase mutant R318L and its application fields, and can solve the problems of low diastereoselectivity and hindering applications

Active Publication Date: 2020-07-10
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 R318L and application thereof
  • L-threonine aldolase mutant R318L and application thereof
  • L-threonine aldolase mutant R318L and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Preparation of L-threonine aldolase R318L mutant

[0040] 1. Design of L-threonine aldolase mutants

[0041] 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.

[0042] The amino acid sequence (343aa) of the wild-type L-threonine aldolase is:

[0043] MYSFKNDYSEGAHPRILETLLRTNLEQCEGYGKDTYCEEAENLIKNKLNNESIEVHFISGGTQTNLIAISAFLRPHEGVISADTGHIFVNEAG...

Embodiment 2

[0090] Example 2. Utilize L-TA R318L mutant to synthesize droxidopa and detect de value

[0091] 1. Synthesis of droxidopa

[0092] 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 R318L enzyme prepared in Example 1 to each milliliter of the substrate solution, mix well, place at 25° C. for 2 hours, and then obtain a sample by ultrafiltration (10 kDa, 4000×g, 30 minutes).

[0093] 2. Detection of droxidopa and calculation of de value

[0094] 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...

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Abstract

The present invention relates to threonine aldolase and particularly to an L-threonine aldolase mutant R318L and an application thereof. An amino acid sequence of the L-threonine aldolase mutant is shown in SEQ ID NO:3 and the L-threonine aldolase mutant is obtained by changing the 318th site amino acid of L-threonine aldolase with an amino acid sequence of SEQ ID NO:1 from Arg to Leu. The mutanthas a diastereoselectivity of 84.9% in catalyzing synthesis of droxidopa [L-threo-(3,4-dihydroxy)phenylserine] from 3,4-dihydroxybenzaldehyde and glycine, the diastereoselectivity of the mutant is 2.8 times that of the wild type, and the mutant has huge application potential in the biosynthesis of the droxidoca.

Description

technical field [0001] The invention relates to threonine aldolase, in particular to an L-threonine aldolase mutant R318L and its application. Background technique [0002] Droxidopa (L-threo-DOPS, that is, L-threo-(3,4-dihydroxy)phenylserine) is a new anti-Parkinson's disease drug, used to improve the symptoms caused by Parkinson's disease Stiffness of gait and orthostatic dizziness; improvement of orthostatic hypotension, orthostatic dizziness and fainting caused by Shy-Drager syndrome or familial amyloid neuropathy; improvement of dizziness and fatigue caused by orthostatic hypotension in hemodialysis patients. [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, heavy metals and highly toxic hydrogen sulfid...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/22C12R1/19
CPCC12N9/88C12N15/70C12P13/225C12Y401/02005
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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