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High-throughput rapid screening method for L-threonine transaldolase mutant

A screening method and threonine technology, applied in the fields of biochemistry and enzyme engineering, can solve problems such as low efficiency, time-consuming and laborious, and achieve the effects of good repeatability, high sensitivity and accuracy, and easy popularization and application.

Pending Publication Date: 2020-03-24
福建昌生生物科技发展有限公司
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Problems solved by technology

[0006] Aiming at the problem that screening L-threonine transaldolase mutants in the prior art is time-consuming, laborious, and inefficient, and not suitable for high-throughput rapid screening of L-threonine transaldolase mutants, the present invention provides a A high-throughput rapid screening method for L-threonine transaldolase (LTTA) mutants, as follows:

Method used

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  • High-throughput rapid screening method for L-threonine transaldolase mutant

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Embodiment Construction

[0030] Preferred embodiments of the present invention are provided below to help further understanding of the present invention. Those skilled in the art should understand that the descriptions of the embodiments of the present invention are only exemplary, and are not intended to limit the solution of the present invention.

[0031] Step 1. Reagent preparation

[0032] L-threonine transaldolase whole cell reaction solution: Randomly pick mutant single clones from the L-threonine transaldolase mutation library, inoculate into 48-well deep-well plates (0.3mL LB liquid medium, containing 50μg / mL kanamycin) at 37°C, 180rpm overnight. The next day, transfer 50 μL of cell culture solution to another 48-well deep-well plate (containing 950 μL LB liquid medium, 50 μg / mL kanamycin), and culture at 37° C. for 2-3 hours with shaking at 200 rpm. Add IPTG to a final concentration of 0.1 mM, and stop culturing after induction at 30°C for 8 hours. Centrifuge at 4000 rpm for 10 min at 4°...

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Abstract

The invention discloses a high-throughput rapid screening method for L-threonine transaldolase mutant, and belongs to the fields of biochemistry and enzyme engineering. According to the invention, acetaldehyde and ethanol dehydrogenase reaction solutions are taken and then placed in a microplate reader to detect the change of OD340nm with time, and the average reaction rate VOD340nm of OD340nm decline is calculated. Taking acetaldehyde concentration as the independent variable x and VOD340nm as the dependent variable y, the regression equation y=ax and the acetaldehyde standard curve are obtained. After fully mixing and reaction of a transaldehyde reaction solution and an L-threonine transaldolase mutant whole cell reaction solution, the cell precipitate is removed by centrifugation, the supernatant and ethanol dehydrogenase reaction solution are taken and then placed into a microplate reader, the change of OD340nm with time is detected, and the average rate of OD340nm decline VOD340nmis calculated. The concentration of acetaldehyde in the reaction solution is calculated by VOD340nm, and the L-threonine transaldolase mutant is screened by the concentration of acetaldehyde. The method is not interfered by impurities in the reaction liquid, has high sensitivity and accuracy and good repeatability, is simple to operate, and is convenient for promotion and application.

Description

technical field [0001] The invention belongs to the fields of biochemistry and enzyme engineering, and in particular relates to a high-throughput rapid screening method for L-threonine transaldolase mutants. Background technique [0002] L-threonine transaldolase (LTTA) is a serine hydroxymethyltransferase family protein, which can asymmetrically catalyze the synthesis of (2S,3R)-p-thiamphenicol benzaldehyde and L-threonine . (2S,3R)-P-thiamphenicol phenylserine is a key chiral intermediate for the synthesis of β-aminoalcohol antibiotics thiamphenicol and florfenicol. In recent years, the demand for florfenicol has been increasing, ranking among One of the top 10 export drugs. [0003] The catalytic efficiency of LTTA that has been reported so far is low (the maximum conversion substrate concentration is only 20mM), which cannot meet the requirements of industrial production. Natural enzymes generally have problems such as poor stereo / regioselectivity, narrow substrate sp...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C12Q1/32
CPCC12Q1/32C12Q1/48G01N2333/9104
Inventor 林娟许炼王力超许鑫琦陈承滔赖凌燕
Owner 福建昌生生物科技发展有限公司
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