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Method for screening stereoselective alpha-hydroxy acid dehydrogenase

A hydroxyacid dehydrogenase, stereoselective technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of time-consuming, laborious, and no stereoselective α-hydroxyacid dehydrogenase screening. Model, low work efficiency and other problems, to achieve the effect of simple screening process, fast speed and strong versatility

Inactive Publication Date: 2012-09-12
ZHEJIANG UNIV OF TECH
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  • Claims
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Problems solved by technology

[0010] At present, the screening of stereoselective α-hydroxyacid dehydrogenase biocatalysts still uses the traditional GC or HPLC method to detect a large number of microorganisms or enzymes one by one, which is time-consuming and laborious, resulting in low work efficiency; on the other hand, with the With the continuous development of protein directed evolution technology, in order to obtain mutants faster, it is particularly urgent to establish a method for rapid and effective characterization of these catalysts
So far, no screening model for stereoselective α-hydroxyacid dehydrogenase has been reported

Method used

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  • Method for screening stereoselective alpha-hydroxy acid dehydrogenase
  • Method for screening stereoselective alpha-hydroxy acid dehydrogenase
  • Method for screening stereoselective alpha-hydroxy acid dehydrogenase

Examples

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Embodiment 1

[0040] Example 1: Screening of microorganisms containing R-mandelate dehydrogenase

[0041] After culturing each microorganism in the microbial library to be screened, centrifuge, wash with physiological saline, and evenly disperse in phosphate buffer solution with pH=8.0 to prepare OD 600 =10.0 bacterial suspension, the reaction temperature is 30° C., carried out in a stoppered Erlenmeyer flask (50 mL).

[0042] The composition of the reaction solution: 1. Sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (10ml, 100mM, pH7.5); 2. Bacteria dispersed in the buffer solution (10ml, OD 600 =10.0); ③(R)-phenyllactic acid or S-phenyllactic acid (10ml, 10mM), the reaction was carried out in a water-bath shaker at 30°C and 150r / min, and 800μL of samples were taken every 30min, and the bacteria were removed by centrifugation. Pipette 10 μL of transformation solution into a 96-well plate, add 240 μL of chromogenic reagent (FeCl 3 ·6H 2 (2mmol / L, DMSO 400ml / L, gl...

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Abstract

The invention provides a method for screening stereoselective alpha-hydroxy acid dehydrogenase, comprising the following steps: (1) dissolving a sample to be detected containing alpha-hydroxy acid dehydrogenase in a buffer with pH of 6-9, adding an alpha-hydroxy acid chiral monomer as a substrate, and carrying out conversion reaction in water-bath at 20-50 DEG C; and (2) after the conversion reaction, adding the conversion solution in an FeCl3 developer solution to conduct color development reaction for 5-30 min, when the color development reaction finishes, judging the result according to the color appeared in the reaction solution. The method has no need to carry out derivatization on the substrate which is intend to screen, can rapidly identify the dehydrogenase activity and optical selectivity of the selected microbe by using simple colourimetry, has the advantages of simple screening flow, fast speed, low request for devices, strong versatility and the like, and offers great conveniences to the obtainment of optically pure products by using racemic mandelic acid and related derivatives as substrates to carry out resolution through biological enzymatic method.

Description

(1) Technical field [0001] The present invention relates to a screening method for stereoselective alpha-hydroxyacid dehydrogenase. (2) Background technology [0002] Stereoselective α-hydroxyacid dehydrogenase is a special class of oxidoreductase, which can stereoselectively catalyze one isomer in racemic α-hydroxycarboxylic acid compounds to generate the corresponding α-ketoacid, the reaction Another isomer (see the formula below) is left in the liquid, and the optically active α-hydroxyacid and ketoacid in the product have a large difference in properties and are easy to separate. The enzyme can be used to produce chiral α-hydroxyacids and ketoacids, and has good development and application prospects. [0003] [0004] It is of great significance to use α-hydroxyacid dehydrogenase to resolve racemic α-hydroxyacids to produce chiral α-hydroxyacids and broaden the production methods of optically active compounds. In recent years, some studies have focused on the separa...

Claims

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Application Information

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IPC IPC(8): C12Q1/32
Inventor 郑裕国薛亚平王威沈寅初
Owner ZHEJIANG UNIV OF TECH
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