Method for synthesizing (R)-2-hydroxy acid by biological enzyme method

Abstract

The invention discloses a method for synthesizing (R)-2-hydroxy acid by a biological enzyme method. The method comprises the following steps of taking a wet cell mass obtained by fermenting a recombinant genetic engineering strain as a catalyst, racemization 2-hydroxy acid shown in a formula (I) as a substrate, glucose as a cosubstrate, and a buffer solution of which the pH value is 6.0 to 9.0 as a reaction medium to form a reaction system; performing a conversion reaction under the conditions that the temperature is 20 to 50DEG C and the rotating speed is 150rpm; after the reaction is complete, separating and purifying a reaction solution to obtain the (R)-2-hydroxy acid, wherein the recombinant genetic engineering strain is recombinant escherichia coli containing 2-hydroxy acid dehydrogenase (HADH) genes, carbonyl reductase KAR genes and glucose dehydrogenase (GDH) genes. According to a single-bacteria, double-plasmid and trienzyme co-expression system disclosed by the invention, the conversion between the racemization 2-hydroxy acid and single-configuration (R)-2-hydroxy acid through an engineering strain is innovatively and successfully realized, the culture of multiple bacteria is avoided, the total concentration of bacteria is reduced, the reaction process is simplified, and extraction steps of an intermediate product are reduced; in addition, the catalytic efficiency is also remarkably improved; the system is more suitable for industrial application of a cascade catalysis system.

Description

(1) Technical field [0001] The present invention relates to a preparation method of (R)-2-hydroxyacid, in particular to a method for producing (R)-2 from racemized 2-hydroxyacid by co-expression of single bacterium double plasmid and three enzymes in series redox cascade system - Hydroxy acid method. (2) Background technology [0002] 2-hydroxycarboxylic acid compounds (2-hydroxyacids) are a class of compounds with similar structures, and their structural feature is that there is a hydroxyl group at the C1 position of the carboxyl side. 2-Hydroxycarboxylic acid is very active because it has two functional groups of ketone and hydroxyl. Optically pure 2-hydroxyacid chiral modules are important fine chemical intermediates and chiral drug precursors, and 2-hydroxyacid enantiomers are also important chiral resolution agents and chiral catalysts. Catalysts for the asymmetric conversion of aliphatic and aromatic aldehydes to the corresponding chiral alcohols. Optically active 2...

AI Technical Summary

Problems solved by technology

[0008] ①Diastereomeric salt crystallization resolution method, the common problem faced is that it is expensive and has certain toxicity, which causes waste of resources and environmental pollution to a certain extent. At present, R-mandelic acid produced on a large scale in my country uses The method produces

[0009] ② Chromatographic separation method, the equipment cost of this method is too high, the consumption is large, the cost is too high, and the processing capacity is small, so it is limited to detection and laboratory preparation, and cannot be used for commercial production

[0011] ④The capillary electrophoresis separation method is widely used in the separation of various drug enantiomers due to its high efficiency, rapidity, and economy. This method has disadvantages such as high cost

[0013] ① Enzymatic resolution, enzymatic resolution is a kind of kinetic resolution method, the most commonly used is to degrade one enantiomer in the racemate, leaving another configuration to achieve resolution Purpose, since this method obtains the desired enantiomer by selectively degrading one of the configurations, the conversion rate is limited and cannot exceed 50%, which has certain limitations

The advantage of this method is high theoretical yield, simple operation, etc. The disadvantage is that the reaction process generally requires the addition of coenzyme, which is expensive and greatly increases production costs, which is not conducive to industrial applications.

Images