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Application of L-threonine transaldolase to synthesis of florfenicol chiral intermediates

A technology of threonine and transaldolase, which is applied in the fields of biotechnology and chemical engineering, can solve the problems of cumbersome production steps, large environmental pollution, and high production costs, and achieve the effects of short reaction time, reduced production costs, and mild reaction conditions

Active Publication Date: 2019-12-06
福建昌生生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide an application of L-threonine transaldolase in the synthesis of florfenicol chiral intermediates, and to use the L-threonine transaldolase to catalyze the synthesis of florfenicol chiral intermediates The method step, will solve the technical problems such as the complex production steps of (2S,3R)-p-thymphenylphenylserine in the prior art, low efficiency, high production cost, big environmental pollution

Method used

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  • Application of L-threonine transaldolase to synthesis of florfenicol chiral intermediates
  • Application of L-threonine transaldolase to synthesis of florfenicol chiral intermediates
  • Application of L-threonine transaldolase to synthesis of florfenicol chiral intermediates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] In this example, L-threonine aldolase derived from recombinant Escherichia coli was expressed by means of genetic engineering, and L-threonine aldolase was obtained; at the same time, whole cells were used as carriers to asymmetrically catalyze p-thiamphenicol Synthesis of (2S,3R)-p-thymphenylphenylserine from formaldehyde and L-threonine, by optimizing the reaction conditions, enantiomeric excess ee>99.9%, diastereomeric excess de=94.5%, substrate p-methylsulfone The conversion rate of phenylbenzaldehyde was 67.1%, and a set of L-threonine transaldolase was established to asymmetrically catalyze the synthesis of (2S,3R)-p-thiamphenylphenylserine from p-thiamphenylbenzaldehyde and L-threonine process conditions. The specific operation steps include:

[0036] (1) Obtain whole cells of L-threonine transaldolase by genetic engineering

[0037] A new L-threonine transaldolase gene (GenBankNo.CVTX01000156), SEQ ID NO: 2, was screened through gene mining, and the gene sequenc...

experiment example 1

[0060] The influence of experimental example 1 different metal ions (10mM) on LTTA catalytic activity

[0061] In order to explore the effect of different metal ions on the catalytic activity of LTTA, the following experiments were designed. The reaction system and conditions were the same as those in Example 1, except that the types of metal ions added were different, and the concentration of each metal ion was 10 mM. The experimental results are shown in Table 1 below. It can be seen that Mg, Cs, Li, Ca, and Ba can promote the catalytic activity of LTTA, and Mg has the best effect, and the enzymatic activity of LTTA has increased by 24%; Fe, Ni, Zn, and Cu can inhibit LTTA, and LTTA has different Under the condition of metal ions, the de value is 17%-81%.

[0062] Table 1 Effect of different metal ions on the activity of LTTA

[0063]

experiment example 2

[0064] Experimental Example 2 Effects of Different Substrate Concentrations and Cell Concentrations on LTTA Conversion Rate and De Value

[0065] Taking p-thiamphenicyl benzaldehyde and L-threonine as substrates, the effects of different substrate p-thiamphenic benzaldehyde concentrations (10-40mM) and cell concentrations (3.0-50mg / mL) on LTTA conversion and de Value, the reaction system and conditions are the same as in Example 1, the difference is that the reaction temperature in this experimental example is 30 ° C, and the addition of organic solvent is 10% acetonitrile. The experimental results are attached image 3 shown. It can be seen that the de value of the product decreases with the increase of the cell concentration. When the cell concentration is 3.0 and 6.25mg / mL, the de value of the product does not change significantly (about 85%). When the cell concentration is greater than 6.25mg / mL , the de value of the product begins to decline (from 85% to 73%); when the ...

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Abstract

The invention provides application of L-threonine transaldolase to synthesis of florfenicol chiral intermediates. The L-threonine transaldolase is selected from any one of the following groups that (1) polypeptide contains an amino acid sequence shown in SEQ ID NO:1; (2) polypeptide contains an amino acid sequence greater than or equal to 90% homology shown in SEQ ID NO:1, and the polypeptide hascatalytic activity; and (3) derived polypeptide is formed by substitution, deletion or addition of 1-5 amino acid residues to the amino acid sequence shown in SEQ ID NO:1 and with the retention of catalytic activity. According to the application, new L-threonine transaldolase genes are screened through gene mining, the L-threonine transaldolase derived from recombinant escherichia coli is expressed by adopting a genetic engineering method, methylsulfonyl benzaldehyde and the L-threonine transaldolase are used as raw materials, (2S,3R)-methylsulfonyl phenylserine is synthesized through a wholecell catalyzed reaction, fluorfenicol key chiral synthesis blocks can be obtained by a one-step reaction under the room temperature and pressure, environmental protection is achieved, and the application is an environment-friendly biosynthetic pathway.

Description

technical field [0001] The invention belongs to the fields of biotechnology and chemical engineering, and in particular relates to the application of L-threonine transaldolase in the synthesis of florfenicol chiral intermediates. Background technique [0002] (2S,3R)-P-thymphenylphenylserine is the key chiral building block for the synthesis of Florfenicol. Florfenicol is a β-aminoalcohol antibiotic. It has the advantages of rapid absorption, wide distribution in the body, long half-life, no aplastic anemia side effects, no drug resistance, no residue, and no cross-resistance. It is an alternative to chloramphenicol. and thiamphenicol, a new generation of chloramphenicol antibiotics. It is mainly used to treat bacterial diseases and mycoplasma infections in pigs, chickens and fish caused by sensitive bacteria, and has unique curative effects on diseases caused by Escherichia coli in livestock and poultry, porcine infectious pleuropneumonia and panting disease. In recent ye...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P13/04
CPCC12N9/1014C12P13/04C12Y201/02001
Inventor 林娟许炼王力超陈承滔赖凌燕
Owner 福建昌生生物科技发展有限公司
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