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L-threonine aldolase mutant and application of L-threonine aldolase mutant in synthesis of L-synn-p-methylsulfonylphenylserine

A technology of thiamphenylphenylserine and threonine aldolase, which is applied in the field of enzyme engineering, can solve the problems of complex production process, complex process, and low resolution yield, and achieve simple production process, simple separation and purification, and atomic The effect of high utilization

Active Publication Date: 2022-03-04
宁波泓森生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process mainly has the following disadvantages: the highest theoretical yield is only 50%, the invalid enantiomer is difficult to apply mechanically, the process is more complicated, and an equimolar amount of chiral resolving agent needs to be used at the same time, and the single resolution yield is low. Recycling and reacting copper salts will generate a lot of waste water and waste salt
This process has the following disadvantages: the production process is complicated, the utilization rate of atoms is not high, the theoretical maximum is only 50%, and there will also be problems such as copper salts polluting the environment.
However, the activity of L-threonine aldolase reported so far is not high, and the catalytic efficiency is not ideal, which limits the large-scale application of biological preparation of L-syn-p-thymphenylphenylserine
Therefore, there is currently no research report on the direct application of highly active L-threonine aldolase to synthesize L-syn-p-thymphenylphenylserine with high chiral purity

Method used

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  • L-threonine aldolase mutant and application of L-threonine aldolase mutant in synthesis of L-synn-p-methylsulfonylphenylserine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] 1. Construction of wild enzyme engineering bacteria

[0044] Enter keywords such as threoninealdolase in the National Coalition Building Institute (NCBI) database to retrieve and select the amino acid sequence WP_016204489.1 (derived from Bacillus nealsonii (BnLTA)) encoding L-threonine aldolase. The amino acid sequence was converted into a nucleotide sequence according to the codon preference of Escherichia coli (the nucleotide sequence is shown in SEQ ID NO.1), and the amino acid sequence was shown in SEQ ID NO.2. The nucleotide sequence was fully synthesized by chemical method (Anhui General Biology), and integrated between the multiple cloning sites BamH I and HindIII of the expression vector pET-28a. Finally, the constructed plasmid was introduced into Escherichia coli BL21 (DE3) to construct wild-type L-threonine aldolase engineering bacteria.

[0045] 2. Construction of mutant enzymes

[0046] 2.1 Activation of engineering bacteria and plasmid extraction

[00...

Embodiment 2

[0075] Example 2 Preparation of L-syn-p-thymphenylphenylserine by L-threonine aldolase and its mutants in aqueous solution

[0076] The engineering bacteria capable of expressing L-threonine aldolase and its mutants were cultivated according to the method in Example 1 to obtain crude enzyme liquid. Quantitatively weigh 0.1M p-thymphenylbenzaldehyde and 1M glycine into a 1L reactor, use 100mM pH 8.0NaOH-Gly buffer solution to make up to 1L, the final concentration of pyridoxal phosphate is 1μM, and the concentration of wet bacteria is 5g / L. The reaction temperature was controlled by a water bath at 30°C and magnetically stirred. After 10 minutes of reaction, the concentrations of substrates and products were detected to determine the enzyme activity and conversion rate, and the de value of L-syn-p-thymphenylphenylserine was determined. The data at the end of the reaction are shown in Table 1.

Embodiment 3

[0077] Example 3 Effects of different concentrations of DMF on the enzyme activity of L-threonine aldolase mutant 8H / 31H / 143R / 305R

[0078] The engineering bacteria capable of expressing L-threonine aldolase and its mutants were cultivated according to the method in Example 1 to obtain crude enzyme liquid. Quantitatively weigh 0.1M p-thymphenylbenzaldehyde and 1M glycine into a 1L reactor, the volume fraction of DMF in the reaction system is 0%-30% (with a gradient of 5%), and use 100mM pH 8.0NaOH-Gly buffer solution The volume was adjusted to 1 L, the final concentration of pyridoxal phosphate was 1 μM, and the concentration of wet bacteria was 5 g / L. The reaction temperature was controlled at 30° C. by a water bath, magnetically stirred, and the concentration of the substrate and the product were detected after 10 minutes of reaction to determine the enzyme activity. The effect of different concentrations of DMF on enzyme activity is shown in Figure 6 .

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Abstract

The invention discloses an L-threonine aldolase mutant and an application of the L-threonine aldolase mutant in synthesis of L-syn-p-methylsulfonyl phenyl serine. The L-threonine aldolase mutant is obtained by mutating wild type L-threonine aldolase, and L-syn-p-methylsulfonyl phenyl serine can be generated by a catalytic condensation reaction by taking glycine and p-methylsulfonyl benzaldehyde as substrates and taking pyridoxal phosphate as a coenzyme. When the L-threonine aldolase mutant is used for producing L-synn-p-methylsulfonylphenyl serine, the L-threonine aldolase mutant has the following advantages: 1, the production process is simple, and the reaction conditions are mild; 2, the selectivity of the L-threonine aldolase is high, the optical purity of the product is high, and resolution is not needed; 3, the atom utilization rate is high and can reach 100% theoretically; the production process is environmentally friendly, pollution is small, and the green chemistry concept is met; 5, the product is simple to separate and purify.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to an L-threonine aldolase mutant, a gene and a method for preparing L-syn-p-thymphenylphenylserine. Background technique [0002] L-syn-p-thiamphenicol phenylserine is an important pharmaceutical intermediate, used in the synthesis of a variety of broad-spectrum antibiotics, such as thiamphenicol, florfenicol, etc., research on thiamphenicol and florfenicol The new high-efficiency green synthetic route has always been the focus and difficulty of organic synthesis research. [0003] At present, there are two main types of L-syn-p-thymphenylphenylserine. [0004] One is a chemical synthesis method, using copper sulfate as a catalyst, utilizing the complexation of metal ions, and selectively reacting p-thiamphenicol benzaldehyde and glycine to generate two cis products: L-syn-p-thiamphenicyl phenylserine Copper and D-syn-copper p-thiamphenicyl phenylserine, the acid canno...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12N15/70C12Y401/02005C12P13/04
Inventor 徐刚方赛吴坚平楼其向
Owner 宁波泓森生物科技有限公司
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