High-temperature-resistant L-threonine aldolase and application thereof to synthesis of p-methylsulfonyl phenyl serine

A technology of thiamphenylphenylserine and threonine aldolase, which is applied in the field of enzyme engineering, can solve problems such as difficult synthesis, and achieve the effects of simple steps, mild reaction conditions and high catalytic activity

Active Publication Date: 2021-08-31
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since β-hydroxy-α-amino acids have two chiral centers and four is

Method used

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  • High-temperature-resistant L-threonine aldolase and application thereof to synthesis of p-methylsulfonyl phenyl serine
  • High-temperature-resistant L-threonine aldolase and application thereof to synthesis of p-methylsulfonyl phenyl serine
  • High-temperature-resistant L-threonine aldolase and application thereof to synthesis of p-methylsulfonyl phenyl serine

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preparation example Construction

[0038] The preparation method of the whole cell of above-mentioned recombinant escherichia coli comprises the following steps:

[0039] S1: Cloning the target gene having the nucleotide sequence shown in SEQ ID NO: 1 into the restriction site between BamHI and HindIII of the vector pET-28a to construct the recombinant expression plasmid vector PsLTA-pET-28a;

[0040] S2: Transform the recombinant expression plasmid vector PsLTA-pET-28a described in S1 into Escherichia coli BL21 competent cells, and cultivate the recombinant strain E.coli BL21 / PsLTA-pET-28a;

[0041] S3: Collect the recombinant E. coli BL21 / PsLTA-pET-28a cultured in S2, and obtain the whole cells of the recombinant E. coli.

[0042] The application of the above-mentioned L-threonine aldolase in the synthesis of p-thymphenylphenylserine.

[0043] Preferably, the whole cell of recombinant Escherichia coli containing the nucleotide sequence shown in SEQ ID NO:1 is used as the catalyst, and the L-threonine aldolas...

Embodiment 1

[0046] Example 1: Construction of recombinant expression plasmid vector PsLTA-pET-28a

[0047] The construction of the plasmid was codon-optimized by Nanjing GenScript Co., Ltd. from the LTA gene (nucleotide sequence shown in SEQ ID NO: 1) derived from Pelosinus sp., and then cloned into the BamHI and HindIII of the vector pET-28a Between the restriction enzyme sites, the recombinant expression plasmid vector PsLTA-pET-28a was constructed ( figure 1 ).

Embodiment 2

[0048] Embodiment 2: Construction and cultivation of recombinant bacteria E.coli BL21 / PsLTA-pET-28a The plasmid carrier PsLTA-pET-28a constructed by the present invention is transformed into competent cells of E.coli DH5α for cloning, and then the plasmid is extracted and transformed into In E.coliBL21 competent cells, an expression strain was constructed for protein expression. Pick a single colony from the plate and inoculate it into a test tube of LB liquid medium containing 50 μg / mL kanamycin. After cultivating at a constant temperature and shaking at 37°C and 220 rpm for about 12 hours, inoculate the tube containing 50 μg / mL kanamycin according to 1% inoculation amount. Namycin in TB liquid medium. Determination of OD of culture medium by ultraviolet spectrophotometry 600 When reaching 0.6-0.8, add 0.2mM inducer IPTG to induce protein expression. The expression was induced for 22 hours at 16°C and 220rpm.

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Abstract

The invention relates to the technical field of enzyme engineering, and discloses a high-temperature-resistant L-threonine aldolase and application thereof to synthesis of p-methylsulfonyl phenyl serine. After the L-threonine aldolase, to which the invention relates, of a L-threonine aldolase (LTA) gene derived from Pelosinus sp. is expressed in escherichia coli, glycine and p-methylsulfonyl benzaldehyde are catalyzed to synthesize L-p-methylsulfonyl phenyl serine under the condition that a pH value is 7-10, the ee value is greater than 99%, and the process has the advantages of being mild in reaction condition, simple in step and the like. Moreover, the L-threonine aldolase PsLTA has relatively high catalytic activity at the temperature of 65 DEG C, still keeps 90% of the catalytic activity after the L-threonine aldolase PsLTA is incubated for 2h at the temperature of 55 DEG C, and has high thermal stability and catalytic activity and has industrial significance.

Description

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a high-temperature-resistant L-threonine aldolase and its application in the synthesis of p-thymphenylphenylserine. Background technique [0002] β-hydroxy-α-amino acid plays an important role in chemical synthesis and drug production, for example, phenylserine and its derivatives can be used as important intermediates in the synthesis of antibiotics such as florfenicol and thiamphenicol. However, since β-hydroxy-α-amino acids have two chiral centers and four isomers, it is difficult to synthesize optically pure β-hydroxy-α-amino acids. Using chemical methods such as glycine and copper sulfate to generate copper glycine under alkaline conditions, derivatized by Schiff base and then condensed with p-thiamphenyl benzaldehyde aldol to generate DL-p-thiamphenyl phenylserine copper, and then through chemical chiral resolution After separation, the final esterification is o...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12N15/70C12P13/04C12Y401/02005
Inventor 吴杰群李淑英刘峰帆施志豪
Owner ZHEJIANG UNIV OF TECH
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