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Directed evolution and biocatalytic application of N-deoxyribosyltransferase II

A deoxyribose and transferase technology, applied in the direction of glycosyltransferase, transferase, biochemical equipment and methods, etc., can solve the problem of low catalytic efficiency

Active Publication Date: 2019-02-01
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The present invention aims at the low catalytic efficiency of existing N-deoxyribose transferase II (NDT) to nucleoside analogues with glycosyl modifications, especially 2',3'-dideoxyribose analogues, and provides a method through Preparation method and application of N-deoxyribosyltransferase Ⅱ mutant that improves enzyme activity by directed evolution

Method used

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  • Directed evolution and biocatalytic application of N-deoxyribosyltransferase II

Examples

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Embodiment 1

[0046] Embodiment 1, the acquisition of N-deoxyribosyltransferase II (NDT) mutant

[0047] 1. Construction of N-deoxyribosyltransferase Ⅱ (NDT) recombinant engineering strain

[0048] Lactobacillus helveticus (CGMCC 1.1877) bacterial solution was used as a template to perform PCR amplification to obtain a fragment of the target gene ndt (the nucleotide sequence is sequence 1 in the sequence listing), and the amino acid sequence of the protein encoded by the gene is sequence 2 in the sequence listing.

[0049] According to the nucleic acid sequence of Lactobacillus helveticus N-deoxyribosyltransferase II gene (ndt) found in Genbank, a pair of amplification primers were designed:

[0050] ndt(+):5'–CGCCATATGATGAACAAGAAAAAAGAC-3'

[0051] ndt(-):5'–CGGGAATTCTTAATATACAGCTCCG-3'

[0052] The recovered and purified PCR product was ligated with the pMD18-T plasmid to obtain the cloning plasmid pMD18-T-ndt. The ligation product pMD18-T-ndt was transformed into Escherichia coli Trans5...

Embodiment 2

[0068] Example 2, Preparation and functional testing of NDT mutant THNDT

[0069] 1. Preparation of NDT mutant THNDT

[0070] 1. Recombinant bacteria expressing NDT mutant THNDT

[0071] The recombinant bacterium expressing the NDT mutant THNDT is a recombinant bacterium obtained by introducing the recombinant plasmid pET-28a(+)-THNDT into the expression strain E.coli BL21(DE3). The above-mentioned recombinant plasmid pET-28a(+)-THNDT is obtained by replacing the fragment between the Nde I and EcoR I restriction sites of the pET-28a(+) vector with the NDT mutant THNDT coding gene shown in Sequence 1 in the sequence listing A plasmid expressing the NDT mutant THNDT.

[0072] The recombinant bacterium expressing NDT is the recombinant bacterium obtained by introducing the recombinant plasmid pET-28a(+)-NDT into the expression strain E.coli BL21(DE3). The above-mentioned recombinant plasmid pET-28a(+)-NDT is a plasmid obtained by replacing the fragment between the Nde I and Ec...

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Abstract

The invention discloses a directed evolution and biocatalytic application of N-deoxyribosyltransferase II. The invention provides an N-deoxyribosyltransferase II (NDT) mutant. The tenth Gly of the amino acid residue as shown in the Sequence 2 of the sequence table is mutated to Ser, but other amino acid residues are not changed so as to obtain a protein. by sequence alignment of Lactobacillus helveticus-derived NDT (Lh NDT) and Lactobacillus Leichamanni-derived NDT (Ll NDT), homologous modeling is conducted, a saturated mutant library is established, and high throughput screening of 600 mutants is carried out to obtain a Lactobacillus helveticus-derived N-deoxyribosyltransferase II mutant with enzyme activity improved by 10.4 times. The N-deoxyribosyltransferase II mutant provided by the invention can efficiently catalyze the preparation of an anti-AIDs drug zalcitabine.

Description

technical field [0001] The invention belongs to the technical field of directed evolution modification and biocatalysis application of enzymes, and in particular relates to an N-deoxyribose transferase II directed evolution modification and biocatalysis application. Background technique [0002] Nucleoside analogues are widely used in the treatment of viruses and tumor diseases clinically, and nearly 50% of the currently used antiviral drugs are nucleoside drugs. Such as acyclovir and famciclovir for the treatment of herpes virus infection; adenosine vidarabine for the treatment of herpes simplex and encephalitis; ribavirin and sofosbuvir for the treatment of hepatitis C; and HIV reverse transcriptase inhibitors for the treatment of AIDS. Dovudine, zalcitabine, stavudine, lamivudine, new generation antineoplastic drugs decitabine, gemcitabine, nelarabine, etc. all belong to nucleoside drugs. Among them, zalcitabine (DDC), chemical name 2', 3'-dideoxycytidine, is converted i...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N1/21C12N15/74C12P19/38C12R1/225
CPCC12N9/1077C12N15/74C12P19/385C12Y204/02006
Inventor 王洪钟李京美
Owner TSINGHUA UNIV
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