Enzyme and application thereof

Patent metadata

Field	Value
Publication number	US-12516299-B2
Application number	US-202217700237-A
Country	US
Kind code	B2
Filing date	Mar 21, 2022
Priority date	Apr 1, 2016
Publication date	Jan 6, 2026
Grant date	Jan 6, 2026

Abstract

Provides an artificial enzyme obtained by improving upon a sequence of a natural nicotine dehydrogenase, wherein the improvement comprises replacing at least one amino acid hindering product release with an amino acid with smaller side chains, thereby improving a catalytic rate.

First claim

The invention claimed is:

1 . An artificial enzyme, wherein the artificial enzyme is produced based on modification of a sequence of a natural enzyme and has nicotine dehydrogenase activity, the natural enzyme having an amino acid sequence as shown in SEQ ID NO: 1,
wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355, tryptophan at position 364, phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with same or different amino acids with smaller side chains; wherein the amino acids with smaller side chains are selected from the group consisting of alanine, valine, glycine, proline, serine and threonine.

2 . The artificial enzyme as in claim 1 , wherein the amino acids with smaller side chains are alanine or valine.

3 . The artificial enzyme as in claim 1 , wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with a first small-molecular-weight amino acid, and/or substituting one or more of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with a second small-molecular-weight amino acid,
wherein the first small-molecular-weight amino acid is alanine, valine, glycine, proline, serine or threonine, and the second small-molecular-weight amino acid is alanine.

4 . The artificial enzyme as in claim 3 , wherein the first small-molecular-weight amino acid is alanine or valine, and the second small-molecular-weight amino acid is alanine.

5 . The artificial enzyme as in claim 1 , wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with valine, and/or substituting one or more of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine.

6 . The artificial enzyme as in claim 1 , wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with glycine, and/or substituting one or more of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine or glycine.

7 . The artificial enzyme as in claim 1 , wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with proline, and/or substituting one or more of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine or proline.

8 . The artificial enzyme as in claim 1 , wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with serine, and/or substituting one or more of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine or serine.

9 . The artificial enzyme as in claim 1 , wherein the modification is substituting one or more of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with threonine, and/or substituting one or more of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine or threonine.

10 . The artificial enzyme as in claim 1 , wherein the modification is substituting all of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with glycine, and/or substituting all of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine.

11 . The artificial enzyme as in claim 1 , wherein the modification is substituting all of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with proline, and/or substituting all of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine.

12 . The artificial enzyme as in claim 1 , wherein the modification is substituting all of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with serine, and/or substituting all of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine.

13 . The artificial enzyme as in claim 1 , wherein the modification is substituting all of phenylalanine at position 353, phenylalanine at position 355 and tryptophan at position 364 of the amino acid sequence SEQ ID NO: 1 with threonine, and/or substituting all of phenylalanine at position 163, tyrosine at position 214, tyrosine at position 218, tyrosine at position 242, methionine at position 246 and glutamic acid at position 249 of the amino acid sequence SEQ ID NO: 1 with alanine.

14 . A nucleic acid encoding the artificial enzyme as in claim 1 .

15 . A method for treating nicotine addiction in a subject in need, comprising administration of a therapeutically effective amount of a medicament comprising the artificial enzyme of claim 1 to the subject.

Assignees

• Univ Shanghai Jiaotong

Inventors

• Xu Ping
• Tang Hongzhi
• Zhang Kunzhi
• Wu Geng
• Tao Fei

Classifications

• A61K31/405

  Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• A61K31/401

  Proline; Derivatives thereof, e.g. captopril · CPC title

• A61P43/00 Primary

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K31/198

  Alpha-amino acids, e.g. alanine or edetic acid [EDTA] (betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00) · CPC title