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US-12618056-B2 - Modified enzyme and method of producing imidazole dipeptide using same

US12618056B2US 12618056 B2US12618056 B2US 12618056B2US-12618056-B2

Abstract

Described herein is an enzyme useful for producing a target peptide, capable of producing the target peptide without requiring ATP. More specifically, provided herein is a modified enzyme includes an amino acid sequence such as a modified amino acid sequence including a mutation of one or more of the following amino acid residues: E81, I127, I136, T139, F140, G142, W143, I147, I181, I201, Q219, T229, M244, A249, P255, E256, I260, S293, N294, Y295, and I299 in an amino acid sequence of SEQ ID NO: 1, and having the following property (a) or (b) enhanced relative to an enzyme of the amino acid sequence of SEQ ID NO: 1: (a) imidazole dipeptide production activity; or (b) thermal stability.

Inventors

  • Mizuki Tamori
  • Uno Tagami
  • Hiroyuki Nozaki

Assignees

  • AJINOMOTO CO., INC.

Dates

Publication Date
20260505
Application Date
20240327
Priority Date
20210930

Claims (13)

  1. 1 . A modified enzyme comprising: (A) an amino acid sequence of SEQ ID NO. 1, wherein the amino acid sequence additionally comprises a mutation of an amino acid residue selected from the group consisting of E81, I127, I136, T139, F140, G142, W143, I147, I181, I201, Q219, T229, M244, A249, P255, E256, I260, S293, N294, Y295, I299, and combinations thereof; (B) the amino acid sequence of (A) comprising an additional mutation of substitution, deletion, insertion, and addition of one or more amino acid residues; or (C) an amino acid sequence having 90% or more identity to said amino acid sequence of (A) or (B); and wherein an imidazole dipeptide production activity or thermal stability is enhanced in the modified enzyme relative to an enzyme of the amino acid sequence of SEQ ID NO: 1.
  2. 2 . The modified enzyme according to claim 1 , wherein the mutation of (A) is: (1) E81D; (2) I127V or I127L; (3) I136V; (4) T139S or T139A; (5) F140A, F140Y, F140H, F140W, F140Q, F140N, or F140D; (6) G142A; (7) W143F or W143A; (8) I147A; (9) I181V; (10) I201F; (11) Q219A; (12) T229A; (13) M244L; (14) A249F or A249L; (15) P255A; (16) E256A or E256D; (17) I260A; (18) S293F; (19) N294H; (20) Y295A, Y295W, or Y295V; or (21) I299V or I299F.
  3. 3 . The modified enzyme according to claim 1 , wherein the imidazole dipeptide production activity is an activity of producing an imidazole dipeptide from an amino acid ester and an L-amino acid having an imidazole group in a side chain.
  4. 4 . The modified enzyme according to claim 3 , wherein the amino acid ester is a compound represented by the following formula (I): wherein R 1 is an alkyl group having 1 to 3 carbon atoms, and n is an integer of 1 to 4; the L-amino acid having an imidazole group in a side chain is an L-form of a compound represented by the following formula (II): wherein R 2 and R 3 are each independently absent, a hydrogen atom, or an alkyl group having 1 to 3 carbon atoms; and the imidazole dipeptide is represented by the following formula (III): wherein R 2 and R 3 are the same as in said formula (II), and n is the same as in said formula (I).
  5. 5 . The modified enzyme according to claim 3 , wherein the imidazole dipeptide is carnosine or anserine.
  6. 6 . A polynucleotide encoding the modified enzyme according to claim 1 .
  7. 7 . An expression vector comprising the polynucleotide according to claim 6 .
  8. 8 . A host cell comprising an expression unit containing a polynucleotide encoding the modified enzyme according to claim 1 and a promoter operably linked thereto.
  9. 9 . A method of producing an imidazole dipeptide or a salt thereof, comprising contacting an amino acid ester or a salt thereof and an L-amino acid having an imidazole group in a side chain or a salt thereof with the modified enzyme according claim 1 to form the imidazole dipeptide or the salt thereof.
  10. 10 . The method according to claim 9 , wherein said contacting is performed using said modified enzyme.
  11. 11 . The method according to claim 9 , wherein said contacting is performed using a host cell comprising an expression unit containing a polynucleotide encoding the modified enzyme and a promoter operably linked thereto.
  12. 12 . The method according to claim 9 , wherein the amino acid ester is a compound represented by said formula (I), the L-amino acid having an imidazole group in a side chain is an L-form of a compound represented by said formula (II), and the imidazole dipeptide is represented by said formula (III).
  13. 13 . The method according to claim 9 , wherein the imidazole dipeptide is carnosine or anserine.

Description

This application is a Continuation of, and claims priority under 35 U.S.C. § 120 to, International Application No. PCT/JP2022/036782, filed Sep. 30, 2022, and claims priority therethrough under 35 U.S.C. § 119 to Japanese Patent Application No. 2021-160738, filed Sep. 30, 2021, the entireties of which are incorporated by reference herein. Also, the Sequence Listing filed electronically herewith is hereby incorporated by reference (File name: 2024-03-27T_US-659_Seq_List.xml; File size: 33,423 bytes; Date recorded: Mar. 19, 2024). BACKGROUND Technical Field The present invention relates to a modified enzyme, a method of producing an imidazole dipeptide using the same, and the like. Background Art Carnosine (β-alanyl histidine) is an imidazole dipeptide composed of β-alanine and histidine. Carnosine is abundantly present in muscles, brains, and hearts of mammals including humans, and has been reported to have pH-regulating effects, anti-inflammatory effects, tissue repair effects, immunomodulatory effects, antioxidant effects, anti-protein glycation effects, and the like. As a method of producing carnosine, a method using an enzyme that catalyzes a reaction of producing β-alanyl histidine from β-alanine and histidine (β-Ala+His→β-Ala-His) is known (Non Patent Literature 1, Non Patent Literature 2, and Patent Literature 1). However, these enzymes have required ATP in the reaction. On the other hand, Patent Literature 2 describes that some enzymes derived from multiple microorganisms can produce β-alanyl amino acids or derivatives thereof from β-alanyl esters or β-alanyl amides and amino acids or derivatives thereof without requiring ATP, and examples of such enzymes include wild-type RhDmpA3 enzymes derived from Rhodotorula minuta. Patent Literature Patent Literature 1: United States Patent Application Publication US 2005/0287627APatent Literature 2: International Publication WO 2009/139392A Non Patent Literature Non Patent Literature 1: Skaper S D et al., J Neurochem. 1973 December; 21(6): 1429-45.Non Patent Literature 2: Horinishi H et al., J Neurochem. 1978 October; 31(4): 909-19. SUMMARY It is an aspect of the present invention to provide an enzyme useful for producing a target peptide, capable of producing the target peptide without requiring ATP. As a result of an extensive study, the present inventors have found that a modified enzyme of wild-type RhDmpA3 derived from Rhodotorula minuta exhibits properties useful for production of a target peptide. More specifically, the present inventors have found that such a modified enzyme is excellent in imidazole dipeptide production activity and/or excellent in thermal stability without requiring ATP. The prior art neither describes nor suggests such identification. It is an aspect of the present invention to provide a modified enzyme comprising: (A) an amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence additionally comprises a mutation of an amino acid residue selected from the group consisting of E81, I127, I136, T139, F140, G142, W143, I147, I181, I201, Q219, T229, M244, A249, P255, E256, I260, S293, N294, Y295, I299, and combinations thereof;(B) the amino acid sequence of (A) comprising an additional mutation of substitution, deletion, insertion, and/or addition of one or more amino acid residues; or(C) an amino acid sequence having 90% or more identity to said amino acid sequence of (A) or (B); andwherein an imidazole dipeptide production activity or thermal stability is enhanced in the modified enzyme relative to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1. It is a further aspect of the present invention to provide the modified enzyme as described above, wherein the mutation of (A) is: (1) E81D;(2) I127V or I127L;(3) I136V;(4) T139S or T139A;(5) F140A, F140Y, F140H, F140W, F140Q, F140N, or F140D;(6) G142A;(7) W143F or W143A;(8) I147A;(9) I181V;(10) I201F;(11) Q219A;(12) T229A;(13) M244L;(14) A249F or A249L;(15) P255A;(16) E256A or E256D;(17) I260A;(18) S293F;(19) N294H;(20) Y295A, Y295W, or Y295V; or(21) I299V or I299F. It is a further aspect of the present invention to provide the modified enzyme as described above, wherein the imidazole dipeptide production activity is an activity of producing an imidazole dipeptide from an amino acid ester and an L-amino acid having an imidazole group in a side chain. It is a further aspect of the present invention to provide the modified enzyme as described above, wherein the amino acid ester is a compound of formula (I),the L-amino acid having an imidazole group in a side chain is an L-form of a compound of formula (II), andthe imidazole dipeptide is a compound of formula (III). It is a further aspect of the present invention to provide the modified enzyme as described above, wherein the imidazole dipeptide is carnosine or anserine. It is a further aspect of the present invention to provide a polynucleotide encoding the modified enzyme as described above. It is a further aspect of t