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PreprintCited by 2Enzyme Catalysis and ImmobilizationEnzyme Production and CharacterizationProtein purification and stability

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Introduction of multiple disulfide bonds increases the thermostability of transglutaminase

Open access
Ono, Takuto, Takahashi, Kazutoshi, Hirao, Yoshinori, Mihara, Yasuhiro, Abe, Isao, Sugiki, Masayuki·2025·Scientific Reports
DOI 10.21203/rs.3.rs-5776787/v1PMC PMC12214910

Preprint — published as 10.1038/s41598-025-07842-5 · Paper page

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Abstract

Microbial transglutaminase (MTG) is an enzyme that catalyzes the cross-linking of glutamine and lysine residues in proteins. Because of its ability to modify proteins, MTG has various applications in the medical and food industries. Most studies have aimed to enhance the thermal stability of MTG by focusing only on point mutations. Introducing a disulfide (S-S) bond in the N-terminal region has been found to be effective, whereas S-S bonds in other regions were considered ineffective. Therefore, this study aimed to evaluate the impact of introducing an additional S-S bond on the thermal stability of an MTG mutant. We found that adding S-S bonds to regions other than the N-terminal, in conjunction with the N-terminal S-S bond, significantly enhanced thermal stability. This finding demonstrates the importance of reinforcing the weakest part of the protein first, followed by strengthening other regions for optimal thermal stability. The MTG variant with two S-S bonds retained its catalytic activity and substrate specificity towards protein substrates, making it a promising candidate for industrial applications. Thus, introducing S-S bonds could be an effective strategy to increase thermal stability of MTG and other industrial enzymes, thereby contributing to their potential industrial applications.

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Figures

Fig. 1 Sequence and structure of MTG mutants. (A) The structure of MTG. Left: α-helix domain 1 is shown in blue, β-sheet domain in green, and α-helix domain 2 in red. Right: Colored according to B-factor values. Warmer colors indicate higher B-factor, while cooler colors indicate lower B-factor. (B) The sequence of MTG. α-helix domain 1 is shown in blue, β-sheet domain in green, and α-helix domain 2 in red. The dashed lines indicate combinations of amino acid residues with S-S bonds introduced. (C) Structure models of the S-S mutant MTGs. Red color indicates the Cys residue introduced to form an S-S bond.
Fig. 1 Sequence and structure of MTG mutants. (A) The structure of MTG. Left: α-helix domain 1 is shown in blue, β-sheet domain in green, and α-helix domain 2 in red. Right: Colored according to B-factor values. Warmer colors indicate higher B-factor, while cooler colors indicate lower B-factor. (B) The sequence of MTG. α-helix domain 1 is shown in blue, β-sheet domain in green, and α-helix domain 2 in red. The dashed lines indicate combinations of amino acid residues with S-S bonds introduced. (C) Structure models of the S-S mutant MTGs. Red color indicates the Cys residue introduced to form an S-S bond.CC BY-NC-ND · PubMed Central
Fig. 2 Thermal stability and temperature dependence of MTG mutants. (A) Residual activity of each mutant after thermal treatment (n = 3). Error bars indicate standard errors. (B) Specific activity of each mutant measured at each temperature (n = 3). Error bars indicate standard errors. (C) Relative activity of each mutant measured at each temperature (n = 3). Error bars indicate standard errors. (D) Melting temperatures of each mutant were determined using nanoDSF, with each sample measured in duplicate.
Fig. 2 Thermal stability and temperature dependence of MTG mutants. (A) Residual activity of each mutant after thermal treatment (n = 3). Error bars indicate standard errors. (B) Specific activity of each mutant measured at each temperature (n = 3). Error bars indicate standard errors. (C) Relative activity of each mutant measured at each temperature (n = 3). Error bars indicate standard errors. (D) Melting temperatures of each mutant were determined using nanoDSF, with each sample measured in duplicate.CC BY-NC-ND · PubMed Central
Fig. 3 Protein substrate reactivity of MTG S-S mutants. The activity after incubation at 37 °C for 60 min (n = 3). Error bars indicate standard errors.
Fig. 3 Protein substrate reactivity of MTG S-S mutants. The activity after incubation at 37 °C for 60 min (n = 3). Error bars indicate standard errors.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Ono, T., Takahashi, K., Hirao, Y., Mihara, Y., Abe, I., & Sugiki, M.
Affiliations
Ajinomoto (Japan) (JP)
Corresponding author
Takahashi, Kazutoshi
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2025-02-10
Volume
15
Issue
1
Pages
21105-21105
ISSN
2045-2322 (ISSN-L)
DOI
10.21203/rs.3.rs-5776787/v1
PMC
PMC12214910
OpenAlex
W4411871565
Cited by
2
References
1
Citations by year
2026: 2
FWCI
0.60
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Ajinomoto
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
microbial transglutaminase, disulfide bonds, thermal stability, protein engineering, protein cross-linking, industrial enzymes, catalytic activity, substrate specificity
MeSH terms
Disulfides, Enzyme Stability, Transglutaminases, Substrate Specificity, Temperature

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