- 【Updated on May 12, 2025】 Integration of CiNii Dissertations and CiNii Books into CiNii Research
- Trial version of CiNii Research Automatic Translation feature is available on CiNii Labs
- Suspension and deletion of data provided by Nikkei BP
- Regarding the recording of “Research Data” and “Evidence Data”
Engineered Active Zymogen of Microbial Transglutaminase
-
- Ariyoshi, Ryutaro
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Matsuzaki, Takashi
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Sato, Ryo
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Minamihata, Kosuke
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Kounosuke Hayashi
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Hayashi, Kounosuke
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Wakabayashi, Rie
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
-
- Goto, Masahiro
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University Division of Biotechnology, Center for Future Chemistry, Kyushu University
-
- Kamiya, Noriho
- Department of Applied Chemistry, Graduate School of Engineering, Kyushu University Division of Biotechnology, Center for Future Chemistry, Kyushu University
Description
Microbial transglutaminase (MTG) has shown to be a powerful biocatalytic glue for site-specific crosslinking of a range of biomolecules and synthetic molecules, those handled with an MTG-reactive moiety. The preparation of active recombinant MTG requires the posttranslational proteolytic digestion of propeptide working as an intramolecular chaperon to assist the correct folding of MTG zymogen (MTGz) in the biosynthesis. Herein, we propose an engineered active zymogen of MTG (EzMTG) that is expressed as soluble form in the host E. coli cytosol and exhibits the cross-linking activity without limited proteolysis. Based on the 3D structure of MTGz and serendipitous findings, saturated mutagenesis of K10 or Y12 in propeptide domain leads to generate several active MTGz mutants. In particular, K10D/Y12G mutant exhibited the catalytic activity comparable with a mature form. However, the expression level was low possibly due to the reduction of chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. By contrast, soluble K10R/Y12A mutant is expressed in the cytosol of host E. coli and exhibited unique substrate-dependent reactivity toward peptidyl substrates. The quantitative analysis of the binding affinity of mutated propeptide to the active site suggested the trade-off relationship of EzMTGs between the binding affinity and the catalytic activity. Our proof-of-concept study provides insights into the design of a new biocatalyst by using the zymogen as a scaffold and will convey a potential route to the high-throughput screening of MTG mutants for bioconjugation applications.
- Tweet
Details 詳細情報について
-
- CRID
- 1050864789554778624
-
- HANDLE
- 2324/7236919
-
- Text Lang
- en
-
- Article Type
- journal article
-
- Data Source
-
- IRDB