2021 Fiscal Year Annual Research Report
Development of single molecule display platform for directed evolution of enzymes
| Project/Area Number |
18K14387
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| Research Institution | Nagoya University |
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Principal Investigator |
ダムナニョヴィッチ ヤスミナ 名古屋大学, 生命農学研究科, 講師 (00754673)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | cDNA display / substrate profiling / protein engineering / enzyme engineering / NGS / transglutaminase / D-amino acid oxidase |
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| Outline of Annual Research Achievements |
The established cDNA display platform was applied to substrate profiling of transglutaminase 1 (TG1) and microbial transglutaminase (MTG). In parallel, the platform has been optimized for the screening of combinatorial enzyme libraries. Specifically, D-amino acid oxidase (DAAO), a microbial enzyme catalyzing the oxidation of D-amino acids was used for enzyme library generation.
cDNA display platform for substrate profiling of TG1 and MTG was first optimized using binary model libraries consisting of known TG substrate and non-substrate peptides. In the following, random libraries of TG1 and MTG substrates were made with 5-7 randomized amino acid positions in the original peptide backbone. Screening of the TG1 substrate library followed by NGS and bioinformatics revealed the preferred amino acid sequence of human TG1. Surprisingly, not only the sequences in positions near the reactive Gln but also the sequences in positions distant from it seem to be important for the TG1 reactivity. Screening of a combinatorial library of MTG substrate is ongoing.
As for DAAO screening, we have established the reporter enzyme unit (additional module with peroxidase which enables reliable detection of DAAO activity during the screening), and confirmed the enzyme display formation. The strategy of attachment of the enzyme reporter unit to the cDNA displayed enzyme has been changed to relieve the steric hindrance between the reporter enzyme and displayed enzyme, DAAO.
Binary model libraries for initial optimization of the cDNA display-based DAAO screening are now being prepared.
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