2019 Fiscal Year Annual Research Report
非天然型アミノ酸生産のためのPLP依存性アミノ基転移酵素の改変
| Project/Area Number |
19F19764
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
LAURINO Paola 沖縄科学技術大学院大学, タンパク質工学・進化ユニット, 准教授 (90812256)
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| Co-Investigator(Kenkyū-buntansha) |
DINDO MIRCO 沖縄科学技術大学院大学, タンパク質工学・進化ユニット, 外国人特別研究員
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| Project Period (FY) |
2019-11-08 – 2022-03-31
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| Keywords | Biochemistry / enzyme / library / screening |
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| Outline of Annual Research Achievements |
During FY19 we have expressed in E.coli, purified, and biochemical characterized the selected DPAT. We then proceed with a deep literature search to understand which is the most impactful reaction to tackle with the engineering. In parallel we have studied the reaction mechanism of another PLP dependent enzyme namely alanine:glyoxylate aminotransferase (ATG), this study concluded in a publication listed in the below section. We have also prepared a small library for this AGT enzyme and screen for thermostability and activity. This library helped to understand the role a critical disordered region for its oligomerization state and activity (manuscript in preparation).
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
We proceed with the initial plan of studying the DPAT enzyme and in parallel we studied another PLP dependent enzyme -namely AGT. This side-project gave us enough results for one already published paper and another in preparation.
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| Strategy for Future Research Activity |
Setting up a high-throughput assay based on detection of ketoglutarate produced in the half transamination reaction using GDH (Glutamate Dehydrogenase), which utilizes NADH as a coenzyme. Based on this assay the recombinant DPAT will be engineered by rounds of directed evolution. Bioinformatic approaches will allow us to identify target amino acid residues that are most likely to influence protein function and to design semi-rational libraries. Libraries will be prepared using a PCR assembly method. The main aims of this part are (i) to change the substrate specificity of the enzyme for production of selected aromatic ncAAs and their derivatives, using inexpensive substrates and (ii) to increase the catalytic activity of DPAT for production of aromatic ncAAs
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