Establishment of molecular design guidelines for enzyme mutants by principal component analysis aiming at improving enzyme electrochemical reaction

2022 Fiscal Year Final Research Report

• Project/Area Number: 21K14782
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 38030:Applied biochemistry-related
• Research Institution: University of Fukui
• Principal Investigator: TAKAMURA Eiichiro 福井大学, 学術研究院工学系部門, 講師 (30843015)
• Project Period (FY): 2021-04-01 – 2023-03-31
• Keywords: 直接電子移動型酵素 / 電気化学スクリーニング / 酵素分子設計 / 多変量解析

Outline of Final Research Achievements

In the present study, we aimed to improve the electroenzymatic reaction of direct electron transfer-type enzymes, which are capable of direct electron transfer between enzyme and electrode. Electrochemical screening, rather than conventional enzyme activity screening, was performed on random mutant libraries. From the screening results, mutant libraries were classified by hierarchical cluster analysis. Mutants exhibiting similar properties to those showing superior electrocatalytic performance had similar mutations introduced. Focusing on the redox potential of the enzyme, the high effect sites on the redox potential due to the introduction of mutations were identified. A saturating mutation library was prepared and evaluated, and mutants with redox potential changes of approximately 70 mV were obtained with only 0.1% of the total sequence space screened.

Free Research Field

生物電気化学

Academic Significance and Societal Importance of the Research Achievements

本研究成果によって、直接電子移動型酵素における電気化学スクリーニングの重要性が示された。また、本研究における高影響部位の特定までの手法は、これまでの指向性進化法における大量のスクリーニングや、部位特異的変異導入法における構造的知見を必要としない。そのため、一次配列と機能さえ明らかであれば少ないコストで性能向上変異体を得ることが可能となり、新規酵素の実用化までの時間短縮などに貢献すると考えられる。