Authors:

Madelyn Clair Watson

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About Paper:

Biocatalysis uses enzymes to catalyze chemical reactions towards small molecules, including food, fuel, firepower, fabrication, and fitness. Computer aided synthesis planning (CASP) tools use machine learning and rule-based methods to analyze target compounds in a retrosynthetic manner. A target is identified, and various pathways are formed in a backwards, piecewise manner before arriving at simple precursors. These CASP tools leverage insights from over 16,000 biocatalysis candidates within various enzymatic reaction databases. As a result, they can use common reaction chemistries used in industrial biocatalysis, including hydrolysis, reverse hydrolysis, redox, C-C bond formation, and C-X bond formation, to suggest pathways towards small molecule commodity chemicals. Notwithstanding these impressive capabilities, very few CASP predicted pathways have been experimentally tested. In this study, we test a computationally predicted pathway towards isopropanol. This reaction involves the reduction of acetone to isopropanol by alcohol dehydrogenase (ADH), coupled with the oxidation of NADPH to NADP+. Reaction samples were run under a plate reader using UV- spectrometry at 340 nm to monitor changes in NADPH concentration over time and to assess reaction kinetics. These ideas were compared with literature to determine whether the reaction was performed successfully. Additional SDS-PAGE analysis was performed to evaluate the purity of the ADH enzyme, and FPLC assays are planned for further characterization. This work demonstrates the potential of validating CASP-predicted biocatalytic pathways experimentally, providing a foundation for future efforts in pathway optimization and scale-up. Keywords: Enzymatic Reaction; Retrosynthesis; Biocatalysis

Source:

Purdue University / 2025

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Co-authors:

Madelyn Clair Watson