Abstract
Biocatalysis is an established technology with significant application in the pharmaceutical industry. Immobilization of enzymes offers significant benefits for commercial and practical purposes to enhance the stability and recyclability of biocatalysts. Determination of the spatial and chemical distributions of immobilized enzymes on solid support materials is essential for an optimal catalytic performance. However, current analytical methodologies often fall short of rapidly identifying and characterizing immobilized enzyme systems. Herein, we present a new analytical methodology that combines non-negative matrix factorization (NMF)-an unsupervised machine learning tool-with Raman hyperspectral imaging to simultaneously resolve the spatial and spectral characteristics of all individual species involved in enzyme immobilization. Our novel approach facilitates the determination of the optimal NMF model using new data-driven, quantitative selection criteria that fully resolve all chemical species present, offering a robust methodology for analyzing immobilized enzymes. Specifically, we demonstrate the ability of NMF with Raman hyperspectral imaging to resolve the spatial and spectral profiles of an engineered pantothenate kinase immobilized on two different commercial microporous resins. Our results demonstrate that this approach can accurately identify and spatially resolve all species within this enzyme immobilization process. To the best of our knowledge, this is the first report of NMF within hyperspectral imaging for enzyme immobilization analysis, and as such, our methodology can now provide a new powerful tool to streamline biocatalytic process development within the pharmaceutical industry.
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