Rapid spectroscopic detection method for microsamples aimed at efficient screening of biological enzymes

Enzymes are extensively employed as biocatalysts owing to their high efficiency and environmental compatibility, playing essential roles in the food industry, pharmaceuticals, environmental remediation, and materials science. Nevertheless, their industrial application is often limited by low catalytic activity, insufficient stability, and poor substrate selectivity. Directed evolution, which involves the construction of large mutant libraries combined with high-throughput screening, has proven to be an effective strategy for overcoming these limitations. However, traditional spectroscopic techniques encounter significant challenges in multi-component catalytic systems, particularly due to spectral overlap and interference from structurally similar compounds. To address these issues, this paper proposes a rapid and accurate spectroscopic detection method based on a 96-well plate platform. Experimental validation confirms that the proposed method, which integrates chemometric analysis with high-throughput capabilities, enables simultaneous quantification of the substrate (2-naphthol, 2-NH), the target product (2,6-dihydroxynaphthalene, 2,6-DNH), and the byproduct (2,7-dihydroxynaphthalene, 2,7-DNH). This method provides reliable analytical support for enzyme screening and directed evolution. Moreover, by detecting relative differences in product composition, it facilitates rapid classification of enzyme variants into high, medium, and low activity groups, offering practical value for microsample-based screening workflows.