Chen_2025_Genes.(Basel)_16_

BACKGROUND/OBJECTIVES: Esterases are widely used in various industrial fields. This study aimed to isolate and characterize esterase genes from Laceyella sacchari HS49-1, a thermophilic bacterium from a hot spring, which can survive at 45-60 degreesC and pH 5-10 with robust esterase activity. METHODS: A genomic shotgun library was constructed to identify three esterase genes: two in family XII (Est2 and Est7) and one in family VIII (Est1). Sequence analysis revealed significant divergence from other genera. Only Est1 was successfully expressed in Escherichia coli. Its activity, optimal conditions, thermostability, and structure were investigated using p-NP butyrate, temperature/pH assays, heating pre-treatment, and fluorescence quenching. RESULTS: Est1 demonstrated high activity (57.43 +/- 0.04 U/mg) towards short-chain p-NP butyrate (C4). Molecular-docking analyses revealed that Est1's catalytic motif (GXSXG) interacts with various p-NP esters, with binding energy and interaction types varying by acyl chain length. The optimal temperature was 60 degreesC, and the optimal pH was 8. Est1 exhibited excellent thermostability, retaining 90% of its activity after pre-treatment at 50 degreesC for 8 h and 69.8% after pre-treatment at 80 degreesC for the same duration. Fluorescence quenching showed that after 1 h at 80 degreesC, the fluorescence was reduced by only 16.6%, indicating remarkable heat resistance. Additionally, Est1 did not require metal ions as cofactors and maintained 74.8% of its activity in the presence of 0.1% SDS. CONCLUSIONS: The unique properties of Est1 from L. sacchari HS49-1 highlight its potential for industrial applications. Further exploration of this thermophilic bacterium could uncover more valuable genes.