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Pseudomonas sp. D01, capable of growing in tributyrin medium, was isolated from the gut microbiota of yellow mealworm. By using in silico analyses, we discovered a hypothesized esterase encoding gene in the D01 bacterium, and its encoded protein, EstD04, was classified as a bacterial hormone-sensitive lipase (bHSL) of the type IV lipase family. The study revealed that the recombinant EstD04-His(6x) protein exhibited esterase activity and broad substrate specificity, as it was capable of hydrolyzing p-nitrophenyl derivatives with different acyl chain lengths. By using the most favorable substrate p-nitrophenyl butyrate (C₄), we defined the optimal temperature and pH value for EstD04 esterase activity as 40 °C and pH 8, respectively, with a catalytic efficiency (k_cat/K_m) of 6.17 × 10³ mM⁻¹ s⁻¹ at 40 °C. EstD04 demonstrated high stability between pH 8 and 10, and thus, it might be capably used as an alkaline esterase in industrial applications. The addition of Mg²⁺ and NH₄⁺, as well as DMSO, could stimulate EstD04 enzyme activity. Based on bioinformatic motif analyses and tertiary structural simulation, we determined EstD04 to be a typical bHSL protein with highly conserved motifs, including a triad catalytic center (Ser¹⁶⁰, Glu²⁵³, and His²⁸³), two cap regions, hinge sites, and an oxyanion hole, which are important for the type IV enzyme activity. Moreover, the sequence analysis suggested that the two unique discrete cap regions of EstD04 may contribute to its alkali mesophilic nature, allowing EstD04 to exhibit extremely distinct physiological properties from its evolutionarily closest esterase. Full article

Bacterial hormone-sensitive lipases (bHSLs), which are homologous to the catalytic domains of human HSLs, have received great interest due to their uses in the preparation of highly valuable biochemicals, such as drug intermediates or chiral building blocks. Here, a novel cold-active HSL from Halocynthiibacter arcticus (HaHSL) was examined and its enzymatic properties were investigated using several biochemical and biophysical methods. Interestingly, HaHSL acted on a large variety of substrates including tertiary alcohol esters and fish oils. Additionally, this enzyme was highly tolerant to high concentrations of salt, detergents, and glycerol. Furthermore, immobilized HaHSL retained its activity for up to six cycles of use. Homology modeling suggested that aromatic amino acids (Trp²³, Tyr⁷⁴, Phe⁷⁸, Trp⁸³, and Phe²⁴⁵) in close proximity to the substrate-binding pocket were important for enzyme activity. Mutational analysis revealed that Tyr⁷⁴ played an important role in substrate specificity, thermostability, and enantioselectivity. In summary, the current study provides an invaluable insight into the novel cold-active HaHSL from H. arcticus, which can be efficiently and sustainably used in a wide range of biotechnological applications. Full article