Gene Cloning, Characterization and Transesterification Reactions of Mgl-C255, a Lipolytic Enzyme from Neobacillus thermocopriae C255 Isolated from Ash from Popocatépetl Volcano

Graciela Espinosa-Luna; Aaron S. Bustos-Baena; Rocio Solis-Palacios; Jonathan Lara-Sanchez; Aurelio Espinosa-Honorato; Rosa María Oliart-Ros

Gene Cloning, Characterization and Transesterification Reactions of Mgl-C255, a Lipolytic Enzyme from Neobacillus thermocopriae C255 Isolated from Ash from Popocatépetl Volcano

2024

Lipases and carboxylesterases are enzymes of biotechnological interest both for their reactions and their specificity. They have wide-ranging applications in the food, pharmaceuticals, biodiesel synthesis, and bioremediation industries. For that reason, the strain Neobacillus thermocopriae C255 was isolated from ash from Popocatepetl volcano and studied as a new source of lipolytic enzymes. It was identified using 16S ribosomal RNA and flagellar protein FliF sequence homology, yielding 100% identity. From the sequencing of its genome, an enzyme with lipolytic activity, classified as a monoacylglycerol lipase, and named Mgl-C255, was cloned in E. coli BL21, and then expressed, biochemically characterized, and tested via transesterification reactions with alcohols and monosaccharides. Based on its sequence and structure, it was placed within family V, having a catalytic triad of S90-D207-H237. Biochemical characterization showed its highest activity at 40 °C, pH 7.5 to 8.5, with C-2 length substrate preference. No metal ions or inhibitors influenced lipolytic activity, except for PMSF, SDS, Cu−2, and Hg−2. Mgl-C255 retained about 50% of its activity in non-polar solvents and showed synthetic activity in organic solvents, making it a good candidate for studying its catalytic potential and selectivity.

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