info:eu-repo/semantics/article
Biochemical characterization of heat-tolerant recombinant L-arabinose isomerase from Enterococcus faecium DBFIQ E36 strain with feasible applications in D-tagatose production
Fecha
2019-06Registro en:
Manzo, Ricardo Martín; Antunes, André Saraiva Leão Marcelo; de Sousa Mendes, Jocélia; Hissa, Denise Cavalcante; Goncalves, Luciana Rocha Barros; et al.; Biochemical characterization of heat-tolerant recombinant L-arabinose isomerase from Enterococcus faecium DBFIQ E36 strain with feasible applications in D-tagatose production; Humana Press; Molecular Biotechnology; 61; 6; 6-2019; 385-399
1073-6085
CONICET Digital
CONICET
Autor
Manzo, Ricardo Martín
Antunes, André Saraiva Leão Marcelo
de Sousa Mendes, Jocélia
Hissa, Denise Cavalcante
Goncalves, Luciana Rocha Barros
Mammarella, Enrique José
Resumen
D-Tagatose is a ketohexose, which presents unique properties as a low-calorie functional sweetener possessing a sweet flavor profile similar to D-sucrose and having no aftertaste. Considered a generally recognized as safe (GRAS) substance by FAO/WHO, D-tagatose can be used as an intermediate for the synthesis of other optically active compounds as well as an additive in detergent, cosmetic, and pharmaceutical formulations. This study reports important features for L-arabinose isomerase (EC 5.3.1.4) (L-AI) use in industry. We describe arabinose (araA) gene virulence analysis, gene isolation, sequencing, cloning, and heterologous overexpression of L-AI from the food-grade GRAS bacterium Enterococcus faecium DBFIQ E36 in Escherichia coli and assess biochemical properties of this recombinant enzyme. Recombinant L-AI (rL-AI) was one-step purified to homogeneity by Ni2+-agarose resin affinity chromatography and biochemical characterization revealed low identity with both thermophilic and mesophilic L-AIs but high degree of conservation in residues involved in substrate recognition. Optimal conditions for rL-AI activity were 50 °C, pH 5.5, and 0.3 mM Mn2+, exhibiting a low cofactor concentration requirement and an acidic optimum pH. Half-life at 45 °C and 50 °C were 1427 h and 11 h, respectively, and 21.5 h and 39.5 h at pH 4.5 and 5.6, respectively, showing the high stability of the enzyme in the presence of a metallic cofactor. Bioconversion yield for D-tagatose biosynthesis was 45% at 50 °C after 48 h. These properties highlight the technological potential of E. faecium rL-AI as biocatalyst for D-tagatose production.