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This study reports an alternative strategy for the expression of a recombinant l-AI from Enterococcus faecium DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual whey lactose as inducer agent. Commercial lactose and isopropyl β-d-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of d-galactose into d-tagatose. l-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant l-AI aiming its high-scale production. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

l-Arabinose isomerase (L-AI, EC 5.3.1.4) catalyzes the isomerization between l-arabinose and l-ribulose, and most of the reported ones can also catalyze d-galactose to d-tagatose, except Bacillus subtilis L-AI. In this article, the L-AI from the psychrotolerant bacterium Pseudoalteromonas haloplanktis ATCC 14393 was characterized. The enzyme showed no substrate specificity toward d-galactose, which was similar to B. subtilis L-AI but distinguished from other reported L-AIs. The araA gene encoding the P. haloplanktis L-AI was cloned and overexpressed in E. coli BL21 (DE3). The recombinant enzyme was purified by one-step nickel affinity chromatography . The enzyme displayed the maximal activity at 40 °C and pH 8.0, and showed more than 75 % of maximal activity from pH 7.5-9.0. Metal ion Mn was required as optimum metal cofactor for activity simulation, but it did not play a significant role in thermostability improvement as reported previously. The Michaelis-Menten constant ( K ), turnover number ( k ), and catalytic efficiency ( k / K ) for substrate l-arabinose were measured to be 111.68 mM, 773.30/min, and 6.92/mM/min, respectively. The molecular docking results showed that the active site residues of P. haloplanktis L-AI could only immobilize l-arabinose and recognized it as substrate for isomerization. [ABSTRACT FROM AUTHOR]