Biochemical Characterization of Heat-Tolerant Recombinant l-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 Strain with Feasible Applications in d-Tagatose Production

Abstract

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 Ni 2+ -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 Mn 2+ , 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. Keywords l-Arabinose isomerase • d-Tagatose • Enterococcus faecium • Virulence gene analysis • d-Galactose

Reagentless D-Tagatose Biosensors Based on the Oriented Immobilization of Fructose Dehydrogenase onto Coated Gold Nanoparticles- or Reduced Graphene Oxide-Modified Surfaces: Application in a Prototype Bioreactor

“…However, the chemical route has several disadvantages such as high temperature and high pressure during the process [ 23 ]. In contrast, recently significant literature has grown up around the theme of D-tagatose biosynthesis [ 24 , 25 , 26 , 27 ].…”

Alternative Heterologous Expression of l-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 By Residual Whey Lactose Induction

“…However, the biological production of d-tagatose has several advantages, such as, the use of mild reaction conditions and high specificity due to the use of l-arabinose isomerase (EC 5.3.1.4, l-AI), which isomerizes d-galactose into d-tagatose [9][10][11]. l-AI can be obtained from various bacterial strains, such as Enterococcus faecium [12], Pediococcus pentosaceus [13], Saccharomyces cerevisiae [14], Thermoanaerobacterium saccharolyticum [15], Geobacillus stearothermophilus [16], Pseudoalteromonas haloplanktis [17], among other sources of origin.…”

“…Heterologous expression of l-AI using Escherichia coli as host is usually done by the use of isopropyl-1-thio-β-dgalactopyranoside (IPTG), a synthetic lactose analog [18], as an inducer agent which was applied in many studies [12,13,15,19]. This inducer acts by removing the repressor protein present in the lac operon, promoting enzyme expression [20][21][22].…”

“…Therefore, in this work, a possible replacement of IPTG as inducer agent was investigated and a promising method for the heterologous expression of Enterococcus faecium l-AI was proposed. This enzyme has shown relevant physicochemical properties with industrial feasibility [12]. In this method, IPTG is replaced by residual whey lactose through an auto-induction process (AI).…”

A Three-Step Process for the Bioconversion of Whey Permeate into a Glucose-Free D-Tagatose Syrup

“…Only 20% of the ingested D-tagatose is absorbed in the small intestine [8]. Since the remaining 80% seems to favourably modulate the composition of the gut microbiota [9], this sugar has been proposed as a potential prebiotic.…”