Search Results (4)

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn²⁺ and/or Co²⁺ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 °C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The K_m values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (k_cat/K_m) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM^–1 min^–1, respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose. Full article

Extracted sugar beet press pulp (SBPP) is a promising agricultural residue for saccharification and further bioconversion. Combining solid-state fermentation of SBPP with engineered Aspergillus niger for enzyme production followed by hydrolysis of additionally added SBPP in the same bioreactor was studied to produce a sugar solution (hydrolysate) in a one-pot process. The initial aerobic solid-state fermentations were carried out in duplicate on non-milled, wet SBPP (moisture content of 72% (w/v)) with an A. niger strain engineered for constitutive pectinase production for 96 h, and this resulted in polygalacturonase activities of up to 256 U mL⁻¹ in the wet media. Afterwards, water was added to the bioreactor, and the remaining solids were suspended by stirring to dissolve the hydrolytic enzymes. Metabolic activities of A. niger were inactivated by a N₂-atmosphere and by increasing the temperature to 50 °C. High solid loads of milled SBPP were added to the stirred-tank reactor with a delay of 24 h to enable sugar yield calculations based on the compositional analysis of the SBPP used. The resulting final sugar concentrations of the hydrolysate after 166 h were 17 g L⁻¹ d-glucose, 18.8 g L⁻¹ l-arabinose, and 12.5 g L⁻¹ d-galacturonic acid, corresponding to sugar yields of 98% d-glucose, 86% l-arabinose, and 50% d-galacturonic acid, respectively. Including the other sugars released during enzymatic hydrolysis in the one-pot process (d-xylose, d-mannose, d-galactose), a total sugar concentration of 54.8 g L⁻¹ was achieved in the hydrolysate. The one-pot process combining hydrolytic enzyme production in solid-state fermentation with high solid loads during enzymatic hydrolysis of the milled SBPP reduces hydrolytic process costs by replacing chemical pre-treatments, enabling the in situ production of SBPP-adapted hydrolytic enzymes, as well as avoiding intermediate enzyme extraction and preparation steps. Full article

As electrode nanomaterials, thermally reduced graphene oxide (TRGO) and modified gold nanoparticles (AuNPs) were used to design bioelectrocatalytic systems for reliable D-tagatose monitoring in a long-acting bioreactor where the valuable sweetener D-tagatose was enzymatically produced from a dairy by-product D-galactose. For this goal D-fructose dehydrogenase (FDH) from Gluconobacter industrius immobilized on these electrode nanomaterials by forming three amperometric biosensors: AuNPs coated with 4-mercaptobenzoic acid (AuNP/4-MBA/FDH) or AuNPs coated with 4-aminothiophenol (AuNP/PATP/FDH) monolayer, and a layer of TRGO on graphite (TRGO/FDH) were created. The immobilized FDH due to changes in conformation and spatial orientation onto proposed electrode surfaces catalyzes a direct D-tagatose oxidation reaction. The highest sensitivity for D-tagatose of 0.03 ± 0.002 μA mM⁻¹cm⁻² was achieved using TRGO/FDH. The TRGO/FDH was applied in a prototype bioreactor for the quantitative evaluation of bioconversion of D-galactose into D-tagatose by L-arabinose isomerase. The correlation coefficient between two independent analyses of the bioconversion mixture: spectrophotometric and by the biosensor was 0.9974. The investigation of selectivity showed that the biosensor was not active towards D-galactose as a substrate. Operational stability of the biosensor indicated that detection of D-tagatose could be performed during six hours without loss of sensitivity. Full article

The transgalactosylations of serine/threonine derivatives were investigated using β-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-β-D-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl L-serine benzyl ester (23.2%) was achieved in heptane:buffer medium (70:30), whereas with the lactose, the highest bioconversion yield (3.94%) was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme’s active site was stronger (−4.6~−7.9 kcal/mol) than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8%) and N-carboxybenzyl serine benzyl ester (3.4%), their binding affinities and the distances between their hydroxyl side chain and the 1′-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2%) demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction. Full article