Search Results (14)

The genera Parageobacillus and Geobacillus comprise thermophilic, spore-forming bacteria. The extraordinary heat resistance of their spores, together with their ability to form biofilms and produce thermostable enzymes, makes them a relevant cause of spoilage in shelf-stable, heat-treated products like dairy and canned foods. However, these same biological traits offer valuable opportunities for the food industry. In this context, the purpose of this review is to describe the challenges posed by (Para)Geobacillus spp. as food spoilage agents, while also highlighting their existing and prospective applications in the food industry. In terms of food safety, G. stearothermophilus spores are used as biological indicators in commercially available tests to detect antibiotic residues in food within a few hours. Additionally, (Para)Geobacillus can be exploited for the fermentation of agri-food residues to produce high-value compounds such as biofuels, food ingredients and technological adjuvants, and compost. Their thermostable enzymes—such as amylases, xylanases, L-arabinose isomerases, β-galactosidases, lipases, proteases, and L-asparaginases—have potential applications in food processing and ingredient production. However, several challenges persist, including limited knowledge on genetic diversity, physiology, and metabolism, as well as low yields of biomass and target compounds. These issues reinforce the need for further studies to unlock their full potential. Full article

As global milk production continues to rise, the disposal of expired milk contributes to environmental pollution and valuable resource wastage. This study presents the development of a novel L-arabinose isomerase, designated BmAIase12, and its application in the enzymatic recycling of expired milk. BmAIase12 exhibited a specific activity of 10.7 U/mg and showed optimal performance at 50 °C and pH 7.0. Furthermore, it exhibited higher activity than most other L-arabinose isomerases. It converted D-galactose into D-tagatose with a high conversion ratio of 53.3% after 48 h at 50 °C. The conversion efficiency of expired milk to D-tagatose was recorded at 40.62%, resulting in a maximum tagatose yield of 1.625 g/L. This was accomplished through the incorporation of β-galactosidase (120 U/mL) and Saccharomyces cerevisiae (30 mg/mL) to hydrolyze lactose and metabolize glucose, followed by the addition of 3 U/mL of BmAIase12. Ultimately, following purification, the purity of tagatose was determined to be 98%, and the final yield was 29.8%. These results suggest that BmAIase12 may serve as a promising enzyme for D-tagatose production due to its high conversion yield. Full article

D-tagatose is a functional sweetener with glucose-regulating and prebiotic properties, but its bioproduction from D-galactose faces many limitations, particularly the high production costs. In particular, the current biosynthesis of D-tagatose suffers from thermal instability and the substrate selectivity issues of L-arabinose isomerase (L-AI) required to convert D-galactose into D-tagatose. In this study, recombinant Escherichia coli BW25113/pQE-80L-araA_F118M/F279I expressing double mutant L-AI was immobilized to enhance its stability and reusability. The optimal conditions for whole-cell catalysis were 60 °C, pH 6.5, 5 mM Mn²⁺, and 20 h, with a yield of 55.2 g/L of D-tagatose. Immobilization with 3% sodium alginate and 2% CaCl₂ retained 90% of the production efficiency displayed by free cells. Notably, the immobilized cells exhibited enhanced heat resistance (60–70 °C) and operational stability, retaining 76% activity after five cycles. The D-tagatose production was further increased to 129.43 g/L by increasing the substrate concentration to 250 g/L. Compared to free cells, immobilized cells retained 83.6% of the initial yield up to 10 batches. This study presents a cost-effective and sustainable method for the production of D-tagatose using optimized whole-cell catalysis through immobilization, which paves the way to solve industrial challenges such as thermal instability and low substrate efficiency. Full article

Agricultural and industrial activities are increasing pollution of water bodies with low doses of xenobiotics that have detrimental effects on aquaculture. The aim of this work was to determine the possibility of using Levilactobacillus brevis 47f culture in fish aquaculture under the influence of low doses of xenobiotics as an adaptogen. An increase in the survival of Danio rerio individuals exposed to the xenobiotic bisphenol A solution and fed with the L. brevis 47f was shown compared to control groups and, at the same time, the cytokine profile in the intestinal tissues of Danio rerio was also investigated. Analysis of differential gene expression of the L. brevis 47f grown under the action of high concentrations of bisphenol A showed changes in mRNA levels of a number of genes, including genes of various transport proteins, genes involved in fatty acid synthesis, genes of transcriptional regulators, genes of the arabinose operon, and the oppA gene. The identification of L. brevis 47f proteins from polyacrylamide gel by mass spectrometry revealed L-arabinose isomerase, Clp chaperone subunit, ATP synthase subunits, pentose phosphate pathway and glycolysis enzyme proteins, which are likely part of the L. brevis 47f strain’s anti-stress response, but probably do not affect its adaptogenic activity toward Danio rerio. Full article

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose. Full article

d-tagatose is a low-calorie alternative to sucrose natural monosaccharide that is nearly as sweet. As a ketohexose, d-tagatose has disease-relieving and health-promoting properties. Due to its scarcity in nature, d-tagatose is mainly produced through chemical and biological methods. Compared to traditional chemical methods, biological methods use whole cells and isolated enzymes as catalysts under mild reaction conditions with few by-products and no pollution. Nowadays, biological methods have become a very important topic in related fields due to their high efficiency and environmental friendliness. This paper introduces the functions and applications of d-tagatose and systematically reviews its production, especially by l-arabinose isomerase (L-AI), using biological methods. The molecular structures and catalytic mechanisms of L-AIs are also analyzed. In addition, the properties of L-AIs from different microbial sources are summarized. Finally, we overview strategies to improve the efficiency of d-tagatose production by engineering L-AIs and provide prospects for the future bioproduction of d-tagatose. Full article

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

d-Tagatose, a functional sweetener, is converted from d-galactose by l-arabinose isomerase, which catalyzes the conversion of l-arabinose to l-ribulose. In this study, the araA gene encoding l-arabinose isomerase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli, and the expressed enzyme was purified and characterized. The purified l-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for d-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 °C in the presence of Mn²⁺ and relative activity for pentoses and hexoses in the order l-arabinose > d-galactose > l-ribulose > d-xylulose > d-xylose > d-tagatose > d-glucose. The thermal stability of recombinant E. coli cells expressing l-arabinose isomerase from K. pneumoniae was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 °C, and 4 g/L cell concentration using 100 g/L d-galactose with 1 mM Mn²⁺. Under these conditions, 33.5 g/L d-tagatose was produced from d-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce d-tagatose using whole-cell reactions, extending its industrial application. Full article

d-tagatose is a popular functional monosaccharide produced from lactose by β-galactosidase and arabinose isomerase. In this study, two d-alanine-deficient heterologous gene expression systems were constructed, B. subtilis 168 D1 and B. subtilis 168 D2, using overlapping extension PCR and the CRE/loxP system. The lacZ gene for β-galactosidase was integrated into a specific locus of the chassis B. subtilis 168 D2. A mutually complementary plasmid pMA5 with the alanine racemase gene alrA attached to it was constructed and used to assemble recombinant plasmids overexpressing β-galactosidase and arabinose isomerase. Afterward, an integrated recombinant was constructed by the plasmid expressing the arabinose isomerase gene araA of E. coli transform-competent B. subtilis 168 D2 cells. The co-expressing plasmids were introduced into alanine racemase knockout B. subtilis 168 D1. Whole-cell bioconversion was performed using the integrated recombinant with a maximum yield of 96.8 g/L d-tagatose from 500 g/L lactose, and the highest molar conversions were 57.2%. B. subtilis 168 D1/pMA5-alrA-araA-lacZ is capable of single-cell one-step production of d-tagatose. This study provides a new approach to the production of functional sugars. 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 Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED) pathways are considered the most abundant catabolic pathways found in microorganisms, and ED enzymes have been shown to also be widespread in cyanobacteria, algae and plants. In a large number of organisms, especially common strains used in molecular biology, these pathways account for the catabolism of glucose. The existence of pathways for other carbohydrates that are relevant to biomass utilization has been recognized as new strains have been characterized among thermophilic bacteria and Archaea that are able to transform simple polysaccharides from biomass to more complex and potentially valuable precursors for industrial microbiology. Many of the variants of the ED pathway have the key dehydratase enzyme involved in the oxidation of sugar derived from different families such as the enolase, IlvD/EDD and xylose-isomerase-like superfamilies. There are the variations in structure of proteins that have the same specificity and generally greater-than-expected substrate promiscuity. Typical biomass lignocellulose has an abundance of xylan, and four different pathways have been described, which include the Weimberg and Dahms pathways initially oxidizing xylose to xylono-gamma-lactone/xylonic acid, as well as the major xylose isomerase pathway. The recent realization that xylan constitutes a large proportion of biomass has generated interest in exploiting the compound for value-added precursors, but few chassis microorganisms can grow on xylose. Arabinose is part of lignocellulose biomass and can be metabolized with similar pathways to xylose, as well as an oxidative pathway. Like enzymes in many non-phosphorylative carbohydrate pathways, enzymes involved in L-arabinose pathways from bacteria and Archaea show metabolic and substrate promiscuity. A similar multiplicity of pathways was observed for other biomass-derived sugars such as L-rhamnose and L-fucose, but D-mannose appears to be distinct in that a non-phosphorylative version of the ED pathway has not been reported. Many bacteria and Archaea are able to grow on mannose but, as with other minor sugars, much of the information has been derived from whole cell studies with additional enzyme proteins being incorporated, and so far, only one synthetic pathway has been described. There appears to be a need for further discovery studies to clarify the general ability of many microorganisms to grow on the rarer sugars, as well as evaluation of the many gene copies displayed by marine bacteria. Full article

We have developed a sustainable three-stage process for the revaluation of cheese whey permeate into D-tagatose, a rare sugar with functional properties used as sweetener. The experimental conditions (pH, temperature, cofactors, etc.) for each step were independently optimized. In the first step, concentrated whey containing 180–200 g/L of lactose was fully hydrolyzed by β-galactosidase from Bifidobacterium bifidum (Saphera^®) in 3 h at 45 °C. Secondly, glucose was selectively removed by treatment with Pichia pastoris cells for 3 h at 30 °C. The best results were obtained with 350 mg of cells (previously grown for 16 h) per mL of solution. Finally, L-arabinose isomerase US100 from Bacillus stearothermophilus was employed to isomerize D-galactose into D-tagatose at pH 7.5 and 65 °C, in presence of 0.5 mM MnSO₄. After 7 h, the concentration of D-tagatose was approximately 30 g/L (33.3% yield, referred to the initial D-galactose present in whey). The proposed integrated process takes place under mild conditions (neutral pH, moderate temperatures) in a short time (13 h), yielding a glucose-free syrup containing D-tagatose and galactose in a ratio 1:2 (w/w). Full article

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg⁻¹, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve. Full article

The use of ketohexose isomerases is a powerful tool in lactose whey processing, but these enzymes can be very sensitive and expensive. Development of immobilized/stabilized biocatalysts could be a further option to improve the process. In this work, β-galactosidase from Bacillus circulans, l-arabinose (d-galactose) isomerase from Enterococcus faecium, and d-xylose (d-glucose) isomerase from Streptomyces rubiginosus were immobilized individually onto Eupergit C and Eupergit C 250 L. Immobilized activity yields were over 90% in all cases. With the purpose of increasing thermostability of derivatives, two post-immobilization treatments were performed: alkaline incubation to favor the formation of additional covalent linkages, and blocking of excess oxirane groups by reacting with glycine. The greatest thermostability was achieved when alkaline incubation was carried out for 24 h, producing l-arabinose isomerase-Eupergit C derivatives with a half-life of 379 h and d-xylose isomerase-Eupergit C derivatives with a half-life of 554 h at 50 °C. Preliminary assays using immobilized and stabilized biocatalysts sequentially to biotransform lactose at pH 7.0 and 50 °C demonstrated improved performances as compared with soluble enzymes. Further improvements in ketohexose productivities were achieved when the three single-immobilizates were incubated simultaneously with lactose in a mono-reactor system. Full article