Search

BACKGROUND: Cellobiose 2‐epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3‐1 (IfCE) was successfully expressed in Escherichia coli and characterized. RESULTS: Unlike other CEs, the purified IfCE shows only epimerization activity toward β‐1,4‐glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, β‐1,4‐glycosidic linkages of trisaccharides and α‐1,4‐glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low‐calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL−1 lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L−1, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl− and Na+ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen. CONCLUSION: The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Epilactose is a disaccharide composed of galactose and mannose, and it is currently considered an "under development" prebiotic. In this study, we described the prebiotic potential of epilactose by in vitro fermentation using human fecal inocula from individuals following a Mediterranean diet (DM) or a Vegan diet (DV). The prebiotic effect of epilactose was also compared with lactulose and raffinose, and interesting correlations were established between metabolites and microbiota modulation. The production of several metabolites (lactate, short-chain fatty acids, and gases) confirmed the prebiotic properties of epilactose. For both donors, the microbiota analysis showed that epilactose significantly stimulated the butyrate-producing bacteria, suggesting that its prebiotic effect could be independent of the donor diet. Butyrate is one of the current golden metabolites due to its benefits for the gut and systemic health. In the presence of epilactose, the production of butyrate was 70- and 63-fold higher for the DM donor, when compared to lactulose and raffinose, respectively. For the DV donor, an increase of 29- and 89-fold in the butyrate production was obtained when compared to lactulose and raffinose, respectively. In conclusion, this study suggests that epilactose holds potential functional properties for human health, especially towards the modulation of butyrate-producing strains. [ABSTRACT FROM AUTHOR]

The discovery that certain cellobiose 2-epimerases (C2E) are capable of synthesizing lactulose and epilactose with high yield and selectivity has renewed the interest in their biological synthesis. In order to provide a valuable tool for the design and optimization of the synthesis of lactulose and epilactose with C2E as biocatalyst, the reaction is modeled here for the first time. Two competing reaction mechanisms, so called extended and reduced mechanisms, were proposed and their corresponding models developed. The extended mechanism was discarded because of the overfitting of the resulting model. The parameters of the reduced model were successfully estimated by no linear regression at 30, 40, 50, 60 and 70 °C, and the temperature dependence of the kinetic parameters was modeled by fitting them to Arrhenius and Vant Hoff-type equations. The reduced model showed a good fit to experimental data within the studied temperature range, well describing the course of reactions. [ABSTRACT FROM AUTHOR]

Epilactose is a disaccharide composed of galactose and mannose, and it is currently considered an “under development” prebiotic. In this study, we described the prebiotic potential of epilactose by in vitro fermentation using human fecal inocula from individuals following a Mediterranean diet (DM) or a Vegan diet (DV). The prebiotic effect of epilactose was also compared with lactulose and raffinose, and interesting correlations were established between metabolites and microbiota modulation. The production of several metabolites (lactate, short-chain fatty acids, and gases) confirmed the prebiotic properties of epilactose. For both donors, the microbiota analysis showed that epilactose significantly stimulated the butyrate-producing bacteria, suggesting that its prebiotic effect could be independent of the donor diet. Butyrate is one of the current golden metabolites due to its benefits for the gut and systemic health. In the presence of epilactose, the production of butyrate was 70- and 63-fold higher for the DM donor, when compared to lactulose and raffinose, respectively. For the DV donor, an increase of 29- and 89-fold in the butyrate production was obtained when compared to lactulose and raffinose, respectively. In conclusion, this study suggests that epilactose holds potential functional properties for human health, especially towards the modulation of butyrate-producing strains.

Epilactose, a lactose derivative known for its prebiotic properties and potential health benefits, has garnered significant interest. Cellulose 2-epimerase (CEase) is responsible for catalyzing the conversion of lactose to epilactose. In this study, the enhancement of food-grade CEase expression in Bacillus subtilis WB600 was systematically investigated. Among seven selected epilactose-producing CEases, Rhodothermus marinus CEase (RmCE) exhibited the highest epimerization activity when expressed in B. subtilis. Translational and transcriptional regulations were employed to enhance CEase expression by screening effective N-terminal coding sequences (NCSs) and promoters. The final strain demonstrated efficient production of CEase, with epimerization activity reaching 273.6 ± 6.5 U/mL and 1255 ± 26.4 U/mL in shake-flask and fed-batch cultivation, respectively. Utilizing only 0.25 % (V /V) of the fed-batch cultivation broth for lactose biotransformation, epilactose was efficiently produced from 300 g/L of lactose within 4 h, achieving a yield of 29.5 %. These findings provide significant support for the potential industrialization of enzymatic epilactose production. • CEase was effectively expressed in Bacillus subtilis for epilactose production. • Translational and transcriptional regulations were used to enhance CEase expression. • Fermentation activity reached 1255 U/mL by fed-batch cultivation. • Epilactose was efficiently produced from 300 g/L of lactose with a yield of 29.5 %. [ABSTRACT FROM AUTHOR]

Bioconversion of lactose to functional lactose derivatives attracts increasing attention. Lactulose is an important high-value lactose derivative, which has been widely used in pharmaceutical, nutraceutical, and food industries. Lactulose can be enzymatically produced from lactose by cellobiose 2-epimerase (CEase). Several studies have already focused on the food-grade expression of CEase, but they are all aimed at the biosynthesis of epilactose. Herein, we reported for the first time the biosynthesis of lactulose using the recombinant food-grade Bacillus subtilis. Lactulose biosynthesis was optimized by varying lactulose-producing CEases and expression vectors. Caldicellulosiruptor saccharolyticus CEase and pP43NMK were determined to be the optimal CEase and expression vector. Fine-tuning of CEase expression was investigated by screening a beneficial N-terminal coding sequence. After fed-batch cultivation, the highest fermentation isomerization activity reached 11.6 U/mL. Lactulose was successfully produced by the broth of the engineered B. subtilis with a yield of 52.1 %. • CEase was effectively expressed in Bacillus subtilis for lactulose production. • N-terminal coding sequence was used to increase the expression level of CEase. • Isomerization activity of the engineered B. subtilis reached 11.6 U/mL. • Lactulose yield of the engineered B. subtilis reached 52.1 %. [ABSTRACT FROM AUTHOR]

BACKGROUND: Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. RESULTS: The encoding gene of cellobiose 2‐epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by‐products, because of high kcat (197.6 s−1) and preferable thermostability. The Rt8‐CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L−1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a β‐galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d‐tagatose was successfully produced from lactose through the co‐expression of l‐arabinose isomerase and β‐galactosidase. CONCLUSION: Our study shed light on the efficient production of epilactose using a food‐grade host expressing a novel CE enzyme. Moreover, an efficient and low‐cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d‐tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The dairy industry is among the most important food industries worldwide. However, the increase in the global dairy industry has led to public environmental problems due to the disposal of whey. A promising approach for the sustainable utilization of whey is converting lactose to valuable lactose derivatives. Epilactose is an interesting bioactive lactose derivative that is enzymatically produced from lactose by cellobiose 2-epimerase (CEase). This review focuses on the health effects and enzymatic production of epilactose. In particular, the enzymatic properties and kinetics, molecular structures, modifications, and practical application of CEases are reviewed. Epilactose is a nondigestible carbohydrate that shows promising prebiotic activity and potentially promotes intestinal mineral absorption, alleviates postgastrectomy osteopenia and anemia, reduces the plasma cholesterol level, and prevents obesity and metabolic disorders. Since 2007, thirty microbial CEases have been identified, and five of their crystal structures have been determined. Most CEases catalyze only the C-2 epimerization of lactose to epilactose, but some hyperthermophilic CEases are bifunctional enzymes that catalyze isomerization and epimerization and produce lactulose as the main product. Food-grade expression of CEases and downstream purification of epilactose have been attempted. However, studies on the molecular modification of CEases for enhanced epilactose productivity remain limited, and enzymatic immobilization and downstream processing should be greatly improved for practical industrialization of epilactose. • Epilactose is one of the most important lactose derivatives with promising functions. • CEase is an important enzyme in dairy industry, which is comprehensively reviewed. • Various approaches for epilactose production and purification are addressed. • Production of epilactose by CEase provides a promising way for lactose utilization. [ABSTRACT FROM AUTHOR]

[Display omitted] • Rare sugars were produced with new mutant cellobiose 2-epimerase (CsCE H356N) • Different substrates were evaluated for isomerization and epimerization reactions. • Isomerization were irreversible while epimerization were reversible reactions. • Mannose and fructose were produced from glucose with CsCE H356N. • Talose and tagatose were produced from galactose with CsCE H356N. Cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) can epimerize and isomerize lactose into epilactose and lactulose respectively. Competition between these reactions reactions has prompted the search for new enzymes to drive the reaction in one direction or the other. The isomerization and epimerization capacity of a novel mutant CsCE (CsCE H356N) was evaluated, obtaining a maximum lactulose yield of 64.3 % and a lactulose selectivity of 9.9. A Michaelis-Menten constant of 551.93 mM and a catalytic efficiency of 0,058 s−1 mM−1 were obtained for lactose epimerization. The ability of CsCE H356N to recognize other substrates was evaluated using lactulose, glucose, mannose, fructose, galactose, talose and tagatose as substrates, assessing the reversibility of such reactions. Yields of 14.8 % mannose and 4.8 % of fructose were obtained from glucose, while talose and tagatose yields of 9.2 % and 5.2 % were obtained from galactose respectively. No significant reaction occurred with lactulose, fructose or tagatose as substrates. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simple and food-grade two-step process to purify epilactose was developed. • The purification process led to the highest epilactose yield obtained so far. • Epilactose was recovered with a yield of 76.4% and a purity of 87% • Epilactose was resistant to digestion processes in the upper gastrointestinal tract. Epilactose is a functional sugar that can be produced from lactose using cellobiose 2-epimerase and it is considered a developing prebiotic. In that sense, the development of strategies to produce and purify epilactose is key for its wider use in the food industry. The aim of this work was to establish a food-grade purification strategy suitable to be scaled-up to an industrial level. Firstly, the epilactose was produced by enzymatic epimerization of lactose in a reaction catalyzed by the recombinant cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus produced by Saccharomyces cerevisiae. Then, to remove the unreacted lactose, a screening study was performed to find a suitable β-galactosidase enzyme with high lactose hydrolysis capacity but low ability to convert the epilactose. The elimination of the generated monosaccharides was then attempted by microbial treatment using different microorganisms and using activated charcoal. The baker's yeast S. cerevisiae was proven to be the most suitable microorganism for glucose and galactose removal from the reaction mixture. Overall, an attractive and food-grade two-step process for epilactose recovery was established, resulting in a purity and yield of 87% and 76.4%, respectively. Additionally, the INFOGEST 2.0 static in vitro simulation of gastrointestinal food digestion was used, for the first time, to assess the resistance of epilactose (77% resistance) to the upper gastrointestinal tract conditions, reinforcing its potential to be used as prebiotic. [ABSTRACT FROM AUTHOR]