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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]

Lactose is the most abundant by-product of the dairy industry and is increasingly recognized as an important feedstock to produce value-added compounds. Lactulose and epilactose are valuable prebiotics that can be generated from lactose with cellobiose 2-epimerases (CEases). Here we describe the characterization of the two CEases CbCEP and RfCEP, originated from the thermophilic microorganism Caldicellulosiruptor bescii and the mesophilic microorganism Roseburia faecis, respectively. CbCEP showed exceptional pH and temperature stability, with maximal activity at pH 7.5 and 70 °C. RfCEP exhibited maximum activity at 50 °C and pH 8. Under optimal conditions in small-scale experiments with commercial lactose, CbCEP produced both lactulose and epilactose with yields of 29.8% and 21.6%, respectively, while RfCEP produced only epilactose with a yield of 19.3%. Furthermore, we evaluated the application of CbCEP for lactulose and epilactose production in stirred fermenters at two different temperatures (70 and 30 °C) at a scale of 5 L volume and a concentration of cheese-whey permeate of 50 g/L. At 70 °C, lactulose was the predominant product of CbCEP-mediated lactose conversion, with a final yield of 30% (12.8 g/L) lactulose and 24.7% (10.6 g/L) epilactose. At 30 °C, lactose was mainly converted into epilactose, with a final yield of 35% (14.9 g/L), and a minor amount of lactulose (final yield of 4.3%, 1.8 g/L). The findings presented here may guide the design of an industrial strategy, based on the temperature-tunable activity of CbCEP, for production of valuable lactose derivatives at high yields directly from whey permeate.

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]

Cellobiose 2-epimerase (CE) is a promising industrial enzyme that can catalyze bioconversion of lactose to its high-value derivatives, namely epilactose and lactulose. A need exists in the dairy industry to catalyze lactose bioconversions at low temperatures to avoid microbial growth. We focused on the discovery of cold-active CE in this study. A genome mining method based on computational prediction was used to screen the potential genes encoding cold-active enzymes. The CE-encoding gene from Roseburia intestinalis , with a predicted high structural flexibility, was expressed heterologously in Escherichia coli. The catalytic property of the recombinant enzyme was extensively studied. The optimum temperature and pH of the enzyme were 45°C and 7.0, respectively. The specific activity of this enzyme to catalyze conversion of lactose to epilactose was measured to be 77.3 ± 1.6 U/mg. The kinetic parameters, including turnover number (k cat), Michaelis constant (K m), and catalytic efficiency (k cat / K m) using lactose as a substrate were 117.0 ± 7.7 s−1, 429.9 ± 57.3 m M , and 0.27 m M −1s−1, respectively. In situ production of epilactose was carried out at 8°C: 20.9% of 68.4 g/L lactose was converted into epilactose in 4 h using 0.02 mg/mL (1.5 U/mL, measured at 45°C) of recombinant enzyme. The enzyme discovered by this in silico method is suitable for low-temperature applications. [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]

Abstract Background Over the past decade, substantial advances have been made in the study of cellobiose 2-epimerase (CE). CE participates in the recently discovered mannan metabolism and catalyzes the interconversion of D-glucose residues at the reducing end of mannooligosaccharides. This enzyme also exhibits different degrees of epimerization and isomerization activities towards different saccharides. Scope and approach This review addresses the latest research on the enzymatic properties, structural information, and technical advances relating to CE. Key findings and conclusions CE is attractive because this enzyme can convert lactose into its epimeric and isomeric forms, epilactose and lactulose, respectively. Epilactose and lactulose are candidate functional food materials that confer significant economic value to CE in the dairy industry. Although a broad range of CEs from different mesophilic and thermophilic microorganisms have been characterized, the present knowledge about the relationship between the function and structure of CE is limited and not connected to further genetic modifications for potential CE applications. Thus, more attention has been paid to the midstream and downstream processes for industrial applications of CE. A number of studies on CE have been performed to seek out better industrial applications. However, further efforts are needed to understand the underlying enzymatic reaction mechanism and protein engineering to fulfill the full potential of CE. Highlights • Cellobiose 2-epimerase (CE) is an important tool for functional disaccharides production. • The enzymatic properties of all reported CEs are comprehensively discussed. • Discussed the epimerization and isomerization mechanisms of CEs based on crystal structures. • Reported the properties and production methods of two primary CE-involving products, epilactose and lactulose. [ABSTRACT FROM AUTHOR]

Lactose is a main by-product in the cheese industry. Many attempts have been made to convert the lactose to high value-added products, including epilactose. Epilactose is a valuable prebiotic and can be epimerized from lactose with cellobiose 2-epimerase (CEase). The objective of the present work was to construct a food-grade recombinant Bacillus subtilis that produces CEase from Thermoanaerobacterium saccharolyticum. The CEase was expressed in B. subtilis without antibiotic resistance genes. After fermentation, the maximum volumetric activity of the fermented broth was more than 7 U/mL. The activity of the recombinant B. subtilis was increased by up to 3.7 fold after ethanol permeabilization. Then, 66.9 ± 0.7 g/L of epilactose was produced from 300 g/L of whey powder solution in 1 h with 13.3 U/mL of permeabilized biocatalyst. In addition, an enzymatic route including degradation of the lactose, yeast fermentation, and cation exchange chromatography was described to further purify the produced epilactose from lactose. Finally, epilactose with a purity >98% was produced from 300 g/L of lactose with a yield of 24.0%. In conclusion, neither antibiotics nor pathogenic bacteria were used throughout the epilactose production and purification procedure. [ABSTRACT FROM AUTHOR]

BACKGROUND Lactulose, a bioactive lactose derivative, has been widely used in food and pharmaceutical industries. Isomerisation of lactose to lactulose by cellobiose 2-epimerase ( CE) has recently attracted increasing attention, since CE produces lactulose with high yield from lactose as a single substrate. In this study, a new lactulose-producing CE from Caldicellulosiruptor obsidiansis was extensively characterised. RESULTS The recombinant enzyme exhibited maximal activity at pH 7.5 and 70 °C. It displayed high thermostability with Tm of 86.7 °C. The half-life was calculated to be 8.1, 2.8 and 0.6 h at 75, 80, and 85 °C, respectively. When lactose was used as substrate, epilactose was rapidly produced in a short period, and afterwards both epilactose and lactose were steadily isomerised to lactulose, with a final ratio of 35:11:54 for lactose:epilactose:lactulose. When the reverse reaction was investigated using lactulose as substrate, both lactose and epilactose appeared to be steadily produced from the start. CONCLUSION The recombinant CE showed both epimerisation and isomerisation activities against lactose, making it an alternative promising biocatalyst candidate for lactulose production. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Epilactose (4- O- β- d-galactopyranosyl- d-mannopyranose) is a rare disaccharide derived from lactose (4- O- β- d-galactopyranosyl- d-glucopyranose) and possesses prebiotic properties. Epilactose is needed for biological studies in high amounts and purified form. The purification of epilactose is challenging because it behaves similarly to lactose, the starting substance of the epilactose production process. Epilactose can be produced both chemically and enzymatically, using cellobiose 2-epimerases for the latter. In this study, a process for semi-preparative epilactose purification is presented. Firstly, epilactose was produced in a buffered system using recombinant cellobiose 2-epimerase from Flavobacterium johnsoniae. After the enzymatic conversion, some of the residual lactose was removed by crystallisation at 6 °C for 72 h. Subsequently, the lactose still remaining in the conversion mixture was separated from epilactose by ligand-exchange chromatography. The conditions for the most effective chromatographic separation of lactose and epilactose were determined using a design of experiments approach with surface response methodology. The best conditions led to an epilactose purity of 99 % with a total yield of 51 %. [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]

The valuable lactose derivatives lactulose and epilactose can be derived from lactose either by the Lobry de Bruyn-Alberda van Ekenstein transformation during heat treatments or by enzymatic conversion using cellobiose 2-epimerases (EC 5.1.3.11). The chromatographic determination of lactose, lactulose, and epilactose in milk is challenging, due to the variable ratio of the three saccharides and their similar retention properties. In this work, a dual high-performance liquid chromatography (HPLC) analysis for the quantification of lactose, lactulose, and epilactose in milk samples was developed and validated. The samples originated from an enzymatic lactose conversion using the cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. Application of this enzyme led to the formation of high lactulose concentrations (28.0 g/L) in milk. The dual HPLC analysis utilized a combination of two chromatographic separation techniques, configured in two parallel systems. After precolumn derivatization, the samples were analyzed as follows: Method 1 determined the concentration of lactose and epilactose using a C column with an ion-pair reagent as eluent, coupled with a UV detector. Method 2 determined the concentration of lactulose using a trimodal stationary phase (hydrophilic interaction, anion- and cation-exchange properties) with acetonitrile/ammonium formiate buffer as eluent, coupled with an evaporative light scattering detector. Both methods were validated in terms of linearity, precision and recovery. The revealing detection limits in the milk samples were 3.32 mg/L for lactose, 4.73 mg/L for epilactose and 139 mg/L for lactulose. The dual HPLC analysis presented allows accurate lactose, lactulose, and epilactose separation in complex food matrices such as milk. [ABSTRACT FROM AUTHOR]

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing endd-glucose residue of β-(1→4)-linked disaccharides tod-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)6-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide. Reactions and substrate recognition of cellobiose 2-epimerase (CE) and mannoside phopshorylases. CE produces prebiotic epilactose. CE epimerizes β-d-Manp-(1→4)-d-Manpfor further phosphorolysis. [ABSTRACT FROM AUTHOR]

Obesity is one of the major health problems throughout the world. The present study investigated the preventive effect of epilactose – a rare non-digestible disaccharide – on obesity and metabolic disorders in mice fed high-fat (HF) diets. Feeding with HF diets increased body weight gain, fat pad weight and adipocyte size in mice (P<0·01), and these increases were effectively prevented by the use of supplemental epilactose without influencing food intake (P<0·01). Caecal pools of SCFA such as acetic and propionic acids in mice fed epilactose were higher compared with mice not receiving epilactose. Supplemental epilactose increased the expression of uncoupling protein (UCP)-1, which enhances energy expenditure, to 2-fold in the gastrocnemius muscle (P=0·04) and to 1·3-fold in the brown adipose tissue (P=0·02) in mice fed HF diets. Feeding HF diets induced pro-inflammatory macrophage infiltration into white adipose tissue, as indicated by the increased expression of monocyte chemotactic protein-1, TNF-α and F4/80, and these increases were attenuated by supplemental epilactose. In differentiated myogenic-like C2C12 cells, propionic acid, but not acetic or n-butyric acids, directly enhanced UCP-1 expression by approximately 2-fold (P<0·01). Taken together, these findings indicate that the epilactose-mediated increase in UCP-1 in the skeletal muscle and brown adipose tissue can enhance whole-body energy expenditure, leading to effective prevention of obesity and metabolic disorders in mice fed HF diets. It is suggested that propionic acid – a bacterial metabolite – acts as a mediator to induce UCP-1 expression in skeletal muscles. [ABSTRACT FROM PUBLISHER]

The enzymatic production of lactulose was described recently through conversion of lactose by a thermophilic cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE). In the current study, we examined the application of CsCE for lactulose and epilactose production in milk (1.5% fat). The bioconversions were carried out in stirred reaction vessels at 2 different temperatures (50 and 8°C) at a scale of 25 mL volume. At 50°C, 2 highly different CsCE amounts were investigated for the time course of formation of lactulose and epilactose. The conversion of milk lactose (initial lactose content of 48.5 ± 2.1 g/L) resulted in a final yield of 57.7% (28.0 g/L) lactulose and 15.5% (7.49 g/L) epilactose in the case of the approximately 9.5- fold higher CsCE amount (39.5 μkatepilactose, 50°C) after 24 h. Another enzymatic lactose conversion was carried out at low 8°C, an industrially relevant temperature for milk processing. Although the CsCE originated from a thermophilic microorganism, it was still applicable at 8°C. This enzymatic lactose conversion resulted in 56.7% (27.5 g/L) lactulose and 13.6% (6.57 g/L) epilactose from initial milk lactose after 72 h. The time courses of lactose conversion by CsCE suggested that first epilactose formed and afterward lactulose via epilactose. To the best of our knowledge, this is the first time that an enzyme has produced lactulose directly in milk in situ at industrially relevant temperatures. [ABSTRACT FROM AUTHOR]

A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity was approximately 6 times higher because the continuous epilactose formation was carried out for about 6 d without further addition of biocatalyst. Within this time, 24 g of epilactose in 2.8 L of permeate were produced. The batch and the EMR process showed that the milk ultrafiltrate, which is a sidestream of the milk protein production, might be upgraded to a dairy product of higher value by the enzymatic in situ production of epilactose. [ABSTRACT FROM AUTHOR]

In this study, a recombinant cellobiose 2-epimerase (GenBank accession number, YP_006392930.1) was characterized from a thermophilic bacterium, Thermoanaerobacterium saccharolyticum JW/SL-YS485. The enzyme was metal independent, showed maximal epimerization activity at pH 7.0 and 60 °C, and displayed 29.8, 15.48, and 13.5 U mg −1 for mannobiose, cellobiose, and lactose under optimum conditions, respectively. It exhibited promising thermostability under incubation below 60 °C. The K m , turnover number ( k cat ), and catalytic efficiency ( k cat / K m ) for lactose were 124.7 mM, 30.9 s −1 , and 0.248 mM −1 s −1 , respectively. At pH 7.0 and 60 °C, 50 mM epilactose was produced from 200 mM lactose by a 0.6 μM of enzyme concentration after reaction for 4 h. [ABSTRACT FROM AUTHOR]

It was reported recently that cellobiose 2-epimerases (CE) from various aerobic microorganisms convert lactose to epilactose in defined buffer systems. In this study, we showed that CE from 2 mesophilic microorganisms, Flavobacterium johnsoniae and Pedobacter heparinus, were capable of converting lactose to prebiotic epilactose not only in buffer but also in a complex milk system. First, the 2 enzymes were separately cloned, recombinantly expressed in Escherichia coli, and purified by column chromatography. The production of F. johnsoniae CE was carried out in a stirred-tank reactor, indicating that future upscaling is possible. The bioconversions of milk lactose were carried out at an industrially relevant low temperature of 8°C to avoid undesired microbial contaminations or chemical side reactions. Both enzymes were reasonably active at this low temperature, because of their origin from mesophilic organisms. The enzymes showed different operational stabilities over a 24-h time-course. A conversion yield of about 30 to 33% epilactose was achieved with both enzymes. No side products were detected other than epilactose. Therefore, CE may introduce an added value for particular dairy products by in situ production of the prebiotic sugar epilactose. [ABSTRACT FROM AUTHOR]

Epilactose (4- O-β- d-galactopyranosyl- d-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed. [ABSTRACT FROM AUTHOR]

The article presents a study which characterized and identified cellobiose 2-epimerases (CE)-like proteins from different aerobes. The study used polymerized chain reaction (PCR) to obtain CE-like genes of various bacteria, including Pedobacter (P.) heparinus, Flavobacterium (F.) johnsoniae, and Herpetosiphon (H.) aurantiacus. Results revealed that all CEs, except in P. heparinus CE, demonstrated highest activity at weak alkaline pH values.

The article discusses the findings of a study which showed the immobilization of thermophilic cellobiose 2-epimerase (CE) from Rhodothermus marinus (RmCE). Duolite A568 resin in the immobilization process and a packed bed reactor of the immobilized RmCE were used to achieve continuous production of epilactose. The experiment reportedly proved the efficacy of immobilized RmCE in reducing enzymes during lactose processing.

Abstract: The conditions for maximum lactulose production from lactose, as a single substrate, by a thermostable recombinant cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus were determined to be pH 7.5, 80°C, 700gl−1 lactose, and 150Uml−1 of enzyme. Under the conditions, the enzyme produced the two bifidus factors lactulose at 408gl−1 and epilactose at 107gl−1 after 2h. The yields of lactulose and epilactose from lactose and the productivities of lactulose and epilactose were 58%, 15%, 204gl−1 h−1, and 54gl−1 h−1, respectively. The yield and productivity of both lactulose and epilactose from lactose were 74% and 258gl−1 h−1, respectively. The yield, concentration, and productivity of lactulose in the present study are the highest among enzymatic syntheses. This is the first trial of enzymatic synthesis of lactulose using the single substrate lactose. [Copyright &y& Elsevier]

The article presents a study on the thermophilic cellobiose 2-epimerase (CE) activity on cell free extract of Rhodothermus marinus JCM9785. The study analyses CE activity through incubation of the cell-free extract of R. marinus JCM9785 with lactose and high performance liquid chromatography (HPLC). Result shows that the CE activity only acts on β-1,4-linked oligosaccharide, lactose, epilactose, and cellobiose. Moreover, the CE has 1.4 to 3.4-fold higher keat than other CEs.

The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4- O-β- d-glucopyranosyl- d-mannose and 4- O-β- d-galactopyranosyl- d-mannose (epilactose). Based on the sequence alignment with N-acetyl- d-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (α/α)6 core barrel structure. [ABSTRACT FROM AUTHOR]

The article focuses on a study regarding gene sequence and catalytic properties of celloboise 2-epiramese in Barcteroides fragilis. Computer analysis, enzyme assay and thin-layer chromatography (TLC) are some of the methods used by the researchers. Results show that B. fragilis cells possessed CE activity and that it exhibited 44-63% identities to N-acyl-D-glucosamine 2-epimerase-like hypothetical proteins of unknown function in bacterial genome sequences.

We recently reported that cellobiose 2-epimerase from Ruminococcus albus effectively converted lactose to epilactose. In this study, we examined the biological effects of epilactose on intestinal microbiota, bile acid metabolism, and postadministrative plasma glucose by animal tests. Dietary supplementation with epilactose or fructooligosaccharide (4.5% each) increased cecal wall weight and cecal contents and decreased the pH of the cecal contents in Wistar-ST rats. The number of total anaerobes tended to be greater in rats fed epilactose and fructooligosaccharide than in those fed the control diet. Lactobacilli and bifidobacteria were more numerous in rats fed epilactose and fructooligosaccharide diets than in those fed the control diet. Analysis of clone libraries of 16S rRNA suggests that supplementation with epilactose did not induce the proliferation of harmful bacteria belonging to classes Clostridia or Bacteroidetes. Epilactose, as well as fructoo.ligosaccharide, inhibited the conversion of primary bile acids to secondary bile acids, which are suggested to be promoters of colon cancer. In addition, oral administration of epilactose did not elevate the plasma glucose concentration in ddY mice. These results clearly indicate that epilactose is a promising prebiotic. We also showed that cellobiose 2-epimerase converted lactose in cow milk and a spray-dried ultrafiltrate of cheese whey to epilactose. Cellobiose 2-epimerase may increase the value of dairy products by changing lactose to epilactose possessing prebiotic properties. [ABSTRACT FROM AUTHOR]

Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4- O-β-d-glucopyranosyl-d-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44–54% identity to N-acyl-d-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 °C and pH 7.0–8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4- O-β-d-galactopyranosyl-d-mannose). [ABSTRACT FROM AUTHOR]

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30°C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4- O-β- d-galactopyranosyl- d-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The K m and k cat/ K m values toward lactose were 33 mM and 1.6 s−1 mM−1, and those toward cellobiose were 13.8 mM and 4.6 s−1 mM−1, respectively. N-Acetyl- d-glucosamine, uridine 5′-diphosphate-glucose, d-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic. [ABSTRACT FROM AUTHOR]