Enzymatic production of rare sugars with a new mutant of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus.

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.

Acidophilic heterotrophs: basic aspects and technological applications.

Acidophiles comprise a group of microorganisms adapted to live in acidic environments. Despite acidophiles are usually associated with an autotrophic metabolism, more than 80 microorganisms capable of utilizing organic matter have been isolated from natural and man-made environments. The ability to reduce soluble and insoluble iron compounds has been described for many of these species and may be harnessed to develop new or improved mining processes when oxidative bioleaching is ineffective.

Amination of naringinase to improve citrus juice debittering using a catalyst immobilized on glyoxyl-agarose.

A naringinase complex was chemically aminated prior to its immobilization on glyoxyl-agarose to develop a robust biocatalyst for juice debittering. The effects of amination on the optimal pH and temperature, thermal stability, and debittering performance were analyzed. Concentration of amino groups on catalysts surface increased in 36 %.

Ultrasound-assisted enzymatic synthesis of galacto-oligosaccharides using native whey with two commercial β-galactosidases: Aspergillus oryzae and Kluyveromyces var lactis.

Native whey obtained during casein micelle microfiltration was used as a novel source to produce galacto-oligosaccharides (GOS). Since the presence of macromolecules and other interferers reduces biocatalyst performance, this work evaluated the effect of different ultrasound processing conditions on GOS synthesis using concentrated native whey. Ultrasonic intensities (UI) below 11 W/cm tended to increase the activity in the enzyme from Aspergillus oryzae for several minutes but accelerated the inactivation in that from Kluyveromyces lactis.

Trends in lactose-derived bioactives: synthesis and purification.

Lactose obtained from cheese whey is a low value commodity despite its great potential as raw material for the production of bioactive compounds. Among them, prebiotics stand out as valuable ingredients to be added to food matrices to build up functional foods, which currently represent the most active sector within the food industry. Functional foods market has been growing steadily in the recent decades along with the increasing awareness of the World population about healthy nutrition, and this is having a strong impact on lactose-derived bioactives.

Enzymatic production of lactulose by fed-batch and repeated fed-batch reactor.

The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values ​​of lactulose yield (Y), lactulose productivity (π) and selectivity of transgalactosylation (S/). Maximum Y of 0.

Effect of product partition on the synthesis of butyl-β-D-galactoside from Aspergillus oryzae.

The effect of donor substrate and products partitioning on the performance of butyl-β-galactoside synthesis with Aspergillus oryzae β-galactosidase was studied. Firstly, the partition coefficient of the donor substrate (lactose) and the reaction products (glucose, galactose and butyl-β-galactoside) were determined in the aqueous and organic phases of the reaction medium. In the temperature range studied (30 to 50 °C), butyl β-galactoside was roughly 130 and 30-fold more soluble in the organic phase than lactose and the monosaccharides, respectively.

Immobilization of Aspergillus oryzae β-galactosidase in cation functionalized agarose matrix and its application in the synthesis of lactulose.

Aspergillus oryzae β-galactosidase was immobilized in in-house quaternary ammonium agarose (QAA) and used for the first time in the synthesis of lactulose. A biocatalyst was obtained with a specific activity of 24,690 IU∙g; protein immobilization yield of 97% and enzyme immobilization yield of 76% were obtained at 30 °C in 10 mM phosphate buffer pH 7 for standard size agarose at 100 mg∙g which the maximum protein load of QAA. Highest yield and specific productivity of lactulose were 0.

Synthesis of Butyl-β-D-Galactoside in the Ternary System: Acetone/1-Butanol/Aqueous Solution.

The enzymatic synthesis of short-tailed alkyl glucosides is generally carried out in an aqueous-organic biphasic reaction medium with a rather low fatty alcohol concentration in the aqueous phase (where the synthesis occurs). Thus, hydrolytic reactions have a significant impact on the synthesis performance. Given this background, the use of acetone as cosolvent was studied for the synthesis of butyl-β-galactoside with β-galactosidase.

Synthesis of Lactulose in Continuous Stirred Tank Reactor With β-Galactosidase of Immobilized in Monofunctional Glyoxyl Agarose Support.

Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized β-galactosidase is reported for the first time. The effect of operational variables: inlet concentrations of sugar substrates, temperature, feed substrate molar ratio, enzyme loading and feed flow rate was studied on reactor performance. Even though the variation of each one affected to a certain extent lactulose yield (Y ), specific productivity (π ) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (S ), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio.

Metabolic Costs of a 58-minute Multi-Intensity Exercise Session with and Without Music and Cueing.

It is unclear if the presence or absence of music and cueing influence total energy expenditure (TEE) during a multi-intensity exercise program. The purpose of this study was to determine the difference between TEE with or without music and cueing during a 58-minute exercise session using heart rate estimation (HRe) and indirect calorimetry (IC). Using a randomized crossover design, 22 participants (6 males; 16 females; 27.

Improvements in the production of Aspergillus oryzae β-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose.

Aspergillus oryzae β-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IU g were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%.

Conventional and non-conventional applications of β-galactosidases.

β-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of β-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide.

Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae β-galactosidase.

Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae β-galactosidase is reported for the first time. Alternative strategies to conventional batch synthesis have been scarcely explored for lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing material mass ratio (M/M) were studied on reactor performance.

β-Galactosidase from Exiguobacterium acetylicum: Cloning, expression, purification and characterization.

The main goal of this work was to evaluate the performance of β-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-β-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-β-d-glucopyranoside, butyl- and pentyl-β-d-galactosides.

Co-immobilized β-galactosidase and Saccharomyces cerevisiae cells for the simultaneous synthesis and purification of galacto-oligosaccharides.

Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides (GOS) from lactose was conducted using a combi-biocatalyst formed by crosslinked enzyme aggregates of Aspergillus oryzae β-galactosidase and Saccharomyces cerevisiae cells co-immobilized by entrapment in calcium alginate gel particles. Product yield obtained with the combi-biocatalyst was similar than obtained with the soluble enzyme (23.3%), having a final purity of 25.

Immobilization of Aspergillus oryzae β-galactosidase in an agarose matrix functionalized by four different methods and application to the synthesis of lactulose.

Aspergillus oryzae β-galactosidase was immobilized in monofunctional glyoxyl-agarose and heterofunctional supports (amino-glyoxyl, carboxy-glyoxyl and chelate-glyoxyl agarose), for obtaining highly active and stable catalysts for lactulose synthesis. Specific activities of the amino-glyoxyl agarose, carboxy-glyoxyl agarose and chelate-glyoxyl agarose derivatives were 3676, 430 and 454IU/g biocatalyst with half-life values at 50°C of 247, 100 and 100h respectively. Specific activities of 3490, 2559 and 1060IU/g were obtained for fine, standard and macro agarose respectively.

Fed-batch operation for the synthesis of lactulose with β-galactosidase of Aspergillus oryzae.

Fed-batch synthesis of lactulose from lactose and fructose with Aspergillus oryzae β-galactosidase was evaluated, obtaining a concentration of 40.4g·L, which is 20% higher than obtained in batch, while the concentration of transgalactosylated oligosaccharides (TOS) was reduced by 98%. Therefore, selectivity of lactulose synthesis can be significantly higher by operating in fed-batch mode.

Synthesis of lactulose in batch and repeated-batch operation with immobilized β-galactosidase in different agarose functionalized supports.

Lactulose synthesis was done in repeated-batch mode with Aspergillus oryzae β-galactosidase immobilized in glyoxyl-agarose (GA-βG), amino-glyoxyl-agarose (Am-GA-βG) and chelate-glyoxyl-agarose (Che-GA-βG), at fructose/lactose molar ratios of 4, 12 and 20. Highest yields of lactulose in batch were obtained with Che-GA-βG (0.21, 0.

Synthesis and purification of galacto-oligosaccharides: state of the art.

Lactose-derived non-digestible oligosaccharides are prominent components of functional foods. Among them, galacto-oligosaccharides (GOS) outstand for being prebiotics whose health-promoting effects are supported on strong scientific evidences, having unique properties as substitutes of human milk oligosaccharides in formulas for newborns and infants. GOS are currently produced enzymatically in a kinetically-controlled reaction of lactose transgalactosylation catalyzed by β-galactosidases from different microbial strains.

Performance of an ultrafiltration membrane bioreactor (UF-MBR) as a processing strategy for the synthesis of galacto-oligosaccharides at high substrate concentrations.

The performance of an ultrafiltration membrane bioreactor for galacto-oligosaccharides (GOS) synthesis using high lactose concentrations (470 g/L) and β-galactosidase from Aspergillus oryzae was assessed. Tested processing variables were: transmembrane-pressure (PT), crossflow-velocity (CFV) and temperature. Results showed that processing variables had significant effect on the yield, the enzyme productivity and the flux but did not on GOS concentration and reaction conversion obtained.

Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation.

Lactulose and galacto-oligosaccharides (GOS) are well recognized prebiotics derived from lactose. In the synthesis of lactulose with β-galactosidases GOS are also produced, but the ratio of lactulose and GOS in the product can be tuned at will, depending on the operation conditions, so to obtain an optimal product distribution in terms of prebiotic potential. The selectivity of fermentation of each carbohydrate alone as well as mixtures of both was determined using pH-controlled anaerobic batch cultures with faecal inoculum.

Repeated-batch operation for the synthesis of lactulose with β-galactosidase immobilized by aggregation and crosslinking.

Synthesis of lactulose under repeated-batch operation was done with cross-linked aggregates of Aspergillus oryzae β-galactosidase (CLAGs). The effect of the crosslinking agent to enzyme mass ratio and cross-linking time were first evaluated. Best results were obtained at 5.

Transgalactosylation and hydrolytic activities of commercial preparations of β-galactosidase for the synthesis of prebiotic carbohydrates.

β-Galactosidases exhibit both hydrolytic and transgalactosylation activities; the former has been used traditionally for the production of delactosed milk and dairies, while the latter is being increasingly used for the synthesis of lactose-derived oligosaccharides: balance between both activities was highly dependent on the enzyme origin: β-galactosidases from Aspegillus oryzae and Bacillus circulans exhibited high transgalactosylation activity, while those from one from Kluyveromyces exhibited high hydrolytic activity but quite low transgalactosylation activity. Also the affinity for the donors (lactose or lactulose) and the acceptors (lactose, lactulose or fructose) of transgalactosylated galactose was dependent on the enzyme origin, as reflected by the Michaelis constants obtained in the synthesis of galacto-oligosaccharides, fructosyl-galacto-oligosaccharides and lactulose. Finally, the balance between transgalactosylation and hydrolytic activities of β-galactosidases could be tuned by changing the concentration of galactose donor.

Fed-batch synthesis of galacto-oligosaccharides with Aspergillus oryzae β-galactosidase using optimal control strategy.

Fed-batch synthesis of galacto-oligosaccharides (GOS) from lactose with β-galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed-batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L(-1) as consequence of the increase in total sugars concentration from 40 to 58% w/w.