ß-Galactosidase

Galacto-oligosaccharides (GOS) are oligosaccharides with prebiotic properties derived from lactose. The milk has a favorable pH for the action of the β-galactosidase enzyme produced by Kluyveromyces lactis and in this way may favor the hydrolysis of lactose and the synthesis of GOS. This work aimed to investigate the efficiency of the enzymatic conversion of lactose to GOS in skimmed UHT milk to obtain a dairy raw material with the presence of GOS and reduced lactose content. The best conditions of lactose and enzyme concentrations, and temperature, by enzymatic conversion of lactose, through surface response methodology following a 2³ experimental design (data not published), were investigated in reaction systems during the period of time inferior to that previously performed to favor reduced lactose content and the formation of GOS in skim milk. The carbohydrate analysis was performed by HPLC-IR (High Performance Liquid Chromatography-Refractive Index Detector). A skimmed dairy product was obtained with reduced lactose content (0.66% w/v) and GOS (0.96% w/v). The process was executed in 1.5 hours, at 30° C, with 10% lactose (w/v), and 5 U.mL -1 of enzyme. Once the process was controlled, it was possible to develop dairy products with prebiotics that feature health benefits.

Mannanase produced by mutant Penicillium citrinium Egy5 LC368457 isolated from Pharaoh Mummies of Ancient Egyptian Museum, Cairo, Egypt, was covalently immobilized onto activated chitosan beads by Polyethyleneimine, glutaraldehyde and dopamine. Loading time, enzyme's units, pH and temperature were optimized. The immobilized enzyme can keep its maximum activity at a broader range of pH and make it more stable at higher temperatures, including 60 °C, which is preferred to reduce microbial contamination of the enzyme. The reusability test indicated the high stability of immobilized enzymes that were still active at 75.82% of their activity at cycle number 9 and in the production of mannooligosaccharides, which showed antioxidant and prebiotic activity.

β-galactosidase has been immobilized onto novel alginate/tea waste gel beads (Alg/TW) via covalent binding. Alg/TW beads were subjected to chemical modification through amination with polyethyleneimine (PEI) followed by activation with glutaraldehyde (GA). Chemical modification parameters including PEI concentration, PEI pH, and GA concentration were statistically optimized using Response Surface methodology (RSM) based on Box-Behnken Design (BBD). Analysis of variance (ANOVA) results confirmed the great significance of the model that had F value of 37.26 and P value < 0.05. Furthermore, the R 2 value (0.9882), Adjusted R 2 value (0.9617), and predicted R 2 value (0.8130) referred to the high correlation between predicted and experimental values, demonstrating the fitness of the model. In addition, the coefficient of variation (CV) value was 2.90 that pointed to the accuracy of the experiments. The highest immobilization yield (IY) of β-galactosidase (75.1%) was given under optimized conditions of PEI concentration (4%), PEI pH (9.5), and GA concentration (2.5%). Alg/TW beads were characterized by FT-IR, TGA, and SEM techniques at each step of immobilization process. Moreover, the immobilized β-galactosidase revealed a very good reusability as it could be reused for 15 and 20 consecutive cycles keeping 99.7 and 72.1% of its initial activity, respectively. In conclusion, the environmental waste (tea waste) can be used in modern technological industries such as the food and pharmaceutical industry.

The current study aimed to evaluate the hydrolysis of whole fat milk (WFM) and sweet whey (SW) using β-galactosidase (β-gal) after covalent immobilization onto activated alginate/tea waste (Alg/TW) beads as a novel carrier. The optimum temperature for free and Alg/TW/β-gal was 40 • C and the ideal pH was 7.0. However, Alg/TW/β-gal displayed better stabilities at high temperatures and a wide pH range. Additionally, the value of K m and V max for Alg/TW/β-gal was higher than the free enzyme. The Alg/TW/β-gal showed better residual activity (78.6 %) after 90 storage days at 4 • C. The reusability of Alg/TW/β-gal was very good as it conserved its full activity after 15 consecutive cycles and conserved 93 % of its initial activity after 10 cycles with ONPG (O-nitrophenyl-β-D-galactopyranoside) and lactose as a substrate, respectively. The impact of Alg/TW/ β-gal on WFM and SW using HPLC analysis revealed a remarkable decrease in lactose concentration and increase of glucose and galactose concentrations. The SW exhibited higher degree of lactose hydrolysis (97.3 %) compared to WFM (62.4 %). Besides, SW had a prominent increase in total phenolic content (96.8 mg/L) compared to WFM (54.3 mg/L). The antioxidant activity had increased after enzyme treatment in both WFM and SW. The GC-MS analysis for volatile compounds identified twenty-five flavour constituents. Finally, Alg/TW/ β-gal has a potential application for obtaining healthy, acceptable, and commercial dairy products of low lactose.

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. Most of them are produced by enzyme biocatalysis because of molecular precision and environmental sustainability considerations. The current status and outlook of the production of lactose-derived bioactive compounds is presented with special emphasis on downstream operations which are critical because of the rather modest lactose conversion and product yields that are attainable. Even though some of these products have already an established market, there are still several challenges referring to the need of developing better catalysts and more cost-effective downstream operations for delivering high quality products at affordable prices. This technological push is expected to broaden the spectrum of lactose-derived bioactive compounds to be produced at industrial scale in the near future.

Carrageenan is one of the most common carbohydrates utilised in the entrapment industry to immobilise cells and enzymes. However, it lacks functionality. Carrageenan has been grafted to produce fructose by covalently immobilising glucose isomerase (GI). Fructose is one of the most widely used sweeteners in beverages, food production, and the pharmaceutical business. Up to 91.1 U g-1 gel beads are immobilised by the grafted beads. Immobilized GI has a Vmax of 13.8 times that of the free enzyme. The immovability and tolerance of the gel beads immobilised with GI over 15 consecutive cycles were demonstrated in a reusability test, with 88 percent of the enzyme&#39;s original activity retained, compared to 60 percent by other authors. These findings are encouraging for high-fructose corn syrup producers.

The current demands of the world's biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immobilized enzyme systems allows an easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This review summarizes immobilization definition, different immobilization methods, advantages and disadvantages of each method. In addition, it covers some food industries, protein purification, human nutrition, biodiesel production, and textile industry. In these industries, the use of enzymes has become an inevitable processing strategy when a perfect end product is desired. It also can be used in many other important industries including health care and pharmaceuticals applications. One of the best uses of enzymes in the modern life is their application in diagnose and treatment of many disease especially when used in drug delivery system or when used in nanoform.

The production of â-galactosidase by Lactobacillus acidophilus NRRL 4495 was studied. The highest yield of intracellular â-galactosidase was achieved using acid whey (3.5%) as carbon source after 48h fermentation. Ammonium sulphate (3%) was the best nitrogen source. Addition of 2 MnCl at 0.02M optimized the production of the enzyme. In addition, the activity reached 51.46U/g cells when the temperature of the fermentation optimized at 40°C. The highest â-galactosidase activity was obtained by using mechanical disintegration of cells by mortar with sterile sea sand under cooling. By studying some properties of the produced enzyme, the enzyme activity was optimum at 40°C and pH 7.0. In absence of substrate and up to 45°C the enzyme was highly stable, at least for 60 min, retaining 83% of its original activity.

β-galactosidase has been immobilized onto novel alginate/tea waste gel beads (Alg/TW) via covalent binding. Alg/TW beads were subjected to chemical modification through amination with polyethyleneimine (PEI) followed by activation with glutaraldehyde (GA). Chemical modification parameters including PEI concentration, PEI pH, and GA concentration were statistically optimized using Response Surface methodology (RSM) based on Box-Behnken Design (BBD). Analysis of variance (ANOVA) results confirmed the great significance of the model that had F value of 37.26 and P value < 0.05. Furthermore, the R 2 value (0.9882), Adjusted R 2 value (0.9617), and predicted R 2 value (0.8130) referred to the high correlation between predicted and experimental values, demonstrating the fitness of the model. In addition, the coefficient of variation (CV) value was 2.90 that pointed to the accuracy of the experiments. The highest immobilization yield (IY) of β-galactosidase (75.1%) was given under optimized conditions of PEI concentration (4%), PEI pH (9.5), and GA concentration (2.5%). Alg/TW beads were characterized by FT-IR, TGA, and SEM techniques at each step of immobilization process. Moreover, the immobilized β-galactosidase revealed a very good reusability as it could be reused for 15 and 20 consecutive cycles keeping 99.7 and 72.1% of its initial activity, respectively. In conclusion, the environmental waste (tea waste) can be used in modern technological industries such as the food and pharmaceutical industry.

In this work, the structural thermostabilization of the characterized nanomagnetic cross-linked enzyme aggregates of naringinase have been considered. Comparisons have been made between free and immobilized enzyme by the determination of temperature-dependent half-lives (t 1/2), energy barriers of thermal inactivation (E a(in)) process, and thermodynamic parameters (ΔH*, ΔG*, and ΔS*) in a storage thermostability approach. Samples of NM-NGase-CLEAs were treated at different temperatures in the range of 40-80°C for 90 min. The K m values of immobilized enzyme was reduced about 10.7 folds compared to the free one. The catalytic efficiency (k cat /K m) was raised about 10.5 folds after immobilization. Enzyme half-life (t 1/2) of NM-NGase-CLEAs increased from 18.7 to 52.9 min (about 3 folds) at 80°C. The thermodynamics study indicated that E a(in) of the free enzyme increased from 38.51 to 49.14 (KJ•mol −1) and ΔH* increased from 35.57 to 46.20 (KJ•mol −1) after immobilization, which indicates an increase in the thermostability of this multimeric enzyme after nanomagnetic CLEAs fabrication. The NM-CLEAs of naringinase preserved 73% of its original activity after 10 cycles, which implies strong operational stability. Thus, the developed method for nanomagnetic CLEAs preparation has provided an efficient and simple approach for the productive and reusable nanobiocatalyst together with ease in enzyme handling.

Lactulose synthesis from fructose and lactose in continuous stirred tank (CSTR) reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae 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 Lactulose), specific productivity (π Lactulose) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (S Lu/TOS), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum Y Lactulose of 0.54 g•g −1 was obtained at 50 • C, pH 4.5, 50% w/w inlet concentrations of sugar substrates, feed flowrate of 12 mL•min −1 , fructose/lactose molar ratio of 8 and reactor enzyme load of 29.06 IU H •mL −1. At such conditions S Lu/TOS was 3.7, lactose conversion (X Lactose) was 0.39 and total transgalactosylation yield was 0.762 g•g −1 , meaning that 76% of the reacted lactose corresponded to transgalactosylation and 24% to hydrolysis, which is a definite advantage of this mode of operation. Even though X Lactose in CSTR was lower than in other reported modes of operation for lactulose synthesis, transgalactosylation was more favored over hydrolysis which reduced the inhibitory effect of galactose on β-galactosidase.

The excretion of recombinant proteins into the culture medium is a preferred approach over cytoplasmic expression because of the high specific activity and ease of purification. However, cell lysis is one of a major problem during the excretion of recombinant protein due to the pressure build up in the periplasmic space through overproduction of the expressed recombinant protein. Cell immobilization is a promising solution for the improvement of protein excretion with reduction in cell lysis. In this study, the effect of cross linkers concentrations on the process of cell immobilization for the cyclodextringlucanotransferase (CGTase) excretion and cell viability were investigated. The hollow fiber membrane was treated using different concentrations (0.3, 0.67 and 1%) of cross linkers namely glutaraldehyde (GA), poly-L-lysine (PLL) and polyethylenimine (PEI). The optimum concentration of cross linkers was found to be 0.3% with the high excretion of CGTase. Interestingly, the immobilized cell on the non-treated hollow fiber membrane showed up to 15% increment of CGTase excretion with 55% reduction of cell lysis, as compared to the immobilized cells on the treated hollow fiber membrane. It could be concluded that, the low concentration of cross linkers exhibited the highest CGTase excretion. Moreover, the non-treated hollow fiber membrane is a promising approach for attachment of cells to the membrane without requiring treatment with any chemicals for the production of recombinant enzyme.

R-␤-Gal is a cold-adapted ␤-galactosidase that is able to hydrolyze lactose and has the potential to produce low-lactose or lactose-free dairy products at low temperatures (4 • C). Cold-adapted enzymes unfold at moderate temperatures due to the lower intramolecular stabilizing interactions necessary for flexibility at low temperatures. To increase stability and usage-performance, R-␤-Gal was encapsulated in gellan gum by injecting an aqueous solution into two different hardening solutions (10 mM CaCl 2 or 10 mM MgCl 2). Enzyme characteristics of both free and encapsulated R-␤-Gal were carried out, and the different effects of two cations were investigated. R-␤-Gal showed better thermal and pH stability after encapsulation. Ca 2+ gels had higher encapsulation efficiency (71.4%) than Mg 2+ (66.7%) gels, and Ca 2+ formed larger inner and surface pores. R-␤-Gal was released from the Ca 2+ hydrogel beads more rapidly than the Mg 2+ hydrogels during storage in aqueous solution due to the larger inner/surface pores of the matrix.

Natural polymeric gel such as alginate and carrageenan, and carbon nanomaterials, especially MWCNT and graphene are gaining increasing popularity as immobilization support owing to their biocompatibility. Thus, this study attempts to make a relative characterization of the immobilized β-glucosidase on alginate and multiwalled carbon nanotubes (MWCNT) to assess their relative merits as biocatalyst. Acid functionalization of MWCNT lead to the formation of carboxyl groups at the MWCNT surface which seemed to have role in the stable attachment of the enzyme on the MWCNT surface. The effectiveness of the immobilized enzyme was evaluated using hydrolysis of p-nitrophenyl-β-D-glucopyranoside to p-nitrophenol. The optimum immobilization conditions were found to be 17 mg MWCNT and 4 h incubation time for MWCNT, and 3.5 wt% sodium alginate solution and 2 h incubation time for the Ca-alginate beads. The immobilization yield on MWCNT was found to be 96% and that for the Ca-alginate beads was 86...

There has been a recent increase in the exploration of cold-active β-galactosidases, as it offers new alternatives for the dairy industry, mainly in response to the current needs of lactose-intolerant consumers. Since extremophilic microbial compounds might have unique physical and chemical properties, this research aimed to study the capacity of Antarctic bacterial strains to produce cold-active β-galactosidases. A screening revealed 81 out of 304 strains with β-galactosidase activity. The strain Se8.10.12 showed the highest enzymatic activity. Morphological, biochemical, and molecular characterization based on whole-genome sequencing confirmed it as the first Rahnella inusitata isolate from the Antarctic, which retained 41–62% of its β-galactosidase activity in the cold (4 °C–15 °C). Three β-galactosidases genes were found in the R. inusitata genome, which belong to the glycoside hydrolase families GH2 (LacZ and EbgA) and GH42 (BglY). Based on molecular docking, some of these enzy...

A new matrix formulation was devised for catalase immobilization. Carrageenan-alginate beads different ratios were developed and soaked into different ratios of CaCl 2-KCl as a hardening solution. The best formulation for loading capacity was selected, treated with polyethylene imine followed by glutaraldehyde and further studied. The best concentration of catalase for immobilization was 300U/ml and the best loading time was 6 h. The catalytic properties increased after immobilization and the immobilized catalase achieved optimum activity at a temperature range of 30-50 °C that was compared to the optimum activity of free catalase which occurred at 40 °C. Higher catalytic activity of immobilized catalase occurred at alkaline pHs than the free one which achieved optimum catalytic activity at neutral pH. A comparison between the kinetic parameters of immobilized and free catalase showed variation. The K M and Vmax of the immobilized catalase were 2.4 fold and six times higher than those of free catalase. The results of the study indicate that the formulated matrix can be used as a good matrix for catalase enzyme in various industrial applications.

Fructooligosaccharides (FOS) are fructose oligomers that are beneficial to human health and nutrition for being prebiotic sugars. Their production occurs by a transfructosylation reaction in sucrose molecules catalyzed by fructosyltransferase enzymes (FTase, E.C.2.4.1.9) adhered to microbial cells. The purpose of this work was to study the preparation, enzymatic activity, and stability of glutaraldehyde-crosslinked Aspergillus oryzae IPT-301 cells used as biocatalyst for the transfructosylation reaction of sucrose in a packed bed reactor (PBR), aiming at FOS production. The highest transfructosylation activity (AT) was presented by the biocatalyst prepared by crosslinking at 200 rpm and 45 min. The highest AT in the PBR were obtained at 50?C, flow rates from 3 mL min-1 to 5 mL min-1 and sucrose concentrations of 473 g L-1 and 500 g L-1. The enzymatic kinetics was described using the Michaelis-Menten model. Finally, the biocatalyst showed constant AT of approximately 75 U g-1 and 300...

The current demands of the world's biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immobilized enzyme systems allows an easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This review summarizes immobilization definition, different immobilization methods, advantages and disadvantages of each method. In addition, it covers some food industries, protein purification, human nutrition, biodiesel production, and textile industry. In these industries, the use of enzymes has become an inevitable processing strategy when a perfect end product is desired. It also can be used in many other important industries including health care and pharmaceuticals applications. One of the best uses of enzymes in the modern life is their application in diagnose and treatment of many disease especially when used in drug delivery system or when used in nanoform.

b-Galactosidase is a metal-activated enzyme, which breaks down the glucosidic bond of lactose and produces glucose and galactose. Among several commercial applications, preparation of lactose-free milk has gained special attention. The present objective is to demonstrate the activity kinetics of b-galactosidase purified from a non-pathogenic bacterium Arthrobacter oxydans SB. The enzyme was purified by DEAE-cellulose and Sephadex G-100 column chromatography. The purity of the protein was checked by high-performance liquid chromatography (HPLC). The purified enzyme of molecular weight * 95 kDa exhibited specific activity of 137.7 U mg-1 protein with a purification of 11.22-fold and yield 12.42 %. The exact molecular weight (95.7 kDa) of the purified protein was determined by MALDI-TOF. Previously, most of the studies have used Mg ?2 as a cofactor of bgalactosidase. In this present investigation, we have checked the kinetic behavior of the purified b-galactosidase in presence of several bivalent metals. Lowest K m with highest substrate (orthonitrophenyl-b-galactoside or ONPG) affinity was measured in presence of Ca 2? (42.45 lM ONPG). However, our results demonstrated that V max was maximum in presence of Mn ?2 (55.98 lM ONP produced mg-1 protein min-1), followed by Fe ?2 , Zn ?2 , Mg ?2 , Cu ?2 and Ca ?2. A large number of investigations reported Mg ?2 as potential co factor for bgalacosidase. However, b-galactosidase obtained from Arthrobacter oxydans SB has better activity in the presence of Mn ?2 or Fe 2? .

Beta-Galacto-oligosahaarides (β-GalOS) are naturally occurred in milk at low concentration, and are found in all dairy products. s-GalOS are commercially produced enzymatically by trans glalactosylation of milk sugar lactose as a substrate using the microbial enzyme s-galactosidase (EC 3.2.1.23) derived from bacteria, yeasts or molds. s-GalOS are one of functional oligosaccharides and are known by different names as oligogalactosyllactose, oligogalactose, oligolactose, tranas-galactosylated oligo- saccharide, and transgalacto-oligosaccharide. Chemical structures of s-GalOs are chain of galactose units that arise through consecutive transgalactosylation enzymatic reactions, linked together into terminal glucose unit in the disaccharide lactose. The degree of galactose polymerization in s-GalOS are vary and ranging from 2 to 8 galactose units. This degree of polymerization depends on the property and source of the enzyme s-galactosidase used in the process. s-GalOS are pass undigested...

We have developed strategies for immobilizing and stabilizing enxymes with very different and complex structures. As an example we have performed the design of two j?-galactosidase derivatives with excellent activity and stability propertiee: i.e. a derivative of the enzyme from E. coli which is more than 1900-fold more stable than the soluble enzyme. These derivatives have allowed us to use drastic conditions (i.e. high concentration of orgaic cosolvents) for improving the specificity of our enzyme derivatives. In this way, by using these p-galactosidase derivatives as catalysts, we have been able to obtain very interesting results in the enzymatic synthesis of several model disaccharides.

The mixture of Eucalyptus globulus wood (EGW) and cheese whey powder (CWP) was proposed for intensification of simultaneous saccharification and fermentation (SSF) at high temperature and solid loadings using the industrial Saccharomyces cerevisiae Ethanol Red® strain. High ethanol concentration (93 g/L), corresponding to 94% ethanol yield, was obtained at 35 °C from 37% of solid mixture using cellulase and β-galactosidase enzymes (24.2 FPU/g and 20.0 U/g, respectively). The use of CWP mixed with pretreated EGW increased the ethanol concentration in 1.5-fold, in comparison with SSF experiments without CWP for both Ethanol Red® and CEN.PK113-7D strains. Moreover, 1.4-fold higher ethanol concentration was obtained with Ethanol Red®, in comparison with CEN.PK113-7D strain. Ethanol Red® strain was genetically engineered for β-galactosidase production in order to advance towards a fully integrated process. This work shows the feasibility of attaining high ethanol concentrations in second...

Encapsulation of enzymes in hydrogel beads may improve their utilization and activity in foods. In this study, the potential of carrageenan hydrogel beads for encapsulating β-galactosidase was investigated. Hydrogel beads were fabricated by injecting an aqueous solution, containing β-galactosidase (26 U) and carrageenan (1 wt%), into a hardening solution (5% potassium chloride). Around 63% of the β-galactosidase was initially encapsulated in the hydrogel beads. Encapsulated β-galactosidase had a higher activity than that of the free enzyme over a range of pH and thermal conditions, which was attributed to the stabilization of the enzyme structure by K + ions within the carrageenan beads. Release of the enzyme from the beads was observed during storage in aqueous solutions, which was attributed to the relatively large pore size of the hydrogel matrix. Our results suggest that carrageenan hydrogel beads may be useful encapsulation systems, but further work is needed to inhibit enzyme leakage.

Background: Glucoamylase enzyme is one of the most important enzymes. It catalyzes the hydrolysis of starch into soluble sugars. It was covalently immobilized onto κ-carrageenan gel beads after activation by using polyethylenimine (PEI) followed by glutaraldehyde (GA). All parameters in activation process were studied. Results: The immobilized enzyme shows enhancement in temperature profile as the optimum temperature for the free enzyme was 60°C, and it becomes 60-80°C that means broader range and also shows stability in acidic conditions more than free enzyme. The apparent Km of immobilized enzyme, 147.46 mM, becomes higher than Km of the free one, 110 mM and the maximum reaction velocity (Vmax) values for the immobilized enzyme decreased from 2.28 to 1.11 μmol min −1. The immobilized enzyme can be reused and kept its activity (100%) till 11 successive cycles. The immobilization process steps were characterized by FTIR and SEM. Conclusions: These results confirm the economic and biotechnical benefits of enzyme immobilization, particularly with regard to the number of enzyme reuses, which open the possibility of different industrial applications.

Tyrosol β-galactoside (TG) is a phenylethanoid glycoside with proven neuroprotective properties. This work deals with its biocatalytic production from tyrosol and lactose using Aspergillus oryzae β-galactosidase in immobilized form. Six commercial carriers were examined to find the optimal biocatalyst. Besides standard biocatalyst performance characteristics, adsorption of the hydrophobic substrate on immobilization carrier matrices was also investigated. The adsorption of tyrosol was significant, but it did not have adverse effects on TG production. On the contrary, TG yield was improved for some biocatalysts. A biocatalyst prepared by covalent binding of β-galactosidase on an epoxy-activated carrier was used for detailed investigation of the effect of reaction conditions on glycoside production. Temperature had a surprisingly weak effect on the overall process rate. A lactose concentration of 0.83 M was found to be optimal to enhance TG formation. The impact of tyrosol concentration was rather complex. This substrate caused inhibition of all reactions. Its concentration had a strong effect on the hydrolysis of lactose and all products. Higher tyrosol concentrations, 30-40 g/L, were favorable as pseudo-equilibrium concentrations of TG and galactooligosaccharide were reached. Repeated batch results revealed excellent operational stability of the biocatalyst.

Early phase development of industrial immobilized biocatalysts has to address the selection of the best candidates from dozens of available carriers and binding methods. This work presents a simple selection method for the immobilization of industrial-grade Aspergillus oryzae β-galactosidase suitable for the production of galactooligosaccharides. Immobilization efficiency and yield of a variety of immobilized biocatalysts are evaluated using simple activity measurements and mathematical modeling of intraparticle kinetics and mass transfer. Activated carriers and some ion-exchange carriers provided highly active biocatalysts and are therefore selected for the investigation of GOS production in the batch reactor. Biocatalysts exhibited significant differences in the yield of galactooligosaccharides and productivity of GOS formation. Based on these process performance criteria, cost considerations are made that allow further selection of the best biocatalyst for future process optimization. The presented method of biocatalyst selection can be useful for other industrial enzymes applications.

We have developed strategies for immobilizing and stabilizing enxymes with very different and complex structures. As an example we have performed the design of two j?-galactosidase derivatives with excellent activity and stability propertiee: i.e. a derivative of the enzyme from E. coli which is more than 1900-fold more stable than the soluble enzyme. These derivatives have allowed us to use drastic conditions (i.e. high concentration of orgaic cosolvents) for improving the specificity of our enzyme derivatives. In this way, by using these p-galactosidase derivatives as catalysts, we have been able to obtain very interesting results in the enzymatic synthesis of several model disaccharides.

Production of intracellular b-galactosidase has been performed by isolated facultative anaerobic consortium Bacillus safensis (JUCHE1) in lab-scale fermentor by batch mode. Two types of microbial growth media, namely, lactose broth and modified de-Mann Rogosa Sharpe (MMRS) media were tested for microbial growth and b-galactosidase production. It was observed that microbial growth was high in MMRS medium compared with lactose broth. Under the present investigation, growth kinetic parameters, namely maximum specific growth rate (l max), Monod constant (K s), yield coefficient (Y x/s) and maintenance coefficient (m s) of this organism have been determined to be 0.75/h, 12 g/l, 0.1 g biomass/g. substrate and 0.688/h respectively considering lactose as the limiting carbohydrate source. Optimum concentrations of lactose and tryptone, a nitrogen source, in microbial growth medium have been determined to be 20 g/l and 10 g/l, respectively. Optimum temperature, pH and stirrer speed for microbial growth have been found to be 37°C, 7.0 and 170 rpm, respectively. To isolate intracellular b-galactosidase, sonication was adopted and optimum sonication time was found 300 s with inter stage cooling for 10 s after an operating time of 30 s. Intracellular b-galactosidase was characterized by enzymatic reaction kinetics, optimum pH and temperature. Maximum reaction velocity (v max), Michaelis-Menten constant (K m), optimum temperature and pH were found 5.1 lmol/min mg/protein, 20 mM, 45°C and 7, respectively using ortho-nitrophenyl-bgalactoside (ONPG) as a substrate. Keywords Bacillus safensis Á Operating parameters Á Growth kinetics Á b-Galactosidase characteristics Life member, Institute of Engineers (Membership No. M130793-7).

The present study demonstrates the immobilization of β galactosidase from apricots (Prunus armeniaca kaisa) on an inexpensive concanavalin A layered cellulose-alginate hybrid gel. Immobilized β galactosidase retained 78% of the initial activity after crosslinking by glutaraldehyde. It exhibited greater fraction of activity at both acidic and basic pH, and showed broad spectrum temperature optimum as compared to free enzyme. Moreover, immobilized enzyme exhibited higher thermal stability at 60°C and retained 80% of the original enzyme activity in presence of 3% galactose. The crosslinked immobilized enzyme showed improved hydrolysis of lactose from milk and whey in batch processes at 50°C as well as in continuous reactors operated at fl ow rate of 20 mL/h and 30 mL/h even after one month. Moreover, crosslinked adsorbed β galactosidase retained 76% activity even after its sixth repeated use, thereby promoting its use for lactose hydrolysis in various dairy products even for longer dur...

We investigated the immobilization of a tetrameric Kluyveromyces lactis β-galactosidase (EC: 3.2.1.23) (KL-Gal) on Immobead 150 using different support modification strategies. Immobead support was modified using an acid solution of H 2 SO 4 :HNO 3 (3:1) (Immobead-Ac) or 5 % (v/v) glutaraldehyde (Immobead-Glu). Its unmodified form (Immobead) was also tested. Immobilization yields and efficiencies were evaluated by testing protein loads from 10 to 200 mg.g-1 support. The thermal, physico-chemical, textural and catalytic properties of the supports (modified and unmodified) and their derivatives (Immobead-KL-Gal, Immobead-Ac-KL-Gal and Immobead-Glu-KL-Gal) were analyzed. The highest immobilization yields and efficiencies were achieved with a protein load of 100 mg.g-1 support. Surface and pore areas of the Immobead support were greatly decreased after modification. Michaelis constant of the immobilized β-galactosidase increased in the derivatives. Maximum velocity decreased approximately 2.8 times for Immobead-KL-Gal and Immobead-Glu-KL-Gal, and approximately 1.4 times for Immobead-Ac-KL-Gal. In batch processes, the three derivatives could be reused successfully at least 15 times, maintaining high residual enzymatic activity during the lactose hydrolysis (in both cheese whey and milk). The tetrameric K. lactis β-galactosidase immobilized on Immobead supports via the tested treatments was stabilized and is an alternative tool for lactose hydrolysis in the dairy industry.

In this study, b-galactosidase has been immobilized on tannic acid stabilized silver nanoparticles (AgNPs). Tannic acid is a phytochemical and it is advantageous to use it as a linker molecule for immobilization because of its antidiarrheal and antimicrobial properties, and very low toxicity. AgNPs with immobilized b-galactosidase were characterized for particle size and catalytic properties. The AgNPs consisted of almost monodispersed particles of average diameter of~20 nm. b-galactosidase immobilized on tannic acid stabilized AgNPs (83.6% Immobilization yield) exhibited good activity with a high enzyme to carrier ratio as compared to the previous reports. Immobilization did not affect the optimum pH (pH 4.5) of the enzyme, however it retained greater fraction of activity in both alkaline and acidic pH range. The immobilized enzyme exhibited greater fraction of activity at higher temperatures as compared to the soluble enzyme, and its optimum temperature increased by 5 C. The immobilized enzyme retained almost 60% of its activity after 10th successive use. The immobilized enzyme hydrolyzed 258 and 474 mM lactose from 1% lactose and from milk lactose, respectively, whereas the soluble enzyme hydrolyzed 235 and 424 mM lactose from 1% lactose and from milk lactose, respectively. Excellent activity and stability of b-galactosidase immobilized on AgNPs provides a cost-effective industrial application of this enzyme. b-galactosidase immobilized on tannic acid stabilized AgNPs are free from toxicity hazards of the linker molecules. Hence, it may find constructive enzyme based applications in food technology.

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. Most of them are produced by enzyme biocatalysis because of molecular precision and environmental sustainability considerations. The current status and outlook of the production of lactose-derived bioactive compounds is presented with special emphasis on downstream operations which are critical because of the rather modest lactose conversion and product yields that are attainable. Even though some of these products have already an established market, there are still several challenges referring to the need of developing better catalysts and more cost-effective downstream operations for delivering high quality products at affordable prices. This technological push is expected to broaden the spectrum of lactose-derived bioactive compounds to be produced at industrial scale in the near future.

A method for immobilization of β-galactosidase from Kluyveromyces lactis on an adsorptive membrane for the continuous synthesis of galacto-oligosaccharides from lactose was carried out. The immobilization was performed at 4, 10, 15 and 40°C. Two strongly basic anion exchange membranes with tradename Mustang TM Q and Sartobind TM Q were investigated. In static experiments, the highest enzyme activity was measured on Mustang TM Q membranes at the immobilization temperature of 10°C. The synthesis of GOS was performed in a Continuous Membrane-Chromatography-Reactor-System (CMCRS) at 40°C and pH 7.0 using 20 wt% initial lactose concentration. The investigated membranes proved to be a good support for the continuous process at high convective flow rates in the enzyme reactor system. Up to 82% lactose conversion with 24% GOS yield was achieved at different fluxes. The corresponding reactor productivity for the production of GOS from lactose in the CMCRS was 98.7 grams GOS per hour and cubic centimeter membrane volume, which significantly exceeds previously reported results.

The continuous enzymatic production of galactosyl-oligosaccharides (GOS) from lactose as a substrate using a new type of ceramic membrane reactor system was investigated. GOS are non-digestible oligosaccharides and have recently attracted interest as prebiotics. However, the composition of oligosaccharides fraction and the variability in ß-glycosidic linkages depends on the enzyme source. In the study presented below, native, physically immobilized, ß-galactosidase from Klyveromyces lactis (EC 3.2.1.23) was used as enzyme to catalyse transgalactosylation reaction to produce GOS, competed against the hydrolysis of lactose into its two component monosaccharides, glucose and galactose. To optimize GOS yielded, process conditions were varied: The average residence time of the enzyme was varied in the range of 13 to 24 min, the trans-membrane pressure (TMP) was in the range of 1 to 2 bar and the initial concentration of substrate was varied from 10 to 30 % (w/w). Regarding the conditions investigated here, the maximum oligosaccharide concentration exceeded 38% (w/w) when the average residence time was 24 min, the TMP was 2 bar and an initial lactose concentration of 30% (w/w) was adjusted.

We set out to isolate alpha-amylase producers from soil samples, optimize the production, and immobilize the enzyme on chitosan-loaded barium ferrite nanoparticles (CLBFNPs). Alpha-amylase producers were isolated on starch agar plates and confirmed by dinitrosalicylic acid assay. The potent isolate was identified by phenotypic methods, 16S-rRNA sequencing, and phylogenetic mapping. Sequential optimization of α-amylase production involved the use of Plackett–Burman (P–BD) and central composite designs (CCD), in addition to exposing the culture to different doses of gamma irradiation. Alpha-amylase was immobilized on CLBFNPs, and the nanocomposite was characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, and scanning electron microscopy, with energy-dispersive analysis of X-ray analysis. Forty-five α-amylase producers were isolated from 100 soil samples. The highest activity (177.12 ± 6.12 U/mg) was detected in the MS009 isolate, which was identified as Bacillus...

The resulting whey from cheese manufacture consists mainly of water and lactose, a sugar with little sweetening and fermentable power and low commercial value. Aiming at valorizing the whey and faced with the challenge to reduce and treat an industrial wastewater present at high volumes and composed of high organic load, several ways to enhance the commercial value lactose have been sought. Thanks to the functional and technological traits of lactose and to the biotechnological advances, several applications have been proposed either for direct use or for the production and application of chemical and enzymatic derivatives or by-products from microbiological processes. Nowadays, lactose has numerous potential applications in food, feed, nutraceutical, pharmaceutical and cosmetic industry, an overview of which is given in this review. On the other hand, many compounds of high added value are produced from lactose, such as oligosaccharides with prebiotic properties, lactobionic acid with antioxidant properties, lactitol as sweetener with dietetic and prebiotic functions, and lactulose as prebiotic ingredient. Nevertheless, the implementation of industrial processes for whey collection and lactose recovery and valorization faces practical difficulties, particularly of logistical and financial investment nature. This communication aims at giving an overview with respect to the problems associated with the high volumes of whey produced in the dairy industry and some of the foremost technological routes for its commercial exploitation through the use of lactose and its derivatives. It also presents a set of considerations regarding the main bottlenecks to overcome towards the creation and implementation of integrated systems tailored for collecting and processing cheese whey in an efficient, sustainable and cost-effective approach. Keywords Whey • Lactose and lactose derivatives • Prebiotics, Probiotics and Symbiotics • Cheese whey by-products • Dairy industry Research highlights • Exploitation of lactose and reduction and treatment of industrial whey effluents • Chemical, enzymatic and microbial derivatives from lactose • Functional and technological traits of lactose and lactose derivatives • Industrial applications of lactose and lactose derivatives • Major bottlenecks found in the industrial implementation of whey collection and lactose recovery. This manuscript has been submitted for publication. It is not to be reproduced or cited without the written permission of the authors.

The kinetic modeling of the lactose hydrolysis and the operational stability of the enzyme β-galactosidase of Kluyveromyces fragilis was determined using as substrate skimmed powdered milk, reconstituted in a way to supply two lactose concentrations: 5% and 10% (w/v). For the operational stability both lactose concentrations were studied in the presence and in the absence of the buffer pH 6.5. Every 40 minutes the residual activity was determined by the method of the initial velocities. The experimental results showed that the buffer leads to enzyme inactivation. However, for lactose solutions without buffer, the residual activity only declines 15% in 6 hours and later it decreases abruptly. In the kinetic modeling the hydrolysis reaction was led to 40 o C/7h, being used enzyme concentrations equivalent to 1500 and 8100 LAU/L. The adjusted model allowed us to conclude that it is necessary to use the quantity of 3450 LAU/L of the enzyme to obtain the hydrolysis from 70% to 80% of the milk lactose in two to three hours of reaction.

R-β-Gal is a cold-adapted β-galactosidase that is able to hydrolyze lactose and has the potential to produce low-lactose or lactose-free dairy products at low temperatures (4°C). Cold-adapted enzymes unfold at moderate temperatures due to the lower intramolecular stabilizing interactions necessary for flexibility at low temperatures. To increase stability and usage-performance, R-β-Gal was encapsulated in gellan gum by injecting an aqueous solution into two different hardening solutions (10mM CaCl2 or 10mM MgCl2). Enzyme characteristics of both free and encapsulated R-β-Gal were carried out, and the different effects of two cations were investigated. R-β-Gal showed better thermal and pH stability after encapsulation. Ca(2+) gels had higher encapsulation efficiency (71.4%) than Mg(2+) (66.7%) gels, and Ca(2+) formed larger inner and surface pores. R-β-Gal was released from the Ca(2+) hydrogel beads more rapidly than the Mg(2+) hydrogels during storage in aqueous solution due to the l...

Bioencapsulation of microbial cells in silica-based matrices has proved to be a good strategy to enhance the biosynthetic capabilities and viability of bioproducers. In the present study, mycelium and pellet cultures of strain Aspergillus oryzae PP were successfully immobilized in solÀgel hybrid matrices composed of tetraethylorthosilicate as an inorganic precursor, 5% (w/v) starch and 10 or 15% (w/v) polyethylene oxide, or 10% (w/v) calcium alginate as organic compounds. Biosynthetic activity of immobilized cultures was investigated by batch and fed-batch cultivation and the obtained results of 3042.04 IU cm ¡3 were comparable with the enzyme activity of the free cell culture. Immobilized cultures retained their viability and biosynthetic capabilities up to the 744th h during fed-batch fermentation processes. Consequently, solÀgel encapsulation in hybrid matrices could be considered as a promising technique for immobilization of Aspergillus oryzae PP in order to increase the a-amylase production.

A newly isolated strain of Bacillus licheniformis E66 was used for the on production of extra cellular β-Galactosidase in a medium containing lactose with concentration 12%. Maximum enzyme production (384U/ml) was obtained when fermentation medium adjusted at pH 6 with cultivation 3% (v/v) inoculum of 24h-old culture and the volume was 50/200 (v/v) with a fermentation time of 48h at shake flask.

Immobilization is the limitation of movement of biocatalysts according to chemical or physical treatment. Immobilized molecules technique using biomaterials and nano-biotechnology is a very interesting topic that is touching almost all aspects of our life. Immobilized enzymes, molecules, and cells have been used in a variety of scientific and industrial applications. Cell immobilization biotechnology is a multidisciplinary area shown to have an important impact on many scientific subdisciplines, including biomedicine, pharmacology, cosmetology, food and agricultural sciences. Many molecules have been immobilized and the majority of them are bio-molecules due to their biological and biomedical applications. Immobilization of enzymes has made them highly applicable to range of evolving biotechnologies. Immobilized enzymes have proven valuable for many medical applications including drug delivery systems, diagnosis and treatment of diseases, as well as in sensors for the management of weight and diabetes. Enzyme immobilization is applied in textile industry. The immobilized microorganism technology offers a multitude of advantages in wastewater treatment. The immobilized cell systems were applied for the production of many organic compounds such as organic acid and ethanol. The immobilization of tissue sections, cells and tissue components for histological and immunohistochemical staining or detection systems is applicable nowadays. Cell immobilization could potentially benefit food industry.

Lactose is a major disaccharide by-product from the dairy industries, and production of whey alone amounts to about 200 million tons globally each year. Thus, it is of particular interest to identify improved enzymatic processes for lactose utilization. Microbial β-glucosidases (BGL) with significant β-galactosidase (BGAL) activity can be used to convert lactose to glucose (Glc) and galactose (Gal), and most retaining BGLs also synthesize more complex sugars from the monosaccharides by transglycosylation, such as galactooligosaccharides (GOS), which are prebiotic compounds that stimulate growth of beneficial gut bacteria. In this work, a BGL from the thermophilic and halophilic bacterium Halothermothrix orenii, HoBGLA, was characterized biochemically and structurally. It is an unspecific β-glucosidase with mixed activities for different substrates and prominent activity with various galactosidases such as lactose. We show that HoBGLA is an attractive candidate for industrial lactose conversion based on its high activity and stability within a broad pH range (4.5-7.5), with maximal β-galactosidase activity at pH 6.0. The temperature optimum is in the range of 65-70°C, and HoBGLA also shows excellent thermostability at this temperature range. The main GOS products from Ho-BGLA transgalactosylation are β-D-Galp-(1→6)-D-Lac (6GALA) and β-D-Galp-(1→3)-D-Lac (3GALA), indicating that D-lactose is a better galactosyl acceptor than either of the monosaccharides. To evaluate ligand binding and guide GOS modeling, crystal structures of HoBGLA were determined in complex with thiocellobiose, 2-deoxy-2-fluoro-D-glucose and glucose. The two major GOS products, 3GALA and 6GALA, were modeled in the substrate-binding cleft of wild-type Ho-BGLA and shown to be favorably accommodated.

The bacterium Pseudomonas fluorescens 07A produces a protease with potential for industrial application. In order to remedy problems associated with the use of free enzymes and allow its reuse, the protease was immobilized on DEAE Sephacel ® resin via three different strategies based on ionic interaction and covalent bonding. The matrix-bound enzymes were characterized in relation to their activity (pH, temperature and stability), reuse and storage. Immobilization raised the optimum temperature of activity from 37 °C to 50 °C, whereas the pH of highest activity changed from 7.5 to 7.0 or 8.0, depending on the immobilization strategy. Immobilization proved to be efficient for successive reuses, even leading to an increase in the enzymatic activity along the use. The immobilized enzyme also presented greater stability to high temperatures and storage conditions and has potential as a biocatalyst for industrial applications due to its high efficiency, stability and easy recovery.

The current demands of the world's biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immobilized enzyme systems allows an easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This review summarizes immobilization definition, different immobilization methods, advantages and disadvantages of each method. In addition, it covers some food industries, protein purification, human nutrition, biodiesel production, and textile industry. In these industries, the use of enzymes has become an inevitable processing strategy when a perfect end product is desired. It also can be used in many other important industries including health care and pharmaceuticals applications. One of the best uses of enzymes in the modern life is their application in diagnose and treatment of many disease especially when used in drug delivery system or when used in nanoform.

Hydrolysis of lactose in milk by β-galactosidase from immobilized bacterial cells has the potential to alleviate the problem of lactose intolerance. The present study was aimed to immobilize cells of L. plantarum strain B134 and evaluate their efficiency in hydrolyzing lactose in ultra high temperature (UHT) milk. Immobilized cells were generated by mixing cell suspensions with solutions of sodium alginate and calcium chloride. The β-galactosidase activity of the immobilized cells was tested by determining their ability in hydrolyzing lactose in UHT milk (whole milk and skimmed milk). Results showed that cells of L. plantarum strain B134 were entrapped optimally using a combination of 1 % sodium alginate, 100 mM calcium chloride and 12 % w/v cell suspension. The highest β-galactosidase activity was achieved at pH 6.5 and a temperature of 45 ºC for 5 minutes incubation time. The immobilization efficiency achieved was 28.95 %. The immobilized cells could reduce lactose by up to 85.45 ...

The current demands of the world's biotechnological industries are enhancement in enzyme productivity and development of novel techniques for increasing their shelf life. Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immobilized enzyme systems allows an easy recovery of both enzymes and products, multiple reuse of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This review summarizes immobilization definition, different immobilization methods, advantages and disadvantages of each method. In addition, it covers some food industries, protein purification, human nutrition, biodiesel production, and textile industry. In these industries, the use of enzymes has become an inevitable processing strategy when a perfect end product is desired. It also can be used in many other important industries including health care and pharmaceuticals applications. One of the best uses of enzymes in the modern life is their application in diagnose and treatment of many disease especially when used in drug delivery system or when used in nanoform.

Background: Glucoamylase enzyme is one of the most important enzymes. It catalyzes the hydrolysis of starch into soluble sugars. It was covalently immobilized onto κ-carrageenan gel beads after activation by using polyethylenimine (PEI) followed by glutaraldehyde (GA). All parameters in activation process were studied. Results: The immobilized enzyme shows enhancement in temperature profile as the optimum temperature for the free enzyme was 60°C, and it becomes 60-80°C that means broader range and also shows stability in acidic conditions more than free enzyme. The apparent Km of immobilized enzyme, 147.46 mM, becomes higher than Km of the free one, 110 mM and the maximum reaction velocity (Vmax) values for the immobilized enzyme decreased from 2.28 to 1.11 μmol min −1. The immobilized enzyme can be reused and kept its activity (100%) till 11 successive cycles. The immobilization process steps were characterized by FTIR and SEM. Conclusions: These results confirm the economic and biotechnical benefits of enzyme immobilization, particularly with regard to the number of enzyme reuses, which open the possibility of different industrial applications.