Aspergillus Oryzae Β-galactosidase Research Articles

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. Afterward, the effect of the 1-butanol/ aqueous phase ratio (α) on the reaction was evaluated in the range from 0.25 to 4. Results show that higher values of α reduce the incidence of secondary hydrolysis by favoring the extraction of butyl-β-galactoside into the organic phase where it is not hydrolyzed, leading to higher yields. Also, major interfacial properties for butyl-β-galactoside were determined at 25 °C.

Optimization and comparison of the production of galactooligosaccharides using free or immobilized Aspergillus oryzae β-galactosidase, followed by purification using silica gel

The aim of this study was to optimize and compare the production of galactooligosaccharides (GOSs) by free and cotton cloth-immobilized Aspergillus oryzae β-galactosidase, and perform economical evaluation of production of GOSs (100%) between them. Using the response surface method, the optimal reaction time (3.9 h), initial lactose concentration (57.13%), and enzyme to lactose ratio (44.81 U/g) were obtained for the free enzyme, which provided a GOSs yield of 32.62%. For the immobilized enzyme, the optimal yield of GOSs (32.48%) was obtained under reaction time (3.09 h), initial lactose concentration (52.74%), and temperature (50.0 ℃). And it showed desirable reusability during five successive enzymatic reactions. The recovery rate of GOSs (100%) is 65% using silica gel filtration chromatography. The economical evaluation showed almost no difference in the manufacturing cost for the GOSs (100%) between these two systems, and that the recovery rate had a great impact on the cost.

Design of immobilized biocatalyst and optimal conditions for tyrosol β-galactoside production.

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 0.83M 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, 30-40g/L, were favorable as pseudo-equilibrium concentrations of TG and galactooligosaccharide were reached. Repeated batch results revealed excellent operational stability of the biocatalyst.

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 Aspergillus oryzae β-galactosidase. The liquid–liquid equilibrium of the reaction mixture components (acetone/1-butanol/aqueous solution) was determined and the single- and two-phase regions were defined at 30, 40, and 50°C. It was observed that the liquid–liquid equilibrium of the ternary system acetone/1-butanol/water differs significantly from the one obtained using an aqueous solution (50 mM McIlvaine buffer pH 4.5; 5 g L–1) instead of water. This is mainly because of the salting-out effect of the buffer; nevertheless, the presence of lactose also altered the equilibrium. Having this in mind, the effects of temperature (30 and 50°C) and reaction mixture composition were assessed. Three general conditions were evaluated: single-phase ternary system (30% acetone), two-phase ternary system (10% acetone) and two-phase binary system (0% acetone). Acetone had a deleterious effect on enzyme stability at 50°C, leading to low reaction yields. However, no enzyme deactivation was detected at 30°C. Moreover, a reaction yield of 0.98 mol mol–1 was attained in the 30/50/20% (w/w) mixture of acetone/1-butanol/aqueous solution. This very high yield can be explained by the huge increase in the concentration of 1-butanol and the reduction of water activity. The synthesis was carried out using also the β-galactosidase immobilized in glyoxal-agarose and amino-glyoxal-agarose, and by aggregation and crosslinking. In the case of agarose-derived catalysts, two average particle diameters were assessed to evaluate the presence of internal mass transfer limitations. Best yield (0.88 mol mol–1) was obtained with glyoxal-agarose derivatives and the particle size had non-effect on yield. The chemical structure of butyl-β-galactoside was determined by NMR and FT-IR.

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

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 (YLactulose), specific productivity (πLactulose) and selectivity of the reaction (lactulose/transgalactosylated oligosaccharides molar ratio) (SLu/TOS), the most significant effects were obtained by varying the inlet concentrations of sugar substrates and the feed substrate molar ratio. Maximum YLactulose 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 IUH⋅mL–1. At such conditions SLu/TOS was 3.7, lactose conversion (XLactose) 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 XLactose 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.

Understanding the Effects of Lactose Hydrolysis Modeling on the Main Oligosaccharides in Goat Milk Whey Permeate.

Enzymatic hydrolysis of lactose is a crucial step to improve the efficiency and selectivity of membrane-based separations toward the recovery of milk oligosaccharides free from simple sugars. Response surface methodology was used to investigate the effects temperature (25.9 to 54.1 °C) and amount of enzyme (0.17 to 0.32% w/w) at 1, 2, and 4 h of reaction on the efficiency of lactose hydrolysis by Aspergillus oryzae β-galactosidase, preservation of major goat whey oligosaccharides, and on the de-novo formation of oligosaccharides. Lactose hydrolysis above 99% was achieved at 1, 2, and 4 h, not being significantly affected by temperature and amount of enzyme within the tested conditions. Formation of 4 Hexose (Hex) and 4 Hex 1 Hex and an increased de-novo formation of 2 Hex 1 N-Acetyl-Neuraminic Acid (NeuAc) and 2 Hex 1 N-Glycolylneuraminic acid (NeuGc) was observed in all treatments. Overall, processing conditions using temperatures ≤40 °C and enzyme concentration ≤0.25% resulted in higher preservation/formation of goat whey oligosaccharides.

Cooperation between β-galactosidase and an isoprimeverose-producing oligoxyloglucan hydrolase is key for xyloglucan degradation in Aspergillusoryzae.

The galactosylation of xyloglucan blocks many of the enzymatic processes targeting this oligosaccharide. We found that the expression of a gene encoding Aspergillusoryzae β-galactosidase (LacA) is induced in the presence of xyloglucan oligosaccharides. With detailed analyses of the substrate specificity of purified recombinant LacA, we show that LacA cleaves galactopyranosyl residues from xyloglucan oligosaccharides, but not from xyloglucan polysaccharide, and plays a vital role in xyloglucan degradation. LacA acts cooperatively with the isoprimeverose-producing oligoxyloglucan hydrolase IpeA to hydrolyze xyloglucan oligosaccharides. Galactosylation of the xylopyranosyl side chain at the nonreducing end of oligoxyloglucan saccharides completely abolishes IpeA activity while LacA efficiently removes the galactopyranosyl residue. Conversely, an isoprimeverose unit at the nonreducing end of the main chain of xyloglucan oligosaccharides blocks LacA activity, while IpeA can still remove the isoprimeverose moiety. This is the first study reporting the cooperative action of β-galactosidase and isoprimeverose-producing oligoxyloglucan hydrolase on xyloglucan oligosaccharide degradation. Our findings shed light on the true role of LacA and the enzymatic coordination between β-galactosidase and other hydrolases on xyloglucan degradation.

BIOTECHNOLOGICAL APPLICATION OF SURFACE MODIFIED CERIUM OXIDE NANOPARTICLES

ABSTRACT Re-engineering of chemical materials at the nanoscale level that employ modification and improvement in their physical and chemical properties has been constantly pursued for application in biomedical and biotechnology industries. Moreover, immobilization of catalysts on these bio/chemically modified nanomaterials improved the performance of enzymes in a plethora of industrial uses. Hence, in this study, cerium oxide nanoparticles (CNPs) were synthesized and their morphology was investigated by TEM and UV-spectra. They were modified by carboxylation and glutaraldehyde to achieve highly efficient surface functionalized nanomatrices for immobilizing Aspergillus oryzae β-galactosidase for producing lactose-free products in dairy industries. Enzyme activity for soluble and immobilized enzyme was observed in different pH and temperature ranges, and on galactose mediated competitive inhibition offered by the substrate. It was observed that all the enzyme preparations exhibited temperature-optima at 50 °C and pH-optima at pH 4.5, respectively. Michaelis-Menten Km (mmole/L) values were 2.40, 5.88, 6.02 and 6.11 for soluble β-galactosidase, and enzyme immobilized on CNPs, carboxylated CNPs and glutaraldehyde modified CNPs, respectively. However, Vmax (mmole/L/min) was found to be 518, 507, 495 and 480 for these enzyme preparations under identical conditions. Immobilized enzyme demonstrated excellent reusability even after seven repeat uses. The bioconversion rates of lactose from solution in continuous batch reactors revealed the remarkable catalytic efficiency of β-galactosidase immobilized on glutaraldehyde modified CNPs in comparison to other enzyme preparations.

Discovery of Novel High-Molecular Weight Oligosaccharides Containing N-Acetylhexosamine in Bovine Colostrum Whey Permeate Hydrolyzed with Aspergillus oryzae β-Galactosidase.

Bovine milk oligosaccharides (BMOs) that resemble human milk oligosaccharides are found in whey permeate, indicating that dairy streams can be used as a potential source of bioactive oligosaccharides. Recovery of oligosaccharides from whey permeate is hindered by their low abundance and high concentration of lactose. In the present work, lactose in bovine colostrum whey permeate was hydrolyzed by Aspergillus oryzae β-galactosidase to facilitate subsequent monosaccharide removal by membrane separation. Chromatographic separation coupled with high-resolution mass spectrometry revealed β-galactosidase degradation of several β-linkage-containing BMOs and production of novel oligosaccharides that ranged in size from 5 to 11 monosaccharide units containing several galactose repeating units and N-acetylhexosamine at their reducing ends. Optimization of BMO hydrolysis and separation methodology could generate high amounts of hetero-oligosaccharides for improved recovery of potentially biotherapeutic oligosaccharides.

Effect of metal ions present in milk on the structure and functional integrity of native and polyaniline chitosan nanocomposites bound β-galactosidase: A multi-spectroscopic approach

β-Galactosidase is vital to dairy industries because it catalyzes the hydrolysis of lactose into glucose and galactose making it useful for lactose intolerant patients to consume milk and its products. The present study demonstrates the effect of metal ions commonly found in milk, namely Zn2+, K+, Ca2+ and Mn2+ on the activity of native and polyaniline chitosan nanocomposites bound Aspergillus oryzae β-galactosidase. In polyaniline chitosan silver nanocomposite adsorbed β-galactosidase, a multi-fold enhancement in catalytic activity was observed in the presence of cocktail of Zn2+, K+, Ca2+ and Mn2+ ions. 3D fluorescence, CD and FTIR studies established significant conformational changes in the secondary structure of polyaniline chitosan silver nanocomposite bound β-galactosidase on addition of metal ions as compared to the native and other bound enzyme. This enhanced catalytic efficiency of the immobilized enzyme in presence of metal ions can promote its economic use for lactose hydrolysis in milk.

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 (MB/MIM) were studied on reactor performance. Increase in any of these variables produced an increase in lactulose yield (YLu) being higher than obtained in batch synthesis at comparable conditions. Maximum YLu of 0.6 g·g−1 was obtained at 50 °C, pH 4.5, 50% w/w total sugars, 15 mL·min−1, fructose/lactose molar ratio of 12 and MB/MIM of 1/8 g·g−1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16 g·g−1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was 28%. Reactor operation with recycle had no significant effect on yield, producing only some decrease in productivity.

Effect of particle size and enzyme load on the simultaneous reactions of lactose hydrolysis and transgalactosylation with glyoxyl-agarose immobilized β-galactosidase from Aspergillus oryzae

Abstract The simultaneous reactions of lactose hydrolysis and transgalactosylation in the production of galacto-oligosaccharides (GOS) were evaluated with immobilized Aspergillus oryzae β-galactosidase in glyoxyl-agarose of different particle sizes (fine and macro) and enzyme loads (1, 10 and 30 mgprotein/gsupport) to produce biocatalysts subjected to different magnitudes of internal diffusional restrictions. The ratio of initial reaction rates depended on the initial concentration of lactose: at values higher than 800 mM, the ratio of hydrolysis to total reaction rate (rhydr/rtotal), which reflects the hydrolytic potential, had values of 16 and 30%, and the ratio of transgalactosylation to total reaction rates (rtransgal/rtotal), which reflects the transgalactosylation potential, had values of 84 and 70% with the biocatalysts of smaller size and enzyme load, and bigger size and enzyme load, respectively. Results obtained highlight the fact that the biocatalyst should be optimized with regard to its intended use; β-galactosidase biocatalysts that have been optimized for lactose hydrolysis can be quite inadequate for performing GOS synthesis.

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β-galactosidase.

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead-Glu) or carboxyl groups through acid solution (Immobead-Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β-galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead-Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10-500 mg g-1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g-1 support. Gal immobilized on Immobead-Glu and Immobead-Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half-lifes than the soluble enzyme, where the half-lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934-943, 2018.

Selective bioconversion with yeast for the purification of raw lactulose and transgalactosylated oligosaccharides

Raw transgalactosylated oligosaccharides (raw TOS) obtained by enzymatic synthesis with Aspergillus oryzae β-galactosidase were purified by selective bioconversion with Saccharomyces cerevisiae ATCC4126 and Kluyveromyces marxianus NRLLY-1109. Monosaccharides were completely removed by S. cerevisiae and K. marxianus from raw TOS without any nutrient supplementation. The effect of the biomass to carbohydrate mass ratio on the purity and specific productivity of removal of sugars was assessed with S. cerevisiae, where both effects were significant. Purity obtained with a collection strain of S. cerevisiae and bakers’ yeast was compared, the latter being higher after 24 h of bioconversion. A product free of monosaccharides was obtained with a purity (0.48; 28% lactulose and 20% oligosaccharides), similar to that of commercial galacto-oligosaccharide preparations. The possibility of yeast recovery and reuse in sequential batch operation was assessed: two batches could be performed before a significant reduction in monosaccharide removal was seen.

Biocatalytic characterization of Aspergillus oryzae β-galactosidase immobilized on functionalized multi-walled carbon nanotubes

The objectives of this work were to immobilize commercial Aspergillus oryzae β-galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs) using different treatments and to characterize the products. Treatments were performed with glutaraldehyde, ethylenediamine and a mixture of concentrated H2SO4:HNO3. The MWCNTs and their derivatives were characterized by thermogravimetric analysis. The immobilized enzymes were evaluated using inactivation kinetics, operating conditions, that is pH and temperature, kinetic parameters and lactose hydrolysis reusability. Immobilization yield and efficiency were significantly higher for β-galactosidase immobilized on MWCNTs functionalized by the acid mixture (Ac-Gal-MWCNTs). These values were 97% and 82%, respectively, after 3 h of immobilization. The activity of the Ac-Gal-MWCNTs was maintained at ∼51% of their initial activity after being stored for 90 days at 4 °C. The Ac-Gal-MWCNTs retained more than 90% of their initial activity up to the fourth recycle. As the acid functionalization was the most efficient method tested for immobilizing A. oryzae β-galactosidase on MWCNTs, this method shows promise for industrial applications.

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−1, 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. The enzyme-limiting substrate mass ratio (E/S) is a critical variable in fed-batch operation. Higher values favor lactose hydrolysis over transgalactosylation, being 400IU/g the limit for proper lactulose synthesis in fed-batch operation. Selectivity of lactulose synthesis increased with E/S being quite high at 800IUH·g−1 or higher. However, this increase was obtained at the expense of lactulose yield. Lactulose synthesis in fed-batch operation was a better option than conventional batch synthesis, since higher product concentration and selectivity of lactulose over TOS synthesis were obtained.

Improvement of recovered activity and stability of the Aspergillus oryzae β-galactosidase immobilized on duolite A568 by combination of immobilization methods

The immobilization and stabilization of Aspergillus oryzae ?-galactosidase on Duolite??A568 was achieved using a combination of physical adsorption, incubation step in buffer at pH 9.0 and cross-linking with glutaraldehyde and in this sequence promoted a 44% increase in enzymatic activity as compared with the biocatalyst obtained after a two-step immobilization process (adsorption and cross-linking). The stability of the biocatalyst obtained by three-step immobilization process (adsorption, incubation in buffer at pH 9.0 and cross-linking) was higher than that obtained by two-steps (adsorption and cross-linking) and for free enzyme in relation to pH, storage and reusability. The immobilized biocatalyst was characterized with respect to thermal stability in the range 55-65 ?C. The kinetics of thermal deactivation was well described by the first-order model, which resulted in the immobilized biocatalyst activation energy of thermal deactivation of 71.03 kcal/mol and 5.48 h half-life at 55.0 ?C.

Synthesis of propyl-β-d-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae

Abstract Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.

Synthesis of Oligosaccharides Derived from Lactulose (OsLu) Using Soluble and Immobilized Aspergillus oryzae β-Galactosidase

β-Galactosidase from Aspergillus oryzae offers a high yield for the synthesis of oligosaccharides derived from lactulose (OsLu) by transgalactosylation. Oligosaccharides with degree of polymerization (DP) ≥ 3 have shown to possess higher in vitro bifidogenic effect than di- and tetrasaccharides. Thus, in this work, an optimization of reaction conditions affecting the specific selectivity of A. oryzae β-galactosidase for synthesis of OsLu has been carried out to enhance OsLu with DP ≥ 3 production. Assays with β-galactosidase immobilized onto a glutaraldehyde–agarose support were also carried out with the aim of making the process cost-effective and industrially viable. Optimal conditions with both soluble and immobilized enzyme for the synthesis of OsLu with DP ≥ 3 were 50 °C, pH 6.5, 450 g/L of lactulose, and 8 U/mL of enzyme, reaching yields of ca. 50% (w/v) of total OsLu and ca. 20% (w/v) of OsLu with DP 3, being 6′-galactosyl-lactulose the major one, after a short reaction time. Selective formation of disaccharides, however, was favored at 60 °C, pH 4.5, 450 g/L of lactulose and 8 U/mL of enzyme. Immobilization increased the enzymatic stability to temperature changes and allowed to reuse the enzyme. We can conclude that the use, under determined optimal conditions, of the A. oryzae β-galactosidase immobilized on a support of glutaraldehyde–agarose constitutes an efficient and cost-effective alternative to the use of soluble β-galactosidases for the synthesis of prebiotic OsLu mixtures.

Development of a continuous membrane reactor process for enzyme-catalyzed lactulose synthesis

The study addresses three main issues which are usually encountered in membrane-assisted enzymatic reactions, such as membrane–enzyme electrostatic interaction, optimum operating conditions (with industrially relevant parameters) and maintenance of catalytic activity for prolonged synthesis. Considering Aspergillus oryzae β-galactosidase charge distribution at pH 4.6 was largely positive, a negatively charged PES membrane was not suited for the synthesis. With this, a rapid increase in transmembrane pressure occurred during continuous lactulose synthesis. The UFX membrane MWCO 10kDa is permanently hydrophilic and exhibited repulsion to the enzyme molecules and in turn, had an insignificant transmembrane pressure increase. Thus, this membrane was selected for further investigations. The operating conditions were optimized successively and were found to be: enzyme concentration=10U/mL, molar ratio of lactose to fructose=1/4, hydraulic residence time=9h and incubation temperature=40°C. With an inaccuracy of enzyme dosing protocol less than 5%, a long-term synthesis of lactulose was performed for 28 days with an average lactulose outlet concentration of 8.67g/L. The enzyme activity was maintained by addition of fresh enzyme every 48h. This is the longest continuous lactulose synthesis ever reported in fully automated membrane reactor.