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Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical industry. At present, production of GOS involves the enzymatic transformation of lactose by transgalactosylation using ß-galactosidase. The yeast Kluyveromyces lactis can utilize lactose as its carbon and energy source. In this species lactose is hydrolyzed by an intracellular ß-galactosidase (EC 3.2.1.23) which is induced by its substrate and related compounds like galactose. The molecular details of gene regulation in kluyveromyces lactis, we have used multiple knockout approaches to study the constitutive expression by which galactose induces ß-galactosidase. The present study involved carrying out to a method of enhancing the constitutive expression of ß-galactosidase through galactose induction and its trans-galactosylation reaction for the production of galacto-oligosaccharides (GOS) in Kluyveromyces lactis (K. Lactis) by applying a knockout based approach on Leloir pathway genes based on fusion-overlap extension polymerase chain reaction and transformation into its genome. The k.lactis strain subjected to Leloir pathway genes knockout, resulted in the accumulation of galactose intracellularly and this internal galactose acts as an inducer of galactose regulon for constitutive expression of ß-galactosidase at early stationary phase was due to the positive regulatory function of mutant gal1p, gal7p and both. These resulted strains used for trans-galactosylation of lactose by ß - galactosidase is characterized for the production of galacto-oligosaccharides. Galactose-induced constitutive expression of ß-galactosidase during the early stationary phase of knockout strains was analysed qualitatively & quantitatively. The activity of ß-galactosidase of wild type, gal1z, gal7k and gal1z & gal7k strains were 7, 8, 9 and 11 U/ml respectively using high cell density cultivation medium. Based on these expression differences in ß-galactosidase, the trans-galactosylation reaction for GOS production and percentage yield of GOS were compared at 25% w/v of lactose. The percentage yield of GOS production of wild type, Δgal1z Lac4+, Δgal7k Lac4++ and Δgal1z Δgal7k Lac4+++mutants strains were 6.3, 13, 17 and 22 U/ml, respectively. Therefore, we propose that the availability of galactose can be used for constitutive over expression of ß - galactosidase in Leloir pathway engineering applications and also for GOS production. Further, increased expression of ß - galactosidases can be used in dairy industry by-products like whey to produce added value products such as galacto-oligosaccharides.

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Few studies expressed the ß-galactosidase encoding gene from L. plantarum in E. coli so far. In the present study, the recombinant ß-galactosidase from L. plantarum FMNP01 was used as a catalyst in transgalactosylation to form tri-GOS and lactosucrose. In the presence of lactose and sucrose, six transfer products were formed in the transgalactosylation reaction with recombinant ß-galactosidase L.pFMNP01Gal as a catalyst. Three transfer products were tri-galacto-oligosaccharides (tri-GOS), lactosucrose, and lactulose; the other three transfer products needed to be identified further. Based on a single factor test and response surface methodological approach, the optimal transgalactosylation conditions of the production of tri-GOS and lactosucrose were determined as initial sugar concentration of 50%, lactose: sucrose ratio of 1:2, enzyme concentration of 3 U/mL, and reaction time of 6 h at 50 °C resulting in a maximum tri-GOS concentration of 47.69 ± 1.36 g/L and a maximum lactosucrose concentration of 8.18 ± 0.97 g/L.

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α-Galactosidase hydrolyzes the α-1,6-linkage present at the non-reducing end of the sugars and results in the release of galactosyl residue from oligosaccharides like melibiose, raffinose, stachyose, etc. In the present study we report, α-galactosidase from Bacillus flexus isolated from Manikaran hot springs (India). Maximum enzyme production was obtained in guar gum and soybean meal after 72 h at 150 rpm. While, the temperature/pH of production was optimized at 50 °C and 7.0, respectively. Isoenzymes (α-gal I and II) were obtained and characterized based on temperature/pH optima along with their stability profile. JS27 α-Gal II was purified with a final purification fold of 11.54. Native and SDS-PAGE were used to determine the molecular weight of the enzyme as 86 and 41 kDa, respectively, indicating its homodimeric form. JS27 α-Gal II showed optimum enzyme activity at 55 °C and pH 7 (10 min). The enzyme displayed Km value of 2.3809 mM and Vmax of 2.0 × 104 µmol/min/ml with pNPG as substrate. JS27 α-Gal II demonstrated substrate hydrolysis and simultaneous formation of transgalactosylation products (α-GOS) with numerous substrates (sugar/sugar alcohols, oligosaccharides, and complex carbohydrates) which were verified by TLC and HPLC analysis. α-GOS are significant functional food ingredients and can be explored as prebiotics.

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A novel ß-galactosidase gene (galM) was cloned from an aquatic habitat metagenome. The analysis of its translated sequence (GalM) revealed its phylogenetic closeness towards Verrucomicrobia sp. The sequence comparison and homology structure analysis designated it a member of GH42 family. The three-dimensional homology model of GalM depicted a typical (ß/α)8 TIM-barrel containing the catalytic core. The gene (galM) was expressed in a heterologous host, Escherichia coli, and the purified protein (GalM) was subjected to biochemical characterization. It displayed ß-galactosidase activity in a wide range of pH (2.0 to 9.0) and temperature (4 to 60 °C). The heat exposed protein showed considerable stability at 40 and 50 °C, with the half-life of about 100 h and 35 h, respectively. The presence of Na, Mg, K, Ca, and Mn metals was favorable to the catalytic efficiency of GalM, which is a desirable catalytic feature, as these metals exist in milk. It showed remarkable tolerance of glucose and galactose in the reaction. Furthermore, GalM discerned transglycosylation activity that is useful in galacto-oligosaccharides' production. These biochemical properties specify the suitability of this biocatalyst for milk and whey processing applications. KEY POINTS: • A novel ß-galactosidase gene was identified and characterized from an aquatic habitat. • It was active in extreme acidic to mild alkaline pH and at cold to moderate temperatures. • The ß-galactosidase was capable to hydrolyze lactose in milk and whey.

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ß-Galactosidase is one of the most important enzymes used in dairy processing. It converts lactose into glucose and galactose, and also catalyzes galactose to form galactooligosaccharides (GOS), so-called prebiotics. However, most of the ß-galactosidases from the starter cultures have low transgalactosylation activities, the process that results in galactose accumulation in yogurt. Here, a site-directed mutation strategy was attempted, to genetically modify ß-galactosidase from Streptococcus thermophilus. Out of 28 Strep. thermophilus strains, a ß-galactosidase gene named bgaQ, encoded for high ß-galactosidase hydrolysis activity (BgaQ), was cloned from the strain Strep. thermophilus SDMCC050237. It was 3,081 bp in size, with 1,027 deduced amino acid residuals, which belonged to the GH2 family. After replacing the Tyr801 and Pro802 around the active sites of BgaQ with His801 and Gly802, the GOS synthesis of the generated mutant protein BgaQ-8012 increased from 20.5% to 26.7% at 5% lactose, and no hydrolysis activity altered obviously. Subsequently, the purified BgaQ or BgaQ-8012 was added to sterilized milk inoculated with 2 starters from Strep. thermophilus SDMCC050237 and Lactobacillus delbrueckii ssp. bulgaricus ATCC11842. The GOS yields with added BgaQ or BgaQ-8012 increased to 5.8 and 8.3 g/L, respectively, compared with a yield of 3.7 g/L without enzymes added. Meanwhile, the addition of the BgaQ or BgaQ-8012 reduced the lactose content by 49.3% and 54.4% in the fermented yogurt and shortened the curd time. Therefore, this study provided a site-directed mutation strategy for improvement of the transgalactosylation activity of ß-galactosidase from Strep. thermophilus for GOS-enriched yogurt making.

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ß-Galactosidases catalyse the hydrolysis of lactose into galactose and glucose; as an alternative reaction, some ß-galactosidases also catalyse the formation of galactooligosaccharides by transglycosylation. Both reactions have industrial importance: lactose hydrolysis is used to produce lactose-free milk, while galactooligosaccharides have been shown to act as prebiotics. For some multi-domain ß-galactosidases, the hydrolysis/transglycosylation ratio can be modified by the truncation of carbohydrate-binding modules. Here, an analysis of BbgIII, a multidomain ß-galactosidase from Bifidobacterium bifidum, is presented. The X-ray structure has been determined of an intact protein corresponding to a gene construct of eight domains. The use of evolutionary covariance-based predictions made sequence docking in low-resolution areas of the model spectacularly easy, confirming the relevance of this rapidly developing deep-learning-based technique for model building. The structure revealed two alternative orientations of the CBM32 carbohydrate-binding module relative to the GH2 catalytic domain in the six crystallographically independent chains. In one orientation the CBM32 domain covers the entrance to the active site of the enzyme, while in the other orientation the active site is open, suggesting a possible mechanism for switching between the two activities of the enzyme, namely lactose hydrolysis and transgalactosylation. The location of the carbohydrate-binding site of the CBM32 domain on the opposite site of the module to where it comes into contact with the catalytic GH2 domain is consistent with its involvement in adherence to host cells. The role of the CBM32 domain in switching between hydrolysis and transglycosylation modes offers protein-engineering opportunities for selective ß-galactosidase modification for industrial purposes in the future.

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ß-galactosidase catalyzes lactose hydrolysis and transfers reactions to produce prebiotics such as galacto-oligosaccharides (GOS) with potential applications in the food industry and pharmaceuticals. However, there is still a need for improved transgalactosylation activity of ß-galactosidases and reaction conditions of GOS production in order to maximize GOS output and reduce production costs. In this study, a ß-galactosidase gene, galA, from Bacillus circulans was expressed in Pichia pastoris, which not only hydrolyzed lactose but also had strong transgalactosylation activity to produce GOS. Response surface methodology was adopted to investigate the effects of temperature, enzyme concentration, pH, initial lactose concentration, and reaction time on the production of GOS and optimize the reaction conditions for GOS. The optimal pH for the enzyme was 6.0 and remained stable under neutral and basic conditions. Meanwhile, GALA showed most activity at 50°C and retained considerable activity at a lower temperature 30-40°C, indicating this enzyme could work under mild conditions. The enzyme concentration and temperature were found to be the critical parameters affecting the transgalactosylation activity. Response surface methodology showed that the optimal enzyme concentration, initial lactose concentration, temperature, pH, and reaction time were 3.03 U/mL, 500 g/L, 30°C, 5.08, and 4 h, respectively. Under such conditions, the maximum yield of GOS was 252.8 g/L, accounting for approximately 50.56% of the total sugar. This yield can be considered relatively high compared to those obtained from other sources of ß-galactosidases, implying a great potential for GALA in the industrial production and application of GOS.

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Trehalose, α-d-glucopyranosyl-(1↔1)-α-d-glucopyranoside, is a disaccharide with multiple effects on the human body. Synthesis of new trehalose derivatives was investigated through transgalactosylation reactions using ß-galactosidase from four different species. ß-galactosidases from Bacillus circulans (B. circulans) and Aspergillus oryzae (A. oryzae) were observed to be the best biocatalysts, using lactose as the donor and trehalose as the acceptor. Galactosyl derivatives of trehalose were characterized using nuclear magnetic resonance spectroscopy. Trisaccharides were the most abundant oligosaccharides obtained followed by the tetrasaccharide fraction (19.5% vs 8.2% carbohydrates). Interestingly, the pentasaccharide [ß-Galp-(1→4)]3-trehalose was characterized for the first time. Greater oligosaccharide production was observed using ß-galactosidase from B. circulans than that obtained from A. oryzae, where the main structures were based on galactose monomers linked by ß-(1→6) and ß-(1→4) bonds with trehalose in the ending. These results indicate the feasibility of commercially available ß-galactosidases for the synthesis of trehalose-derived oligosaccharides, which might have functional properties, excluding the adverse effects of the single trehalose.

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ß-Galactosidases of Mucoromycota are rarely studied, although this group of filamentous fungi is an excellent source of many industrial enzymes. In this study, 99 isolates from the genera Lichtheimia, Mortierella, Mucor, Rhizomucor, Rhizopus and Umbelopsis, were screened for their ß-galactosidase activity using a chromogenic agar approach. Ten isolates from the best producers were selected, and the activity was further investigated in submerged (SmF) and solid-state (SSF) fermentation systems containing lactose and/or wheat bran substrates as enzyme production inducers. Wheat bran proved to be efficient for the enzyme production under both SmF and SSF conditions, giving maximum specific activity yields from 32 to 12,064 U/mg protein and from 783 to 22,720 U/mg protein, respectively. Oligosaccharide synthesis tests revealed the suitability of crude ß-galactosidases from Lichtheimia ramosa Szeged Microbiological Collection (SZMC) 11360 and Rhizomucor pusillus SZMC 11025 to catalyze transgalactosylation reactions. In addition, the crude enzyme extracts had transfructosylation activity, resulting in the formation of fructo-oligosaccharide molecules in a sucrose-containing environment. The maximal oligosaccharide concentration varied between 0.0158 and 2.236 g/L depending on the crude enzyme and the initial material. Some oligosaccharide-enriched mixtures supported the growth of probiotics, indicating the potential of the studied enzyme extracts in future prebiotic synthesis processes.

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BACKGROUND: The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and ß-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract. RESULTS: In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on ß-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as ß-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant ß-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated. CONCLUSIONS: Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and ß-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases.

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Lactobacillus kefiranofaciens contains two types of ß-galactosidase, LacLM and LacZ, belonging to different glycoside hydrolase families. The difference in function between them has been unclear so far for practical application. In this study, LacLM and LacZ from L. kefiranofaciens ATCC51647 were cloned into constitutive lactobacillal expression vector pMG36e, respectively. Furtherly, pMG36n-lacs was constructed from pMG36e-lacs by replacing erythromycin with nisin as selective marker for food-grade expressing systems in Lactobacillus plantarum WCFS1, designated recombinant LacLM and LacZ respectively. The results from hydrolysis of o-nitrophenyl-ß-galactopyranoside (ONPG) showed that the ß-galactosidases activity of the recombinant LacLM and LacZ was 1460% and 670% higher than that of the original L. kefiranofaciens. Moreover, the lactose hydrolytic activity of recombinant LacLM was higher than that of LacZ in milk. Nevertheless, compare to LacZ, in 25% lactose solution the galacto-oligosaccharides (GOS) production of recombinant LacLM was lower. Therefore, two ß-galactopyranosides could play different roles in carbohydrate metabolism of L. kefiranofaciens. In addition, the maximal growth rate of two recombinant strains were evaluated with different temperature level and nisin concentration in fermentation assay for practical purpose. The results displayed that 37°C and 20-40 U/ml nisin were the optimal fermentation conditions for the growth of recombinant ß-galactosidase strains. Altogether the food-grade Expression system of recombinant ß-galactosidase was feasible for applications in the food and dairy industry.

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BACKGROUND: Milk whey, a byproduct of the dairy industry has a negative environmental impact, can be used as a raw material for added-value compounds such as galactooligosaccharides (GOS) synthesis by bgalactosidases. RESULTS: B-gal42 from Pantoea anthophila strain isolated from tejuino belonging to the glycosyl hydrolase family GH42, was overexpressed in Escherichia coli and used for GOS synthesis from lactose or milk whey. Crude cell-free enzyme extracts exhibited high stability; they were employed for GOS synthesis reactions. In reactions with 400 g/L lactose, the maximum GOS yield was 40% (w/w) measured by HPAEC-PAD, corresponding to 86% of conversion. This enzyme had a strong predilection to form GOS with b(1 ? 6) and b (1 ? 3) galactosyl linkages. Comparing GOS synthesis between milk whey and pure lactose, both of them at 300 g/L, these two substrates gave rise to a yield of 38% (60% of lactose conversion) with the same product profile determined by HPAEC-PAD. CONCLUSIONS: B-gal42 can be used on whey (a cheap lactose source) to produce added value products such as galactooligosaccharides.

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Tyrosol ß-galactoside (TG) is a phenylethanoid glycoside with proven neuroprotective properties. This work deals with its biocatalytic production from tyrosol and lactose using Aspergillus oryzae ß-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 ß-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.

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The transgalactosylase activity of ß-galactosidase produces galacto-oligosaccharides (GOSs) with prebiotic effects similar to those of major oligosaccharides in human milk. ß-Galactosidases from Bacillus circulans ATCC 31382 are important enzymes in industrial-scale GOS production. Here, we show the high GOS yield of ß-galactosidase II from B. circulans (ß-Gal-II, Lactazyme-B), compared to other commercial enzymes. We also determine the crystal structure of the five conserved domains of ß-Gal-II in an apo-form and complexed with galactose and an acceptor sugar, showing the heterogeneous mode of transgalactosylation by the enzyme. Truncation studies of the five conserved domains reveal that all five domains are essential for enzyme catalysis, while some truncated constructs were still expressed as soluble proteins. Structural comparison of ß-Gal-II with other ß-galactosidase homologues suggests that the GOS linkage preference of the enzyme might be quite different from other enzymes. The structural information on ß-Gal-II might provide molecular insights into the transgalactosylation process of the ß-galactosidases in GOS production.

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Automated chemical oligosaccharide synthesis is an attractive concept that has been successfully applied to a large number of target structures, but requires excess quantities of suitably protected and activated building blocks. Herein we demonstrate the use of biocatalysis to supply such reagents for automated synthesis. By using the promiscuous NmLgtB-B ß1-4 galactosyltransferase from Neisseria meningitidis we demonstrate fast and robust access to the LacNAc motif, common to many cell-surface glycans, starting from either lactose or sucrose as glycosyl donors. The enzymatic product was shown to be successfully incorporated as a complete unit into a tetrasaccharide target by automated assembly.

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A GH1 ß-glucosidase from the fungus Hamamotoa singularis (HsBglA) has high transgalactosylation activity and efficiently converts lactose to galactooligosaccharides. Consequently, HsBglA is among the most widely used enzymes for industrial galactooligosaccharide production. Here, we present the first crystal structures of HsBglA with and without 4'-galactosyllactose, a tri-galactooligosaccharide, at 3.0 and 2.1 Å resolutions, respectively. These structures reveal details of the structural elements that define the catalytic activity and substrate binding of HsBglA, and provide a possible interpretation for its high catalytic potency for transgalactosylation reaction.

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Enzymatic transgalactosylation, in different concentrated carbohydrate solutions, was investigated using brush border membrane vesicles (BBMV) from the pig small intestine. When lactulose was incubated with BBMV, the hydrolytic activity of the enzyme towards the disaccharide was observed to be very low compared to that towards the lactose, but the linkage specificity ß-(1 â†’ 3), previously observed in lactose solutions, was not significantly affected. As in the case of lactose, lactulose transgalactosylation by BBMV synthesizes the corresponding 3'-galactosyl derivative (ß-Gal-(1 â†’ 3)-ß-Gal-(1 â†’ 4)-ß-Fru). Fructose released during lactulose hydrolysis was found to be good acceptor for the transgalactosylation reaction, giving rise to the synthesis of the disaccharide ß-Gal-(1 â†’ 5)-Fru. When incubating an 80/20 mixture of lactulose/galactose, the presence of galactose did not affect the qualitative composition of the transglycosylated substrate but enhanced the synthesis of ß-Gal-(1 â†’ 5)-Fru and decreased the synthesis of ß-(1 â†’ 3) glycosidic bonds. The marked tendency for synthesizing this linkage indicates that under hydrolytic conditions, ß-Gal-(1 â†’ 3)-Gal- and ß-Gal-(1 â†’ 5)-Fru glycosidic bonds would be preferentially digested.

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Two simple and easily reproducible methods for the immobilization of ß-galactosidase (ß-gal) from Aspergillus oryzae on electrospun gelatin nanofiber mats (GFM) were developed. The process was optimized regarding the electrospinning solvent system and the subsequent cross-linking of GFM in order to increase their stability in water. ß-Gal was covalently immobilized on activated gelatin nanofiber mats with hexamethylenediamine (HMDA) as a bifunctional linker and secondly via entrapment into the gelatin nanofibers during the electrospinning process (suspension electrospinning). Optimal immobilization parameters for covalent immobilization were determined to be at pH 7.5, 40 °C, ß-gal concentration of 1 mg/mL and immobilization time of 24.5 h. For suspension electrospinning, the optimal immobilization parameters were identified at pH 4.5 and ß-gal concentration of 0.027 wt.% in the electrospinning solution. The pH and temperature optima of immobilized ß-gal shifted from 30 °C, pH 4.5 (free enzyme) to pH 3.5, 50 °C (covalent immobilization) and pH 3.5, 40 °C (suspension electrospinning). Striking differences in the Michaelis constant (KM) of immobilized ß-gal compared with free enzyme were observed with a reduction of KM up to 50% for immobilized enzyme. The maximum velocity (vmax) of immobilization by suspension electrospinning was almost 20 times higher than that of covalent immobilization. The maximum GOS yield for free ß-gal was found to be 27.7% and 31% for immobilized ß-gal.

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Present work describes the purification of an acidic ß-galactosidase from Lens culinaris (Lsbgal) to homogeneity via 857 fold with specific activity of 87 U/mg. The molecular mass of purified Lsbgal was estimated ~ 76 kDa by Size Exclusion Chromatography on Superdex-200 (ÄKTA purifier) and on SDS-PAGE, showed hetero-dimeric subunits i.e. 45 kDa and 30 kDa. The purified Lsbgal showed glycoproteinous nature when applied to Con-A Sepharose chromatography. Biochemical studies revealed that optimum condition for purified Lsbgal against o, nitophenyl ß-d-galactopyranoside (ONPG) as a substrate was pH 3.0, 58 °C with an activation energy (Ea) 8.1 kcal/mole and Q10 1.8. Lsbgal hydrolyses ONPG with Km value 1.21 mM and Vmax 90.90 µmoles/min/mg. Purified Lsbgal when incubated with high lactose concentration showed transgalactosylation activity which lead to the formation of trisaccharides as a major product of total GOS. Therefore, the purified Lsbgal could be used as potential alternative in food industry and would be further explicated for trisaccharides synthesis.

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Prebiotics are rising in interest in commercial scale productions due to increasing health awareness of consumers. Under bio-economic aspects, sweet and acid whey provide a suitable feed medium for the enzymatic generation of prebiotic lactulose. Since whey has a broad variation in composition, the influence of the feed composition on the concentration of generated lactulose was investigated. The influence of lactose and fructose concentration as well as enzymatic activity of two commercially available ß-galactosidases were investigated. The results were evaluated via response surface analysis with a quadratic model containing pairwise interaction terms. The optimal feed composition yielding a theoretical maximal amount of lactulose was determined as 1.28 or 0.74 mol/kg fructose and 0.17 or 0.19 mol/kg lactose with an enzymatic activity of 2.0 or 2.8 µkat/kg for acid (pH 4.4) or sweet (pH 6.6) whey. Furthermore, the major reaction product was isolated and subsequently, the structural identity was elucidated and verified via extensive NMR analysis.

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