Optimization of the production conditions of tri-GOS and lactosucrose from lactose and sucrose with recombinant ß-galactosidase.

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.

Production, purification, characterization, and applications of α-galactosidase from Bacillus flexus JS27 isolated from Manikaran hot springs.

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.

An acid-tolerant and cold-active ß-galactosidase potentially suitable to process milk and whey samples.

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.

Site-directed mutation of ß-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making.

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.

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 ß-galactosidase.

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.

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

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.

Lens culinaris ß-galactosidase (Lsbgal): Insights into its purification, biochemical characterization and trisaccharides synthesis.

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.

Enzymatic Building-Block Synthesis for Solid-Phase Automated Glycan Assembly.

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.

Production of galactooligosaccharides using various combinations of the commercial ß-galactosidases.

Galactooligosaccharides (GOS) are currently attracting considerable interest as prebiotic substances and can be prepared by transgalactosylation reactions from lactose using ß-galactosidase. We applied various combinations of the commercial ß-galactosidases, such as Nola Fit 5500, Saphera 2600 L, Maxilact LGI 5000 and Maxilact A4 MG to achieve the highest yield of GOS and reduced lactose content. The combination of the Maxilact LGI 5000 and Nola Fit 5500 resulted in amount of GOS 105 g L-1 with lactose content lower than 5 g L-1, whilst the combination of the Maxilact A4 MG and Maxilact LGI 5000 enzymes led to an increase in GOS to 141,1 g L-1 and decrease of the lactose content to 46,9 g L-1. The combination of enzymes produced a higher yield of GOS, reduced the concentration of lactose, eventually, increases the efficiency of galactooligosaccharides purification that could be potentially used in the further investigations.

A new ß-galactosidase extracted from the infant feces with high hydrolytic and transgalactosylation activity.

A ß-galactosidase (ß-GalINF) was directly isolated from feces of an 8-month-old infant. Mass spectrum analysis showed ß-GalINF with coverage over 50% to the ß-galactosidase from Bifidobacterium longum EK3. Accordingly, the ß-galINF was amplified from the feces metagenomic DNA by degenerate primers. After overexpressed in Escherichia coli, the ß-GalINF was purified and biochemical characterized. ß-GalINF existed as homotetramer and homodimer, whose activity (optimal at 50 °C, pH 6.5) was exhilaratingly increased to 484% by artificial intestinal juice. The Km and Vmax values for oNPG and lactose were 20.95 ± 2.76 mM, 5004.50 ± 318.8 µmol min-1 mg-1 and 140.2 ± 17.7 mM, 293.1 ± 14.7 µmol min-1 mg-1, respectively. The production rate of galacto-oligosaccharides by ß-GalINF from 20% lactose at 50 °C was 33.4 ± 0.67%. These results suggested the ß-GalINF with high hydrolytic and transgalactosylation activity from the infant intestinal has great potential as infant lactase preparation. Moreover, this study provided a new way for exploring undetected enzymes by uncultured-dependent methods.

Synthesis of prebiotic galactooligosaccharides from lactose and lactulose by dairy propionibacteria.

The potential of probiotic bacteria to produce prebiotic oligosaccharides by transgalactosylation has been minimally studied. In this work, we screened the ß-galactosidase (ß-gal) activity of dairy propionibacteria (PAB) isolated from Argentinean foods to select strains for the synthesis of oligosaccharides from lactose (GOS) and lactulose (OsLu). PAB, when grown in a medium with lactose as a carbon source, were disrupted, and the cell-free extracts were assayed for ß-gal activity. Nine strains grew on lactose and showed ß-gal activities from 0.27 to 2.60 U mL-1. Propionibacterium acidipropionici LET 120, the strain with the highest activity, was able to synthesize, using 30% lactose and lactulose at pH 6.5 and 45 °C, 26.8% of LET 120-GOS and 26.1% of LET 120-OsLu after 24 h. When they were tested as carbon sources for growth, P. acidipropionici LET 120 attained higher biomasses, µmax and ß-gal activities at the expense of Aspergillus oryzae-OsLu, Vivinal®-GOS and lactulose compared to lactose or glucose. In addition, LET 120-GOS and LET 120-OsLu synthesized by PAB were prebiotic for some probiotic strains. For the first time, our results show the production of GOS and OsLu by dairy PAB, and these results encourage further studies on the optimization of the synthesis and structure characterization of the obtained oligosaccharides.

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 ß-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.

Combination of two ß-galactosidases during the synthesis of galactooligosaccharides may enhance yield and structural diversity.

In this study, the combination of two ß-galactosidases to synthesize prebiotic galactooligosaccharides (GOS) was evaluated in terms of total GOS yield as well as GOS structures (chain length). Two different combinations of either Aspergillus oryzae and Cryptococcus laurentii or Aspergillus oryzae and Kluyveromyces lactis were tested to examine the influence of enzyme origin. Neither consecutive nor simultaneous synthesis with A. oryzae and C. laurentii led to an increased GOS yield. However, with the latter, synthesis of higher GOS (≥3 monomer units) was enhanced from 38.5% to 40% with special emphasis of tetra- and pentasaccharides, which increased from 6.7% to 12.8% and from 0.4% to 3.3%, respectively. Additionally, due to the different preferences of the two ß-galactosidases in terms of types of glycosidic linkages, the structural diversity of the final GOS product could be increased. Using K. lactis following the synthesis with A. oryzae increased the yield of total GOS from 24.6% to 33.1%, which was mainly due to the formation of GOS disaccharides. On the other hand, applying A. oryzae as the second enzyme led to a degradation of di- and trisaccharides, and thus total GOS yield was diminished, although the yield of tetrasaccharides could be enhanced. In conclusion, with both studied enzyme combinations it was possible to increase the percentage of higher GOS and reduce the residual lactose content of the final mixture, which is beneficial for subsequent purification processes. Thus, using more than one ß-galactosidase during the synthesis of GOS represents an interesting research area, which should be explored in more detail in the future.

Highly active spore biocatalyst by self-assembly of co-expressed anchoring scaffoldin and multimeric enzyme.

We report a spore-based biocatalysis platform capable of producing and self-assembling active multimeric enzymes on a spore surface with a high loading density. This was achieved by co-expressing both a spore surface-anchoring scaffoldin protein containing multiple cohesin domains and a dockerin-tagged enzyme of interest in the mother cell compartment during Bacillus subtilis sporulation. Using this method, tetrameric ß-galactosidase was successfully displayed on the spore surface with a loading density of 1.4 × 104 active enzymes per spore particle. The resulting spore biocatalysts exhibited high conversion rates of transgalactosylation in water/organic emulsions. With easy manufacture, enhanced thermostability, excellent reusability, and long-term storage stability at ambient temperature, this approach holds a great potential in a wide range of biocatalysis applications especially involving organic phases.

Lactulose production by a thermostable glycoside hydrolase from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167.

BACKGROUND: Lactulose has various uses in the food and pharmaceutical fields. Thermostable enzymes have many advantages for industrial exploitation, including high substrate solubilities as well as reduced risk of process contamination. RESULTS: Enzymatic synthesis of lactulose employing a transgalactosylation reaction by a recombinant thermostable glycoside hydrolase (GH1) from the hyperthermophilic archaeon Caldivirga maquilingensis IC-167 was investigated. The optimal pH for lactulose production was found to be 4.5, while the optimal temperature was 85 °C, before it dropped moderately to 83% at 90 °C. However, the relative activity for lactulose synthesis dropped sharply to 35% at 95 °C. At optimal reaction conditions of 70% (w/w) initial sugar substrates with molar ratio of lactose to fructose of 1:4, 15 U mL-1 enzyme concentration and 85 °C, the time course reaction produced a maximum lactulose concentration of 108 g L-1 at 4 h, corresponding to a lactulose yield of 14% and 27 g L-1 h-1 productivity with 84% lactose conversion. The transgalactosylation reaction for lactulose synthesis was greatly influenced by the ratio of galactose donor to acceptor. CONCLUSION: This novel GH1 may be useful for process applications owing to its high activity in very concentrated substrate reaction media and promising thermostability. © 2017 Society of Chemical Industry.

Introducing transgalactosylation activity into a family 42 ß-galactosidase.

Chemo-enzymatic synthesis of oligosaccharides exploits the diversity of glycosidases and their ability to promote transglycosylation reactions in parallel with hydrolysis. Methods to increase the transglycosylation/hydrolysis ratio include site-directed mutagenesis and medium modification. The former approach was successful in several cases and has provided the best synthetic yields with glycosynthases-mutants at the catalytic nucleophile position that promote transglycosylation with high efficiency, but do not hydrolyze the oligosaccharide products. Several glycosidases have proven recalcitrant to this conversion, thus alternative methods to increase the transglycosylation/hydrolysis ratio by mutation would be very useful. Here we show that a mutant of a ß-galactosidase from Alicyclobacillus acidocaldarius in an invariant residue in the active site of the enzymes of this family (glutamic acid 361) carries out efficient transglycosylation reactions on different acceptors only in the presence of external ions with yields up to 177-fold higher than that of the wild type. This is the first case in which sodium azide and sodium formate in combination with site-directed mutagenesis have been used to introduce transglycosylation activity into a glycosidase. These observations will hopefully guide further efforts to generate useful synthases.

Enzymatic synthesis of novel oligosaccharides from N-acetylsucrosamine and melibiose using Aspergillus niger α-galactosidase, and properties of the products.

Two kinds of oligosaccharides, N-acetylraffinosamine (RafNAc) and N-acetylplanteosamine (PlaNAc), were synthesized from N-acetylsucrosamine and melibiose using the transgalactosylation activity of Aspergillus niger α-galactosidase. RafNAc and PlaNAc are novel trisaccharides in which d-glucopyranose residues in raffinose (Raf) and planteose are replaced with N-acetyl-d-glucosamine. These trisaccharides were more stable in acidic solution than Raf. RafNAc was hydrolyzed more rapidly than Raf by α-galactosidase of green coffee bean. In contrast, RafNAc was not hydrolyzed by Saccharomyces cerevisiae invertase, although Raf was hydrolyzed well by this enzyme. These results indicate that the physicochemical properties and steric structure of RafNAc differ considerably from those of Raf.

From by-product to valuable components: Efficient enzymatic conversion of lactose in whey using ß-galactosidase from Streptococcus thermophilus.

ß-Galactosidase from Streptococcus thermophilus was overexpressed in a food-grade organism, Lactobacillus plantarum WCFS1. Laboratory cultivations yielded 11,000 U of ß-galactosidase activity per liter of culture corresponding to approximately 170 mg of enzyme. Crude cell-free enzyme extracts obtained by cell disruption and subsequent removal of cell debris showed high stability and were used for conversion of lactose in whey permeate. The enzyme showed high transgalactosylation activity. When using an initial concentration of whey permeate corresponding to 205 g L-1 lactose, the maximum yield of galacto-oligosaccharides (GOS) obtained at 50°C reached approximately 50% of total sugar at 90% lactose conversion, meaning that efficient valorization of the whey lactose was obtained. GOS are of great interest for both human and animal nutrition; thus, efficient conversion of lactose in whey into GOS using an enzymatic approach will not only decrease the environmental impact of whey disposal, but also create additional value.

Microwave-Assisted Synthesis of Glycoconjugates by Transgalactosylation with Recombinant Thermostable ß-Glycosidase from Pyrococcus.

The potential of the hyperthermophilic ß-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with ß-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galß(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products ß(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates.

Recent advances on prebiotic lactulose production.

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic. The production of lactulose is important in the dairy industry, as it is regarded as a high value-added derivative of whey or lactose. The industrial production of lactulose is still mainly done by chemical isomerization. Due to concerns on the environmental and tedious separation processes, the enzymatic-based lactulose synthesis has been regarded as an interesting alternative. This work aims at comparing chemical and enzyme-catalyzed lactulose synthesis. With an emphasis on the latter one, this review discusses the influences of the critical operating conditions and the suited operation mode on the transgalactosylation of lactulose using microbial enzymes. As an update and supplement to other previous reviews, this work also summarizes the recent reports that highlighted the enzymatic isomerization of lactose using cellobiose 2-epimerase to produce lactulose at elevated yields.