Transgalactosylation Reaction Research Articles

Unravelling the carbohydrate specificity of MelA from Lactobacillus plantarum WCFS1: An α-galactosidase displaying regioselective transgalactosylation

This comprehensive work addresses, for the first time, the heterologous production, purification, biochemical characterization and carbohydrate specificity of MelA, a cold-active α-galactosidase belonging to the Glycoside Hydrolase family 36, from the probiotic organism Lactobacillus plantarum WCFS1. The hydrolytic activity of MelA α-galactosidase on a wide range of p-nitrophenyl glycoside derivatives and carbohydrates of different molecular-weights showed its high selectivity and efficiency towards the α(1 → 6) glycosidic bonds involving the anomeric carbon of galactose and the C6-hydroxyl group of galactose or glucose units. MelA α-galactosidase also presented a high regioselectivity, efficiency and diversity in accommodating donor and acceptor substrates for the synthesis of α-GOS through transgalactosylation reactions. The catalytic mechanism of MelA for the production of α-GOS was elucidated, revealing its great preference for the transfer of galactosyl residues to the C6-hydroxyl group of galactose units to elongate the chain of α-GOS having either a terminal sucrose (raffinose family oligosaccharides, RFOS) or a terminal glucose (melibiose, manninotriose and verbascotetraose). Our findings indicate the feasibility of using MelA α-galactosidase from Lactobacillus plantarum WCFS1 in the hydrolysis of RFOS and in the efficient and versatile synthesis of α-GOS with appealing functional properties in the context of food and nutraceutical applications.

IFST Winning Articles

IFST Winning Articles

Production of galactooligosaccharides using various combinations of the commercial β-galactosidases

Galactooligosaccharides (GOS) are currently attracting considerable interest as prebiotic substances and can be prepared by transgalactosylation reactions from lactose using β-galactosidase. We applied various combinations of the commercial β-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.

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

The main goal of this work was to evaluate the performance of β-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-β-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-β-d-glucopyranoside, butyl- and pentyl-β-d-galactosides. This unique and novel substrate specificity converts the E. acetylicum MF03 β-galactosidase into an ideal catalyst for the formulation of an enzymatic kit for lactulose quantification in thermally processed milk. This is because costly steps to eliminate glucose (resulting from hydrolysis of lactose when a customary β-galactosidase is used) can be avoided.

Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides.

A thermostable β-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme β-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg−1 at 37 °C using ONPG (o-nitrophenyl-β-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3′-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with β-1,4 linkage and β-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with β-1,3 linkage.

Heat-Loving β-Galactosidases from Cultured and Uncultured Microorganisms.

β-galactosidases (EC.3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in the food industry: the production of low-lactose milk and dairy goods for lactose intolerant people, and the generation of galacto-oligosaccharides by transgalactosylation reactions. Due to their thermostability, β-galactosidases from thermophilic microorganisms are very interesting for industrial processes, as high temperatures can increase the initial productivity of the enzyme, provide higher solubility of substrates, and prevent microbial contamination. In the past, it was necessary to cultivate and grow thermophilic microorganisms to discover novel thermozymes, but the development of metagenomic techniques has allowed researchers to access the genomic potential of uncultivated microbes and their enzymes. The present review gives a brief outline of thermophilic β-galactosidases, with a special focus on those obtained through metagenomics. Additionally, the sequences of β-galactosidases found in some public metagenomes from hot springs were studied and compared to other known thermostable β-galactosidases.

Effect of the type of immobilization of β-galactosidase on the yield and selectivity of synthesis of transgalactosylated oligosaccharides

Abstract Aspergillus oryzae β-galactosidase was immobilized by crosslinking and aggregation (CLA), and by covalent attachment to monofunctional glyoxyl agarose (GA) and to the heterofunctional supports: amino-glyoxyl agarose (Am-GA), carboxy-glyoxyl agarose (Cx-GA) and chelate-glyoxyl agarose (Che-GA) with the purpose of determining the effect of immobilization on the performance of the biocatalysts in terms of yield, productivity, kinectic parameters and product distribution in the synthesis of transgalactosylated oligosaccharides (lactulose, galacto-oligosaccharides and fructosyl-galacto-oligosaccharides). In all transgalactosylation reactions, yield was barely affected by the type of immobilization; however, a strong effect on productivity was observed, being the highest with the enzyme immobilized in CLA-βG and Am-GA and the lowest with the enzyme immobilized as Cx-GA and Che-GA. The type of immobilization had a strong influence on the apparent kinetic parameters of the biocatalysts, affecting product composition during the synthesis of transgalactosylated oligosaccharides. Diffusional restrictions of the biocatalysts increased with the degree of crosslinking, particle size and enzyme load; however their effect on the synthesis of the transgalactosylated products was low since reactions are conducted at very high substrate concentrations. Results suggest that, because of the operation conditions, immobilization has a more critical impact on the hydrolysis of lactose than in its transgalactosylation where the use of very high substrate concentrations is mandatory.

Evaluation of β-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization

β-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude β-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that β-galactosidase from L.acidophilus shows a particular specificity towards the formation of β-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86g/l GOS under the previously optimized conditions (lactose concentration 400g/l in 0.1M sodium phosphate buffer, pH 6.8 and temperature 50°C).

Glucoheptonic acid derivative as a new transgalactosylation product

Transgalactosylation is increasingly used for modification of selected compounds because it may introduce new bioactive properties or improve existing ones. This paper presents the application of transgalactosylation activity of Kluyveromyces lactis β-galactosidase (EC 3.2.1.23) for a new derivative of glucoheptonic acid synthesis. The study concerned the impact of following factors on the course of reaction: content and ratio of substrates, enzyme dose, pH of the solution and the presence of salts. In the most favourable conditions (without salt), the final product concentration reached 54.5 g/L, which corresponded to 10.9% of dry matter. Research has shown that higher initial dry matter content results in higher product content (as % dm). The addition of 0.5–0.75 M MgCl2 or 1 M NaCl led to significantly increased yield. In contrast, the presence of MnCl2 or the lowest enzyme dose seemed to slow down the synthesis process. Increasing the pH over the optimal value for hydrolytic activity of β-galactosidase caused inhibition of transgalactosylation reaction. The molar ratio of 1.9:1 (sodium glucoheptonate:lactose) was the best among tested options. The described method allowed to successfully obtain a new compound with satisfactory yield in comparison to other transgalactosylation products.

Synthesis of Galactooligosaccharides in Milk and Whey: A Review.

Galactooligosaccharides (GOS) are synthesized by the enzyme β-galactosidase during the hydrolysis of lactose. In this so-called transgalactosylation reaction the galactosyl moiety is transferred to another sugar molecule instead of water resulting in oligosaccharides of different chain lengths and glycosidic linkages. Because their structures are similar to oligosaccharides present in human breast milk, they act as prebiotics, which has been shown for infants and adults to be alike. While so far most of the research to maximize GOS yield has been carried out using buffered lactose solution as a starting material, more and more work is now conducted with dairy by-products such as whey and whey permeate, or even milk, for direct GOS synthesis in order to develop new GOS-enriched dairy products. This review aims to summarize the results obtained with various dairy liquids, and it rates their suitabilities to act as raw material for GOS production. Most of the studies using whey or milk have been carried out with enzymes from Aspergillus oryzae, Kluyveromyces lactis, Bacillus circulans, Streptococcus thermophilus, and several Lactobacillus species. As the initial lactose concentration (ILC) is known to be a crucial factor for high GOS yield, most of the research has been done with concentrated or supplemented milk and whey. However, a clear dependency on ILC could only be observed for the A. oryzae lactase, indicating a strong influence of milk components like minerals and proteins on the transfer activities of most enzymes.

β-Galactosidase from Lactobacillus brevis PLA28: Purification, Characterization and Synthesis of Galacto-oligosaccharides

Objective: Purification and characterization of β-galactosidase from Lactobacillus brevis PLA28 were carried out and transgalactosylation activity was also studied for the synthesis of galacto-oligosaccharides (GOS). Methods: β-Galactosidase was purified by using ammonium sulphate precipitation method and hydrophobic interaction chromatography. Reaction conditions were optimized for the assay of this β-galactosidase. GOS synthesis was carried out under the optimized reaction conditions in batch mode and the product formed was detected by thin layered chromatography (TLC). Results: β-Galactosidase was purified to 6.6 fold with a yield of 6% and specific activity of 4 U/mg protein. Molecular weight of the purified β-galactosidase was found to be 45 and 60 kDa on SDS PAGE and 105 kDa on native PAGE. The temperature and pH optima of purified enzyme were 30°C and pH 6.5 respectively. The enzyme was found to be stable at 30°C for 6 h. Vmax and KM of the purified β-galactosidase were calculated to be 6.6 U/mg protein and 8.33 mM respectively. GOS synthesis was observed at pH 6.5, 30°C in 6-8 h of incubation by purified enzyme. Conclusion: Lactobacillus brevis PLA28 β-galactosidase has exhibited capability for transgalactosylation reaction with lactose conversion to synthesize GOS.

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

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. 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. 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.

Application of Transgalactosylation Activity of \u03b2-Galactosidase from Kluyveromyces lactis for the Synthesis of Ascorbic Acid Galactoside

In view of a commonly known beneficial role and low stability of ascorbic acid, many efforts are constantly undertaken to produce its improved derivatives. This paper presents results on the synthesis of ascorbic acid galactoside using transgalactosylation properties of β-galactosidase from Kluyveromyces lactis and lactose as a donor of galactosyl moiety. The purpose of this study was to determine the influence of selected factors (concentration and molar ratio of substrates, amount of the enzyme preparation, pH of the solution, presence of different ions) on the course of transgalactosylation reaction. Research has shown that approx. 2.5% dry matter (d.m.; 12.7 g/L) of ascorbic acid galactoside is formed under favourable conditions (50% (w/v) substrates, sodium ascorbate and lactose at the molar ratio of 1.9:1, enzyme dose of 28,600 U/100 g lactose, pH = 7.0). The addition of Mg2+ or K+ ions to the reaction medium caused an increase in the final product content (even up to approx. 3.4% d.m., 17.2 g/L), while Na+ or Mn2+ had an adverse impact on the yield. The gathered data may be valuable for cosmetic or food industry.

Overexpression of both the lactase gene and its transcriptional activator gene greatly enhances lactase production by Kluyveromyces marxianus

Abstract After both a lactase gene (LAC4) and its transcriptional activator gene (LAC9) were expressed in Kluyveromyces marxianus KM-69 which was a glucose-derepressed mutant, one transformant called KM-L9-20 grown in an optimized whey medium for lactase production could produce 77.8 ± 4.0 U/ml of lactase activity at a flask level within 36 h. At the same time, expression of the LAC4 gene and its transcriptional activator gene in the transformant KM-L9-20 were also greatly enhanced compared to that of the LAC4 gene and the LAC9 gene in its parent strain K. marxianus KM-69. 157.9 ± 3.2 U/ml of lactase activity was yielded by the transformant KM-L9-20 within 84 h during a batch fermentation while 274.7 ± 4.3 U/ml of lactase activity was produced within 48 h during a fed-batch fermentation. The produced lactase could actively transform lactose into the galacto-oligosaccharides which contained mainly trisaccharides. The optimal conditions for the transgalactosylation reaction were that the lactose concentration, the reaction temperature, pH and the lactase dose were 400 g/l of lactose, 40 °C, 6.5 and 25 U/g of lactose, respectively. Therefore, the transformant KM-L9-20 and the produced lactase had highly potential applications in biotechnology.

Biochemical characterization of three Aspergillus niger β-galactosidases

β-Galactosidases catalyze both hydrolytic and transgalactosylation reactions and therefore have many applications in food, medical, and biotechnological fields. Aspergillus niger has been a main source of β-galactosidase, but the properties of this enzyme are incompletely studied. Three new β-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned, expressed, and biochemically characterized. In addition to the known activity of LacA encoded by lacA, three putative β-galactosidases, designated as LacB, LacC, and LacE encoded by the genes lacB, lacC, and lacE, respectively, were successfully cloned, sequenced, and expressed and secreted by Pichia pastoris. These three proteins and LacA have N-terminal signal sequences and are therefore predicted to be extracellular enzymes. They have the typical structure of fungal β-galactosidases with defined hydrolytic and transgalactosylation activities on lactose. However, their activity properties differed. In particular, LacB and lacE displayed maximum hydrolytic activity at pH 4–5 and 50°C, while LacC exhibited maximum activity at pH 3.5 and 60°C. All β-galactosidases performed transgalactosylation activity optimally in an acidic environment. Three new β-galactosidases belonging to glycosyl hydrolase family 35 from A. niger F0215 were cloned and biochemically characterized. In addition to the known LacA, A. niger has at least three β-galactosidase family members with remarkably different biochemical properties.

Production of High Galacto-oligosaccharides by Pectinex Ultra SP-L: Optimization of Reaction Conditions and Immobilization on Glyoxyl-Functionalized Silica.

A rational optimization for the synthesis of galacto-oligosaccharides (GOS) from lactose catalyzed by β-galactosidase from Aspergillus aculeatus, included in the commercial product Pectinex Ultra SP-L, has been performed by using experimental design and surface response methodology. This accurate tool optimized empirical production of the most desired high-GOS (tri-GOS and tetra-GOS) up to 16.4% under the following reaction conditions: 59 °C, 4 U/mL free enzyme concentration, pH 6.5, 250 g/L initial lactose concentration, and 20 h of reaction. The statistical analysis revealed temperature and initial lactose concentration as critical parameters. The successful immobilization of the enzyme on a glyoxyl-functionalized porous silica support slightly increased the yield toward high-GOS (17.6%), especially tri-GOS yield (15.3%), under the optimized reaction conditions as compared to the free enzyme. Furthermore, the promotion of the transgalactosylation reaction toward tri-GOS production increased 1.5-fold the productivity of high-GOS as compared to the free enzyme.

Reaction kinetics and galactooligosaccharide product profiles of the β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae.

β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with 13C6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site.

Designing of a novel β-galactosidase for production of functional oligosaccharides

β-galactosidase from L. plantarum FMNP01 was subjected to mutagenesis by domain recombination. A novel β-galactosidase L.pFMNP01Gal-2 was constructed and expressed using the expression vector pET-32a(+) in Escherichia coli BL21 (DE3). The L.pFMNP01Gal-2 was composed of 2406 bp encoding a polypeptide containing 802 amino acids with a predicted molecular mass of 88.22 kDa. When ONPG was used as substrate, the specific activity of L.pFMNP01Gal-2 was 2620 U/g protein, with a Km of 2.87 mmol/L and a Kcat of 62.68/s. The optimal temperature and pH for L.pFMNP01Gal-2 were 40 °C and 7.0, respectively. After pre-incubation for 4 h at 40 and 50 °C, respectively, the residual activity of L.pFMNP01Gal-2 retained over 88%. The metal ions Cu2+ and Na+ had a great inhibitory effect on the activity of L.pFMNP01Gal-2, while other tested metal ions had slight influence. In the presence of lactose and fructose, when L.pFMNP01Gal-2 was used as catalyst, two transfer products were formed during transgalactosylation reaction. It is notable that when L.pFMNP01Gal-2 was used as catalyst with lactose and sucrose as substrates, four transfer products were observed.

Characterization of recombinant β- galactosidase and its use in enzymatic synthesis of lactulose from lactose and fructose

The gene encoding a β- galactosidase was cloned from Lactobacillus plantarum FMNP01 and expressed in Escherichia coli BL21(DE3). The characteristics of this purified recombinant enzyme, L.pFMNP01Gal, were determined, and its transgalactosylation reaction conditions for the production of lactulose were optimized. Using ONPG as substrate, the L.pFMNP01Gal showed specific activity of 980U/g with a Km of 6.86mM and a Kcat of 22.47/s. This enzyme was most stable at 40–50°C, and exhibited optimum catalytic activity at 40°C and pH 7.0. The activity of L.pFMNP01Gal was greatly inhibited by Cu2+, while other tested metal ions had little influence on it. For the optimization of transgalactosylation reaction, high lactulose production was achieved when 60% (W/V) sugars were used as substrates with a lactose/fructose mass ratio of 2:1, and 2U/mL of L.pFMNP01Gal as catalyst. Under these optimum conditions, 18.38±2.17g/L of lactulose was synthesized in 6h at 50°C. This study provides an alternative method for enzymatic synthesis of lactulose.

β-galactosidse 함유하는 재조합 대장균 세포를 이용한 1,2-hexanediol galactoside의 합성

side (HD-G) by a transgalactosylation reaction using β-galactosidase (β-gal)-containing recombinant Escherichia coli cells. The transgalactosylation reaction was carried out under high-lactose conditions for 24 hr. After 12 hr had elapsed, a new spot was identified by thin-layer chromatography (TLC) analysis, and we presumptively designated this new spot as HD-G. The n, we carried out the purification of the presumptive HD-G spot from the reaction mixture by using silica gel chromatography, and its mass was measured by electrospray ionization-mass spectrometry. The purified new spot on the chromatograph was identified a sodium adduct ion ([M+Na] + , m/z = 303.1423) of HD-G. In addition, when purified HD-G was hydrolyzed using commercially available E. coli β-gal, it was observed that a galactose molecule was released from HD-G. That is, it was demonstrated that HD-G is a galactoside derivative of HD. Finally, we confirmed that HD-G was enzymatically synthesized by E. coli β -gal as a new molecular entity. In the future, we plan to determine the minimum inhibitory concentrations of HD-G against different bacterial species. The cytotoxicity of HD-G against human skin cells will also be examined. It is expected hopefully that the galactosylation of HD would improve the functionality of HD-G.