Galactosyl Oligosaccharides Research Articles

An improved method for galactosyl oligosaccharide characterization

Due to beneficial effects of galactosyl oligosaccharides (GOS) on digestive and immune health, their characterization has become increasingly important. This is especially so as GOS are synthesized enzymatically and contain oligosaccharides of different sizes and linkages. High performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) is widely used for GOS characterization. With its high resolving power, it can separate structural isomers. Here we present a significant improvement to currently used methods. Our approach combines high resolution HPAE separation on a CarboPac PA300 column with 4 µm particle size with PAD and Orbitrap mass spectrometry (MS) detections to provide in-depth information on GOS composition. Oligosaccharide resolution, especially in the disaccharide region, is significantly improved and can be routinely achieved. Improvement in technology to remove sodium before MS results in minimal peak dispersion, allowing GOS degrees of polymerization 2 to 6 to be identified based on mass spectra obtained from intact oligosaccharides and confirmed using fragmentation patterns observed in MS/MS data. Combining HPAE with MS led to identification of 28 oligosaccharides in a commercial GOS sample. We attempted to correlate oligosaccharide structure with observed elution behavior. To our knowledge this is first such attempt and can form a basis for a comprehensive structure vs HPAE elution behavior database.

Biosynthesis of Raffinose and Stachyose from Sucrose via an In Vitro Multienzyme System.

Herein, we present a biocatalytic method to produce raffinose and stachyose using sucrose as the substrate. An in vitro multienzyme system was developed using five enzymes, namely, sucrose synthase (SUS), UDP-glucose 4-epimerase (GalE), galactinol synthase (GS), raffinose synthase (RS), and stachyose synthase (STS), and two intermedia, namely, UDP and inositol, which can be recycled. This reaction system produced 11.1 mM raffinose using purified enzymes under optimal reaction conditions and substrate concentrations. Thereafter, a stepwise cascade reaction strategy was employed to circumvent the instability of RS and STS in this system, and a 4.2-fold increase in raffinose production was observed. The enzymatic cascade reactions were then conducted using cell extracts to avoid the need for enzyme purification and supplementation with UDP. Such modification further increased raffinose production to 86.6 mM and enabled the synthesis of 61.1 mM stachyose. The UDP turnover number reached 337. Finally, inositol in the reaction system was recycled five times, and 255.8 mM raffinose (128.9 g/liter) was obtained.IMPORTANCE Soybean oligosaccharides (SBOS) have elicited considerable attention because of their potential applications in the pharmaceutical, cosmetics, and food industries. This study demonstrates an alternative method to produce raffinose and stachyose, which are the major bioactive components of SBOS, from sucrose via an in vitro enzyme system. High concentrations of galactinol, raffinose, and stachyose were synthesized with the aid of a stepwise cascade reaction process, which can successfully address the issue of mismatched enzyme characteristics of an in vitro metabolic engineering platform. The biocatalytic approach presented in this work may enable the synthesis of other valuable galactosyl oligosaccharides, such as verbascose and higher homologs, which are difficult to obtain through plant extraction.

Metagenomic and Metatranscriptomic Analyses of Diverse Watermelon Cultivars Reveal the Role of Fruit Associated Microbiome in Carbohydrate Metabolism and Ripening of Mature Fruits.

The plant microbiome is a key determinant of plant health and productivity, and changes in the plant microbiome can alter the tolerance to biotic and abiotic stresses and the quality of end produce. Little is known about the microbial diversity and its effect on carbohydrate metabolism in ripe fruits. In this study, we aimed to understand the diversity and function of microorganisms in relation to carbohydrate metabolism of ripe watermelon fruits. We used 16S metagenomics and RNAseq metatranscriptomics for analysis of red (PI459074, Congo, and SDRose) and yellow fruit-flesh cultivars (PI227202, PI435990, and JBush) of geographically and metabolically diverse watermelon cultivars. Metagenomics data showed that Proteobacteria were abundant in SDRose and PI227202, whereas Cyanobacteria were most abundant in Congo and PI4559074. In the case of metatranscriptome data, Proteobacteria was the most abundant in all cultivars. High expression of genes linked to infectious diseases and the expression of peptidoglycan hydrolases associated to pathogenicity of eukaryotic hosts was observed in SDRose, which could have resulted in low microbial diversity in this cultivar. The presence of GH28, associated with polygalacturonase activity in JBush and SDRose could be related to cell wall modifications including de-esterification and depolymerization, and consequent loss of galacturonic acid and neutral sugars. Moreover, based on the KEGG annotation of the expressed genes, nine α-galactosidase genes involved in key processes of galactosyl oligosaccharide metabolism, such as raffinose family were identified and galactose metabolism pathway was reconstructed. Results of this study underline the links between the host and fruit-associated microbiome in carbohydrate metabolism of the ripe fruits. The cultivar difference in watermelon reflects the quantum and diversity of the microbiome, which would benefit watermelon and other plant breeders aiming at the holobiont concept to incorporate associated microbiomes in breeding programs.

Human Milk Oligosaccharides and Synthetic Galactosyloligosaccharides Contain 3′-, 4-, and 6′-Galactosyllactose and Attenuate Inflammation in Human T84, NCM-460, and H4 Cells and Intestinal Tissue Ex Vivo

Background: The immature intestinal mucosa responds excessively to inflammatory insult, but human milk protects infants from intestinal inflammation. The ability of galactosyllactoses [galactosyloligosaccharides (GOS)], newly found in human milk oligosaccharides (HMOS), to suppress inflammation was not known.Objective: The objective was to test whether GOS can directly attenuate inflammation and to explore the components of immune signaling modulated by GOS.Methods: Galactosyllactose composition was measured in sequential human milk samples from days 1 through 21 of lactation and in random colostrum samples from 38 mothers. Immature [human normal fetal intestinal epithelial cell (H4)] and mature [human metastatic colonic epithelial cell (T84) and human normal colon mucosal epithelial cell (NCM-460)] enterocyte cell lines were treated with the pro-inflammatory molecules tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) or infected with Salmonella or Listeria. The inflammatory response was measured as induction of IL-8, monocyte chemoattractant protein 1 (MCP-1), or macrophage inflammatory protein-3α (MIP-3α) protein by ELISA and mRNA by quantitative reverse transcriptase-polymerase chain reaction. The ability of HMOS or synthetic GOS to attenuate this inflammation was tested in vitro and in immature human intestinal tissue ex vivo.Results: The 3 galactosyllactoses (3′-GL, 4-GL, and 6′-GL) expressed in colostrum rapidly declined over early lactation (P < 0.05). In H4 cells, HMOS attenuated TNF-α– and IL-1β–induced expression of IL-8, MIP-3α, and MCP-1 to 48–51% and pathogen-induced IL-8 and MCP-1 to 26–30% of positive controls (P < 0.001). GOS reduced TNF-α– and IL-1β–induced inflammatory responses to 25–26% and pathogen-induced IL-8 and MCP-1 to 36–39% of positive controls (P < 0.001). GOS and HMOS mitigated nuclear translocation of nuclear transcription factor κB (NF-κB) p65. HMOS quenched the inflammatory response to Salmonella infection by immature human intestinal tissue ex vivo to 26% and by GOS to 50% of infected controls (P < 0.01).Conclusion: Galactosyllactose attenuated NF-κB inflammatory signaling in human intestinal epithelial cells and in human immature intestine. Thus, galactosyllactoses are strong physiologic anti-inflammatory agents in human colostrum and early milk, contributing to innate immune modulation. The potential clinical utility of galactosyllactose warrants investigation.

Human milk oligosaccharides and galactosyloligosaccharides attenuate inflammation in human intestine

The immature intestinal mucosa is hyperresponsive to exogenous and endogenous inflammatory insults. Human milk provides immune protection to infants, and its oligosaccharides (HMOS) are putative immunomodulators. Of these, galactosyloligosaccharides (GOS) are high in colostrum, but not mature milk. The objective is to determine the ability of GOS to directly attenuate inflammation, and potential mechanisms investigated. Human models of immature and mature intestine were used to investigate the ability of a mixture of HMOS or synthetic GOS to attenuate inflammation induced by proinflammatory stimuli and enteric pathogens. These results were confirmed in the ex vivo immature human intestinal organ cultures. The ability of TNFα and of pathogens to elicit pro‐inflammatory neutrophil chemoattractants IL‐8, MIP‐3α and MCP1 and their mRNA levels was significantly attenuated by HMOS and GOS. TNFα‐induced nuclear translocation of NF‐κB was mitigated by HMOS and GOS. Consistent with previous indications of bioactivity of galactosyllactose from human colostrum, trimeric galactosyloligosaccharides interact with cell receptors to inhibit inflammatory response. HMOS and GOS suppress NF‐κB activation, thereby directly attenuating TNFα‐ and pathogen‐induced inflammation in human intestine. This provides a novel mechanism whereby specific galactosyloligosaccharides found in human milk may prove a novel therapeutic agent against gut inflammation.

Continuous ultrafiltration membrane reactor coupled with nanofiltration for the enzymatic synthesis and purification of galactosyl-oligosaccharides

Continuous enzymatic production of galactosyl-oligosaccharides (GOS) from a lactose substrate in an ultrafiltration membrane reactor (UMR) coupled with a nanofiltration separation system (CPNSS) was investigated. The overall rate of production over 4h of continuous production was 80–104mg GOS formed/U with an average residence time of 66min, an initial lactose concentration of 300g/L, an inlet pressure of 2.0bar inlet pressure, and an outlet pressure of 1.5bar. In the continuous diafiltration (CD) process, the concentration of various sugars and the relationship between the yield and purity of oligosaccharides was well predicted by mathematical models, the increased rate of sugar rejections was less than 10%, and the decreased rate of concentrations in the tank was less than 15%. In the CPNSS, 33.4wt.% GOS was obtained in the UMR, and 1.24kg of high-purity GOS (specifically, GOS purity of 57.2% and lactose content less than 20%) was achieved. This final yield was 80.1% GOS, which meets industry standards.

DIGESTION IN ADULT FEMALES OF THE LEAF‐FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α‐AMYLASE, α‐GALACTOSIDASE, AND α‐GLUCOSIDASE

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is an emerging pest of several crops around the World and up to now very little is known of its digestive system. In this article, glycoside hydrolase (carbohydrase) activities in the adult midgut cells and in the luminal contents of L. zonatus adult females were studied. The results showed the distribution of digestive carbohydrases in adults of this heteropteran species in the different intestinal compartments. Determination of the spatial distribution of α-glucosidase activity in L. zonatus midgut showed only one major molecular form, which was not equally distributed between soluble and membrane-bound isoforms, being more abundant as a membrane-bound enzyme. The majority of digestive carbohydrases were found in the soluble fractions. Activities against starch, maltose and the synthetic substrate NPαGlu were found to show the highest levels of activity, followed by enzymes active against galactosyl oligosaccharides. Based on ion-exchange chromatography elution profiles and banding patterns in mildly denaturing electrophoresis, both midgut α-amylases and α-galactosidases showed at least two isoforms. The data suggested that the majority of carbohydrases involved in initial digestion were present in the midgut lumen, whereas final digestion of starch and of galactosyl oligosaccharides takes place partially within the lumen and partially at the cell surface. The complex of carbohydrases here described was qualitatively appropriate for the digestion of free oligosaccharides and oligomaltodextrins released by α-amylases acting on maize seed starch granules.

Batch Hydrolysis and Rotating Disk Membrane Bioreactor for the Production of Galacto-oligosaccharides: A Comparative Study

Galactosyl-oligosaccharide (GOS) is a prebiotic carbohydrate, produced from lactose hydrolysis, that serves as a value-added functional food element for humans. Now GOS can be produced through enzymatic reaction with lactose either in batch mode or with immobilized enzyme on membrane. In the second case, the main concern is the fouling of the membrane that could reduce both the GOS yield and purity. The present study is thus an attempt to project the superiority of a membrane reactor called the rotating disk membrane bioreactor (RDMBR) over batch mode to obtain purified GOS with high yield. It was found that GOS yield and purity were 32.4% and 77% respectively in batch mode followed by diafiltration-assisted nanofiltration. However, in the immobilized state they were 67.4% and 80.2% at 105 rad s–1 membrane speed. Retention of monosaccharides that inhibit enzyme in the reaction volume of batch mode reduced the yield of GOS. On the contrary, simultaneous production and purification of GOS in RDMBR led to a ...

Galactosyl oligosaccharide purification by ethanol precipitation

Galactosyl oligosaccharides (GOS) are prebiotics commonly manufactured by β-galactosidase conversion of lactose, producing a mixture containing GOS, lactose, glucose and galactose. Enrichment of GOS in this mixture adds value to the product. This study aimed to determine whether the addition of ethanol to aqueous saccharide solutions could be used to selectively precipitate and enrich GOS from a reaction mixture. High concentrations of ethanol (>70% v/v) were required to induce precipitation. The total saccharide concentration was a significant variable, with higher GOS enrichment occurring at lower total saccharide concentrations. Varying the temperature between 10 and 40 °C had less impact than had changes in the concentration of saccharide or ethanol. GOS was enriched 2.3 (±0.1) fold in the precipitate formed in a solution of 90% (v/v) ethanol with 28 g/L of total saccharide at 40 °C. Performing two such precipitations sequentially reduced the monosaccharides from 48% (w/w) of the total saccharides to 4% (w/w). GOS precipitation has potential for industrial application as it is simple in operation and offers levels of purification similar to those by other techniques.

Effect of the Substrate Concentration and Water Activity on the Yield and Rate of the Transfer Reaction of β-Galactosidase from Bacillus circulans

Prebiotic galactosyl oligosaccharides (GOS) are produced from lactose by the enzyme β-galactosidase. It is widely reported that the highest GOS levels are achieved when the initial lactose concentration is as high as possible; however, little evidence has been presented to explain this phenomenon. Using a system composed of the commercial β-galactosidase derived from Bacillus circulans known as Biolacta FN5, lactose and sucrose, the relative contribution of water activity, and substrate availability were assessed. Oligosaccharide levels did not appear to be affected by changes in water activity between 1.0 and 0.77 at a constant lactose concentration. The maximum oligosaccharide concentration increased at higher initial concentrations of lactose and sucrose, while initial reaction rates for transfer increased but remained constant for hydrolysis. This suggests that the high oligosaccharide levels achieved at the raised initial saccharide concentration are due to increases in reactions that form oligosaccharides rather than decreases in concurrent reactions, which degrade oligosaccharides. There were different effects from changing the initial concentration of lactose compared to sucrose, suggesting that the ability of lactose to act as a donor saccharide may be more important for increasing maximum oligosaccharide concentrations than the combined ability of both saccharides to act as galactosyl acceptors.

A Comparative Study on the Production of Galacto-oligosaccharide from Whey Permeate in Recycle Membrane Reactor and in Enzymatic Batch Reactor

The present study primarily is an attempt to establish a technique on the disposal scheme for the wastewater coming out as an effluent from milk industries with proper environmental guides and regulations. Apart from the wastewater treatment methodology development, the present work mainly focuses on the attainment of high value product, such as the production of galactosyl-oligosaccharide (GOS) in batch reactor and as well as in recycle membrane reactor after hydrolyzing recovered lactose from the whey stream with β-galactosidase enzyme originated, from Bacillus circulans. Around 77−78% purity of this GOS was achieved with three-step membrane separation techniques: the first one was the ultrafiltration followed by diafiltration equipped nanofiltration. In recycle membrane reactor, GOS production was found to be 33% higher than that in the batch reactor, and monosaccharide concentration was 78% lower with 23% remaining enzyme activity after 18,000 s of reaction time.

Metabolism of galactosyl-oligosaccharides in Stellaria media – Discovery of stellariose synthase, a novel type of galactosyltransferase

The raffinose family oligosaccharides (RFOs), including raffinose (Gal-α(1 → 6)-Glc-α(1 → 2)β-Fru), stachyose (Gal-α(1 → 6)-Gal-α(1 → 6)-Glc-α(1 → 2)β-Fru) and higher degree of polymerization RFOs are the most widespread galactosyl-oligosaccharides (GOS) in the plant kingdom. Stellaria media is a typical representative of the Caryophyllaceae, a plant family lacking stachyose and the typical galactosyl extensions of stachyose. During cold treatment raffinose, lychnose (Gal-α(1 → 6)-Glc-α(1 → 2)β-Fru-α(1 → 1)-Gal) and stellariose (Gal-α(1 → 6)-[Gal-α(1 → 4)]-Glc-α(1 → 2)β-Fru-α(1 → 1)-Gal) were found to accumulate in S. media stems. Next to these prominent oligosaccharides, two extra GOS were discovered. Biochemical analyses (enzymatic incubations and mild acid hydrolysis) and mass spectrometry identified the first, most abundant oligosaccharide as Glc-α(1 → 2)β-Fru-α(1 → 1)-Gal, a breakdown product of lychnose. The structure of this trisaccharide was confirmed by full NMR characterization. The second, less abundant compound (termed mediose) was identified as Gal-α(1 → 6)-[Gal-α(1 → 4)]Glc-α(1 → 2)β-Fru after biochemical analyses. By partial enzyme purification the presence of discrete lychnose synthase (raffinose:raffinose 1 Fru galactosyltransferase) and stellariose synthase (raffinose:lychnose 4 Glc galactosyltransferase) activities were shown. A model is presented explaining the structural diversity of GOS in S. media. In the absence of stachyose, raffinose is further elongated by lychnose synthase and stellariose synthase to produce lychnose, mediose and stellariose. Most likely, these compounds are also subject to partial trimming by endogenous α-galactosidases.

Facile Pretreatment of Bacillus circulans β-Galactosidase Increases the Yield of Galactosyl Oligosaccharides in Milk and Lactose Reaction Systems

The commercially available preparation of beta-galactosidase from Bacillus circulans , known as Biolacta FN5, has been extensively used in the production of prebiotic galactooligosaccharides (GOS). This study focuses on characterizing the production of GOS in two reaction systems: 10% lactose (w/v) in buffer and skim milk. Analysis of the temperature dependence of the GOS yield along with the relative rates of GOS synthesis and degradation leads to the finding that GOS degradation activity was selectively decreased in Biolacta FN5 above 40 degrees C. Facile heat treatment of Biolacta FN5 solution prior to use allowed for GOS yields to be significantly increased in both reaction systems.

A novel ceramic membrane reactor system for the continuous enzymatic synthesis of oligosaccharides

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

Experimental and Modeling Study of Galactosyl-Oligosaccharides Formation in Continuous Recycle Membrane Reactors (CRMR)

Galactosyl-oligosaccharides (GOS) can be produced with native enzymes in Continuous Recycle Membrane Reactors (CRMR) or with immobilized enzymes in Plug Flow Bed Reactors (PFBR). In this paper a simple three kinetic constants model was implemented and used to define the optimal operation conditions to obtain a higher GOS yield. Ex- perimental GOS yield and productivity obtained with CRMR were in good agreement with simulated results. The advan- tages of using single CRMR, two coupled CRMR and single CRMR coupled with Simulated Moving Bed Chromatogra- phy (SMBC), for product separation and continuous lactose recycling, were investigated with a modeling and experimen- tal study. Experimental studies for single CRMR showed a 89 % higher amount of produced GOS than reports from im- mobilized enzymes in Plug Flow Bed Reactors (PFBR). Two coupled CRMR produced a 25 % higher amount of GOS than single CRMR. Simulated results with CRMR and SMBC for continuous lactose recycling showed 45% higher GOS amount than single CRMR. The results prove the feasibility and advantages of GOS production in single or series CRMR with native enzymes with or without lactose recycling.

Variações dos carboidratos de reserva de sementes de Caesalpinia echinata (pau-brasil) armazenadas sob diferentes temperaturas

Dentre os carboidratos de reserva de sementes destacam-se o amido, a sacarose e os oligossacarídeos galactosilados, que além da função de reserva, podem atuar na estabilidade das membranas durante a dessecação e a retomada do crescimento embrionário. Sementes maduras de Caesalpinia echinata Lam. foram coletadas antes e após a dispersão natural, sendo analisadas imediatamente após a coleta quanto à composição de carboidratos presentes no eixo embrionário e nos cotilédones, e aos 15 e 90 dias após armazenamento a 25 ºC, 7 ºC e -18 ºC. A principal reserva dos eixos embrionários foram os açúcares solúveis, enquanto que o amido foi a dos cotilédones, representando cerca de 30% da massa seca. Sementes recém-dispersas, com 12% de água, armazenadas sob temperaturas baixas (7 ºC e -18 ºC), mantiveram estável o teor de carboidratos totais nos cotilédones, bem como a capacidade germinativa, mas apresentaram redução no teor de açúcares solúveis. Já as sementes maduras não-dispersas, com 22% de água, mantiveram a capacidade germinativa apenas quando mantidas a 7 ºC. Nestas sementes, os açúcares solúveis diminuíram no eixo embrionário e nos cotilédones durante o armazenamento. Análises cromatográficas mostraram que, de modo geral, houve redução nos níveis de hexoses, exceto nos cotilédones de sementes armazenadas sob temperaturas baixas. As variações encontradas nas proporções de sacarose e de hexoses e no teor de amido nos cotilédones e no eixo embrionário das sementes armazenadas sob diferentes temperaturas sugerem que o metabolismo dos carboidratos de reserva é alterado durante o armazenamento. Contudo, as variações encontradas nesses compostos durante o armazenamento não estão diretamente relacionadas com a capacidade germinativa dessas sementes.

Membrane chromatography reactor system for the continuous synthesis of galactosyl-oligosaccharides

Galactosyl-oligosaccharides (GOS) are non-digestible oligosaccharides which are qualified as functional foods. GOS are enzymatically synthesized from lactose by transgalactosylation catalyzed by the enzyme β-galactosidase. In this study, a method for immobilization of β-galactosidase from Kluyveromyces lactis on a chromatography membrane was developed. The immobilization was performed at 4, 15 and 40°C. A strong basic anion exchange membrane was investigated. In static experiments the highest enzyme activity was measured at an immobilization procedure temperature of 15°C. The immobilization on the chromatography membranes was found to be very rapid likely due to ionic adsorption. The synthesis of GOS was carried out in a Continuous Membrane Chromatography Reactor System (CMCRS) at 40°C and pH 7.0 using 20 wt% initial lactose concentration. The chromatography membranes proved to be a good support for the continuous process at high convective flow rates in the enzyme reactor system. Up to 82 % lactose conversion with 24 % GOS yield was achieved at different fluxes. The corresponding reactor productivity for the production of GOS from lactose in the CMCRS was 98.7 g GOS per hour and cubic centimeter membrane volume, which significantly exceeds previously reported results.

Experimental and theoretical study of Galactosyl–Oligosaccharides formation in CRMR by thermostable mesophilic enzymes

Galactosyl–Oligosaccharides (GOS) can beproduced from lactose by enzymatic catalysiswith b-galactosidases. GOS can be produced withfree enzymes compartmentalized in continuousrecycle membrane reactors (CRMR), or withscaffold-immobilized enzymes in plug flow bedreactors (PFBR). For describing GOS productionfrom lactose in a CRMR, rigorous modellingyields complex enzymatic kinetic equations witha large number of constants. Unfortunately, suchmodels are unhandy and are not convenient forpractical purposes.

Membrane supported enzymatic synthesis of galactosyl-oligosaccharides

In the enzymatic hydrolysis of lactose, theenzyme b-galactosidase transfers a galactosemoiety to an acceptor containing a hydroxylgroup. If this acceptor is a water molecule,galactose is liberated (h ydrolysis). When a sugarmoiety acts as the acceptor, galactosyl-oligosac-charides (GOS) are formed (Fig. 1). In addition to hydrolysis, b-galactosidase isalso known to catalyze

Characterization of an Exo-β-1,3-Galactanase from Clostridium thermocellum

A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.