Characterization of a novel inulosucrase from Lactiplantibacillus plantarum.

Fructansucrases produce fructans by polymerizing the fructose moiety released from sucrose. Here, we describe the recombinant expression and characterization of a unique fructansucrase from Lactiplantibacillus plantarum DKL3 that showed low sequence similarity with previously characterized fructansucrases. The optimum pH and temperature of fructansucrase were found to be 4.0 and 35 °C, respectively. Enzyme activity increased in presence of Ca2+ and distinctly in presence of Mn2+. The enzyme was characterized as an inulosucrase (LpInu), based on the production of an inulin-type fructan as assessed byNMR spectroscopy and methylation analysis. In addition to ß-2,1-linkages, the inulin contained a few ß-2,1,6-linked branchpoints. High-performance size exclusion chromatography with refractive index detection (HPSEC-RI) revealed the production of inulin with a lower molecular weight compared to other characterized bacterial inulin. LpInu and its inulin product represent novel candidates to be explored for possible food and biomedical applications.

Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Isolation and characterization of exopolysaccharides from kombucha samples of different origins.

Kombucha is prepared by fermenting sugared green or black tea with a symbiotic culture of bacteria and yeast (SCOBY). Some of the bacteria within the SCOBY are known to form exopolysaccharides (EPS) from sucrose. However, it is yet unknown whether water-soluble EPS are formed in kombucha, and if so, which specific EPS are present. Therefore, different kombucha samples were prepared by fermentation of green and black tea with SCOBYs from different manufacturers. Subsequently, the EPS were isolated and characterized by using various chromatographic methods, partial enzymatic hydrolyses and NMR spectroscopy. It was demonstrated that levans with a varying degree of branching at position O1 (4.3-7.9 %) are present, while only trace amounts of glucans were detected. Furthermore, levans isolated from kombucha had a comparably low molecular weight and the content of levan within the kombucha samples varied from 33 to 562 mg levan/L kombucha. Therefore, our study demonstrated that levans are the main EPS type in kombucha and that levan amounts and structures varied when different starter cultures and ingredients were used. Furthermore, we provide a comprehensive data set on the structural variability of levans from kombucha.

Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high-molecular weight fructans from sucrose.

AIMS: Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. METHODS AND RESULTS: Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l-1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106-107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. CONCLUSIONS: The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

Dextrans of Weissella cibaria DSM14295: Microbial production, structure and functionality.

Lactic acid bacteria of the genus Weissella contribute to spontaneous fermentation in, e.g., sourdough or sauerkraut, but are not registered as starter cultures because of their pending safety assessment. Some strains are able to produce high amounts of exopolysaccharides. This study aims to demonstrate the techno-functionality of five dextrans from W. cibaria DSM14295, produced under varying cultivation conditions, with respect to structural and macromolecular properties. A maximum of 23.1 g/L dextran was achieved by applying the "cold shift" temperature regime. The dextrans differed in molecular mass (9-22∙108 Da, determined by HPSEC-RI/MALLS), intrinsic viscosity (52-73 mL/g), degree of branching (3.8-5.7 % at position O3, determined by methylation analysis) and their side chain length and architecture, determined by HPAEC-PAD after enzymatic hydrolysis. Stiffness of acid gels from milk spiked with these dextrans increased linearly with dextran concentration. Principal component analysis showed that dextrans produced in a semi-defined medium are primarily described by moisture sorption and branching properties, whereas dextrans produced in whey permeate were similar because of their functional and macromolecular properties. Overall, dextrans from W. cibaria DSM14295 have a high potential because of the high production yield and their functionality which can be tailored by the conditions during fermentation.

Influence of ultrasonication and hydrolysis conditions in methylation analysis of bacterial homoexopolysaccharides.

Homoexopolysaccharides (HoEPS) such as α-glucans and ß-fructans are synthesized by lactic and acetic acid bacteria. Methylation analysis is an important and well-established tool for the structural analysis of these polysaccharides, however, multiple steps are required for polysaccharide derivatization. Because ultrasonication during methylation and the conditions during acid hydrolysis may influence the results, we investigated their role in the analysis of selected bacterial HoEPS. The results reveal that ultrasonication is crucial for water insoluble α-glucan to swell/disperse and deprotonate prior to methylation whereas it is not necessary for water soluble HoEPS (dextran and levan). Complete hydrolysis of permethylated α-glucans requires 2 M trifluoroacetic acid (TFA) for 60/90 min at 121 °C while levan is hydrolyzed in 1 M TFA for 30 min at 70 °C. Nevertheless, levan was also detectable after hydrolysis in 2 M TFA at 121 °C. Thus, these conditions can be used to analyze a levan/dextran mixture. However, size exclusion chromatography of permethylated and hydrolyzed levan showed degradation and condensation reactions at harsher hydrolysis conditions. Application of reductive hydrolysis with 4-methylmorpholine-borane and TFA did not lead to improved results. Overall, our results demonstrate that conditions used for methylation analysis have to be adjusted for the analysis of different bacterial HoEPS.

Capsular Exopolysaccharides from Two Streptococcus thermophilus Strains Differ in Their Moisture Sorption Behavior.

Streptococcus thermophilus is a species frequently used in the manufacture of fermented milk. Apart from acid production, some strains additionally synthesize exopolysaccharides (EPS) which contribute to texture improvement and syneresis reduction, both being attributable to the EPS's high water binding capacity. There are two different types of EPS that may be produced, namely free exopolysaccharides (fEPS) which are secreted into the medium, and capsular EPS (cEPS) which remain attached to the bacterial cell wall. This study aims to analyze their individual contribution to techno-functional properties of fermented milk by determining the moisture sorption behavior of isolated fEPS and cell-attached cEPS from two S. thermophilus strains separately: ST-1G, a producer of non-ropy fEPS and cEPS, and ST-2E, a producer of ropy fEPS and cEPS. Differences in moisture load and sorption kinetics, determined for the first time for microbial EPS, were related to structural and macromolecular properties. The observed data are discussed by using previously published data on the physical properties of stirred fermented milk produced with these two strains. ST-1G EPS showed a higher cEPS fraction, a higher moisture load and slower moisture desorption than EPS produced by ST-2E, thus contributing to lower syneresis in fermented milk. For ST-2E, higher gel viscosity was related to a higher intrinsic viscosity and molecular mass of the ropy fEPS. Both strains produced complex EPS or EPS mixtures with clearly different molecular structures.

Influence of Arabinan Fine Structure, Galacturonan Backbone Length, and Degree of Esterification on the Emulsifying Properties of Acid-Extracted Sugar Beet Pectins.

Sugar beet pectins (SBPs) are known for their emulsifying properties, but it is yet unknown which structural elements are most important for functionality. Recent results indicated that the arabinose content has a decisive influence, but the approach applied did not allow causality to be established. In this study, a mostly intact SBP was selectively modified and the obtained pectins were analyzed for their molecular structure and their emulsifying properties. De-esterification only resulted in a moderate increase in droplet size. The length of the pectin backbone only influenced the emulsifying properties when the homogalacturonan backbone was cleaved to a higher extent. By using different arabinan-modifying enzymes, it was demonstrated that both higher portions and chain lengths of arabinans positively influence the emulsifying properties of SBPs. Therefore, we were able to refine the structure-function relationships for acid-extracted SBPs, which can be used to optimize extraction conditions.

The C-Terminal Domain of Liquorilactobacillus nagelii Dextransucrase Mediates the Production of Larger Dextrans Compared to Liquorilactobacillus hordei.

Dextransucrases released by certain lactic acid bacteria form glucose polymers with predominantly α-1,6-linkages and may be exploited biotechnologically for the tailored production of polysaccharides with application potential. Despite releasing two closely related dextransucrases, previous studies showed that water kefir borne Liquorilactobacillus (L.) hordei TMW 1.1822 and L. nagelii TMW 1.1827 produce different amounts of polysaccharides with distinct particle sizes (molecular weight and radius of gyration) and molecular architectures. To investigate where these differences originate and thus to provide deeper insights into the functionally diverse nature of polysaccharide formation during water kefir fermentation, we constructed two variants of the L. nagelii dextransucrase-a full-length enzyme and a truncated variant, devoid of a C-terminal glucan-binding domain that reflects the domain architecture of the L. hordei dextransucrase-and applied them at various enzyme concentrations to form dextran over 24 h. The full-length enzyme exhibited a high activity, forming constant amounts of dextran until a four-fold dilution, whereas the truncated variant showed a gradual decrease in activity and dextran formation at an increasing dilution. The application of the full-length enzyme resulted in higher average particle sizes compared to the truncated variant. However, the dilution of the enzyme extracts also led to a slight increase in the average particle size in both enzymes. Neither the domain architecture nor the enzyme concentration had an impact on the structural architecture of the dextrans. The presented results thus suggest that the comparatively higher processivity of the L. nagelii dextransucrase is predominantly caused by the additional C-terminal glucan-binding domain, which is absent in the L. hordei dextransucrase. The average particle size may be influenced, to some extent, by the applied reaction conditions, whereas the structural architecture of the dextrans is most likely caused by differences in the amino acid sequence of the catalytic domain.

Products Released from Structurally Different Dextrans by Bacterial and Fungal Dextranases.

Dextran hydrolysis by dextranases is applied in the sugar industry and the medical sector, but it also has a high potential for use in structural analysis of dextrans. However, dextranases are produced by several organisms and thus differ in their properties. The aim of this study was to comparatively investigate the product patterns obtained from the incubation of linear as well as O3- and O4-branched dextrans with different dextranases. For this purpose, genes encoding for dextranases from Bacteroides thetaiotaomicron and Streptococcus salivarius were cloned and heterologously expressed in Escherichia coli. The two recombinant enzymes as well as two commercial dextranases from Chaetomium sp. and Penicillium sp. were subsequently used to hydrolyze structurally different dextrans. The hydrolysis products were investigated in detail by HPAEC-PAD. For dextranases from Chaetomium sp., Penicillium sp., and Bacteroides thetaiotaomicron, isomaltose was the end product of the hydrolysis from linear dextrans, whereas Penicillium sp. dextranase led to isomaltose and isomaltotetraose. In addition, the latter enzyme also catalyzed a disproportionation reaction when incubated with isomaltotriose. For O3- and O4-branched dextrans, the fungal dextranases yielded significantly different oligosaccharide patterns than the bacterial enzymes. Overall, the product patterns can be adjusted by choosing the correct enzyme as well as a defined enzyme activity.

Degradation of complex arabinoxylans by human colonic Bacteroidetes.

Some Bacteroidetes and other human colonic bacteria can degrade arabinoxylans, common polysaccharides found in dietary fiber. Previous work has identified gene clusters (polysaccharide-utilization loci, PULs) for degradation of simple arabinoxylans. However, the degradation of complex arabinoxylans (containing side chains such as ferulic acid, a phenolic compound) is poorly understood. Here, we identify a PUL that encodes multiple esterases for degradation of complex arabinoxylans in Bacteroides species. The PUL is specifically upregulated in the presence of complex arabinoxylans. We characterize some of the esterases biochemically and structurally, and show that they release ferulic acid from complex arabinoxylans. Growth of four different colonic Bacteroidetes members, including Bacteroides intestinalis, on complex arabinoxylans results in accumulation of ferulic acid, a compound known to have antioxidative and immunomodulatory properties.

Insights into extracellular dextran formation by Liquorilactobacillus nagelii TMW 1.1827 using secretomes obtained in the presence or absence of sucrose.

Dextrans are α-(1,6)-linked glucose polymers, which are exclusively produced by lactic acid bacteria from sucrose via extracellular dextransucrases. Previous studies suggested that the environmental pH and the presence of sucrose can impact the release and activity of these enzymes. To get deeper insight into this phenomenon, the dextransucrase expressed by water kefir borne Liquorilactobacillus (L.) nagelii TMW 1.1827 (formerly Lactobacillus nagelii) was recovered in supernatants of buffered cell suspensions that had been incubated with or without sucrose and at different pH. The obtained secretomes were used to time-dependently produce and recover dextrans, whose molecular and macromolecular structures were determined by methylation analysis and AF4-MALS-UV measurements, respectively. The initial pH of the buffered cell suspensions had solely a minor influence on the released dextransucrase activity. When sucrose was present during incubation, the secretomes contained significantly higher dextransucrase activities, although the amounts of totally released proteins obtained with or without sucrose were comparable. However, the dextransucrase appeared to be released in lower amounts into the environment if sucrose was not present. The amount of isolable dextran increased up to 24 h of production, although the total sucrose was consumed within the first 10 min of incubation. Furthermore, the sucrose isomer leucrose had been formed after 10 min, while its concentrations decreased over time and the portions of longer isomaltooligosaccharides (IMOs) increased. This indicated that leucrose can be used by L. nagelii TMW 1.1827 to produce more elongated and branched dextran molecules from presynthesized IMOs, while disproportionation reactions on short IMOs may appear additionally. This leads to increasing amounts of high molecular weight dextran in a state of sucrose depletion. These findings reveal new insights into the pH- and sucrose-dependent kinetics of extracellular dextran formation and may be useful for optimization of fermentative and enzymatic dextran production processes.

Analysis of Structural and Functional Differences of Glucans Produced by the Natively Released Dextransucrase of Liquorilactobacillus hordei TMW 1.1822.

The properties of the glucopolymer dextran are versatile and linked to its molecular size, structure, branching, and secondary structure. However, suited strategies to control and exploit the variable structures of dextrans are scarce. The aim of this study was to delineate structural and functional differences of dextrans, which were produced in buffers at different conditions using the native dextransucrase released by Liquorilactobacillus (L.) hordei TMW 1.1822. Rheological measurements revealed that dextran produced at pH 4.0 (MW = 1.1 * 108 Da) exhibited the properties of a viscoelastic fluid up to concentrations of 10% (w/v). By contrast, dextran produced at pH 5.5 (MW = 1.86 * 108 Da) was gel-forming already at 7.5% (w/v). As both dextrans exhibited comparable molecular structures, the molecular weight primarily influenced their rheological properties. The addition of maltose to the production assays caused the formation of the trisaccharide panose instead of dextran. Moreover, pre-cultures of L. hordei TMW 1.1822 grown without sucrose were substantial for recovery of higher dextran yields, since the cells stored the constitutively expressed dextransucrase intracellularly, until sucrose became available. These findings can be exploited for the controlled recovery of functionally diverse dextrans and oligosaccharides by the use of one dextransucrase type.

Production and molecular structure of heteropolysaccharides from two lactic acid bacteria.

In the dairy industry, exopolysaccharides (EPS) produced in situ from lactic acid bacteria are of great interest because of their contribution to product texture. Some EPS cause ropiness which might be linked to specific physical and chemical EPS properties. EPS show a broad variety of chemical structures and, because analysis is rather complex, it is still a major challenge to establish structure-function relationships. The aim of this study was to produce EPS with different degree of ropiness, perform in-depth structural elucidations and relate this information to their behaviour in aqueous solutions. After cultivation of Streptococcus thermophilus DGCC7919 and Lactococcus lactis LL-2A and subsequent EPS isolation, both EPS showed similar macromolecular properties, but pronounced differences in monosaccharide composition and glycosidic linkages. Our data suggests that mainly the side chains in the EPS from LL-2A might be responsible for a higher ropiness than that observed for EPS from DGCC7919.

Size-Dependent Variability in Flow and Viscoelastic Behavior of Levan Produced by Gluconobacter albidus TMW 2.1191.

Levan is a fructan-type exopolysaccharide which is produced by many microbes from sucrose via extracellular levansucrases. The hydrocolloid properties of levan depend on its molecular weight, while it is unknown why and to what extent levan is functionally diverse depending on its size. The aim of our study was to gain deeper insight into the size-dependent functional variability of levan. For this purpose, levans of different sizes were produced using the water kefir isolate Gluconobacter albidus TMW 2.1191 and subsequently rheologically characterized. Three levan types could be identified, which are similarly branched, but differ significantly in their molecular size and rheological properties. The smallest levan (<107 Da), produced without adjustment of the pH, exhibited Newton-like flow behavior up to a specific concentration of 25% (w/v). By contrast, larger levans (>108 Da) produced at pH ≥ 4.5 were shear-thinning, and the levan produced at pH 5.0 showed a gel-like behavior at 5% (w/v). A third (intermediate) levan variant was obtained through production in buffers at pH 4.0 and exhibited the properties of a viscoelastic fluid up to concentrations of 15% (w/v). Our study reveals that the rheological properties of levan are determined by its size and polydispersity, rather than by the amount of levan used or the structural composition.

Thermophilic Degradation of Hemicellulose, a Critical Feedstock in the Production of Bioenergy and Other Value-Added Products.

Renewable fuels have gained importance as the world moves toward diversifying its energy portfolio. A critical step in the biomass-to-bioenergy initiative is deconstruction of plant cell wall polysaccharides to their unit sugars for subsequent fermentation to fuels. To acquire carbon and energy for their metabolic processes, diverse microorganisms have evolved genes encoding enzymes that depolymerize polysaccharides to their carbon/energy-rich building blocks. The microbial enzymes mostly target the energy present in cellulose, hemicellulose, and pectin, three major forms of energy storage in plants. In the effort to develop bioenergy as an alternative to fossil fuel, a common strategy is to harness microbial enzymes to hydrolyze cellulose to glucose for fermentation to fuels. However, the conversion of plant biomass to renewable fuels will require both cellulose and hemicellulose, the two largest components of the plant cell wall, as feedstock to improve economic feasibility. Here, we explore the enzymes and strategies evolved by two well-studied bacteria to depolymerize the hemicelluloses xylan/arabinoxylan and mannan. The sets of enzymes, in addition to their applications in biofuels and value-added chemical production, have utility in animal feed enzymes, a rapidly developing industry with potential to minimize adverse impacts of animal agriculture on the environment.

Structural characterization of mixed-linkage α-glucans produced by mutants of Lactobacillus reuteri TMW 1.106 dextransucrase.

Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.

Enzymatic Synthesis and Characterization of Mono-, Oligo-, and Polyglucosylated Conjugates of Caffeic Acid and Gallic Acid.

Glucansucrases can be used to glucosylate various plant-derived phenolic compounds by using sucrose as donor substrate. We applied Lactobacillus reuteri TMW 1.106 dextransucrase to glucosylate the acceptor substrates caffeic acid and gallic acid. Subsequently, monoglucosylated and in particular oligo- and polyglucosylated conjugates were characterized by using different chromatographic techniques and two-dimensional NMR spectroscopy. Both acceptors were substituted at positions O3 and O4. Under the conditions used, two monoglucosylated products were formed for caffeic acid, whereas only one O3-monosubstituted conjugate was detected for gallic acid. However, both acceptors resulted in O4-substituted oligo- and polyglucosylated conjugates, the amount of which was higher from gallic acid than from caffeic acid. Profile analysis tensiometry suggested that, in contrast to unmodified dextrans, oligo- and polymeric glucoconjugates of gallic acid are highly interfacially active. Overall, we provide the first detailed characterization of enzymatically conjugated oligo- and polymeric dextrans, which may have further potential as functional ingredients.

Comparison and Optimization of Different Protein Nitrogen Quantitation and Residual Protein Characterization Methods in Dietary Fiber Preparations.

Proteins are plant cell wall components but they are not included in the definition of dietary fiber. Therefore, dietary fiber preparations have to be corrected for their residual protein contents. This is commonly done by calculating the residual protein concentrations from the nitrogen contents after Kjeldahl digestion. Here, three different methods to determine nitrogen in Kjeldahl digests were compared: conventional titration with hydrochloric acid after steam distillation, a colorimetric assay (24-well microplates and cuvettes), and the determination by using an ammonia electrode. All assays gave similar results but detection using the ammonia electrode was found to be the most time-efficient approach. Also, an amino-acid profiling method, which is not based on commercial kits and which is suitable for routine analysis of dietary fiber preparations, was established. For this purpose, an HPLC-FLD method following amino acid derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was optimized for fiber samples. Although all commonly used dietary fiber preparation methods involve the application of proteases the amino acid profiles of fiber samples from different sources were shown to be quite diverse. Considering the amino acid composition of the residual protein in various dietary fiber preparations, residual protein is probably not only based on structural proteins.

Chromatographic analysis of alginate degradation by five recombinant alginate lyases from Cellulophaga algicola DSM 14237.

Alginate lyases can be used for alginate oligosaccharide production and for structural characterization or modification of alginates. For these applications it is important to obtain detailed information on mode of action and substrate specificities of alginate lyases. In this study, five alginate lyase genes were cloned from Cellulophaga algicola DSM 14237 genomic DNA, heterologously expressed, and characterized by using HPSEC-RI and HPAEC-PAD/MS. It was demonstrated that these analytical approaches can provide detailed information on preferred substrates, extent of hydrolysis, and the liberated products. The recombinant enzymes cleaved alginates endolytically (CaAly1, CaAly2, CaAly3) or exolytically (CaAly4, CaAly5). The three endolytic alginate lyases predominantly hydrolyzed guluronic acid-rich alginates, only CaAly1 also showed activity on mannuronic acid-rich alginates. The oligosaccharide profiles further demonstrated that the endolytic enzymes have rather narrow but slightly different substrate specificities and that the two exolytic alginate lyases mainly cleaved unsaturated guluronic acid oligosaccharides to monomers.