Preparation Of Oligosaccharides Research Articles

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60°C and over a broad pH range between 5.0 and 9.0, and was stable at 50°C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of β-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

High-level Expression of an Acidic and Thermostable Chitosanase in Pichia pastoris Using Multi-copy Expression Strains and High-cell-density Cultivation

Chitin is a linear homopolymer of acetylated β-(1,4)-linked glucosamine residues and among the most abundant polysaccharides in the world. Here, we identified and purified a novel chitosanase (CCHA) from Aspergillus oryzae NKY2017 obtained from Hu’bei province in China. Construction of a cDNA library from this strain revealed the gene sequence subsequently expressed in Pichia pastoris and subsequent construction of multi-copy expression plasmids (CCHA1/2/3/4). The results demonstrated elevated levels of CCHA expression in multi-copy strains, with strain CCHA4 chosen for high-density fermentation and enzyme-activity experiments. High-density fermentation achieved a CCHA yield of 22,500 U/mL, and temperature and pH optimization resulted in the highest CCHA activity at 40°C and 4.0, respectively. We used this enzyme for a large-scale preparation of oligosaccharides: 4 g enzyme could convert 150 kg chitosan into oligosaccharides in 24 h at 40°C. These results demonstrated abundant CCHA expression in P. pastoris and suggested the efficacy of CCHA for use in industrial applications.

Strategy for Isolation, Preparation, and Structural Analysis of Chondroitin Sulfate Oligosaccharides from Natural Sources.

The structure of chondroitin sulfate oligosaccharides (CSOs), especially their sulfation pattern, has been found to be closely related with many biological pathways and diseases. However, detailed functional analysis such as their interaction with glycan binding proteins (GBPs) has been lagging, presumably due to the unavailability of well-defined, diverse structures. Besides challenging chemical and enzymatic synthesis, this is also due to the challenges in their purification at the isomer level and structural analysis owing to their instability, structural complexity, and low mass spectrometry detection sensitivity. Herein, we first used recycling preparative HPLC to separate and purify shark CS tetrasaccharide component labeled by a bifunctional fluorescent linker 2-amino-N-(2-aminoethyl)benzamide (AEAB) at the isomer level. Then, each isomer was derivatized through a multistage procedure including N-acetylation, carboxyl amidation, permethylation, and desulfation with silylating reagent. Structural analysis of each derivatized isomer was performed with ESI-MSn in positive ion mode. A total of 16 isomers of CSO-AEAB were isolated, with a minimum mass component of 0.007 mg and a maximum mass component of 17.53 mg, of which 10 isomers (>90 μg) were structurally analyzed. This preparation and structure analysis of CSOs lay the foundation for further study of the structure-activity relationship of CSOs.

Structure and Function of Bovine Whey Derived Oligosaccharides Showing Synbiotic Epithelial Barrier Protective Properties.

Commensal gut microbiota and probiotics have numerous effects on the host’s metabolic and protective systems, which occur primarily through the intestinal epithelial cell interface. Prebiotics, like galacto-oligosaccharides (GOS) are widely used to modulate their function and abundance. However, important structure–function relations may exist, requiring a detailed structural characterization. Here, we detailed the structural characterization of bovine whey derived oligosaccharide preparations enriched with GOS or not, dubbed GOS-enriched milk oligosaccharides (GMOS) or MOS, respectively. We explore GMOS’s and MOS’s potential to improve intestinal epithelial barrier function, assessed in a model based on barrier disruptive effects of the Clostridioides difficile toxin A. GMOS and MOS contain mainly GOS species composed of β1-6- and β1-3-linked galactoses, and 3′- and 6′-sialyllactose. Both GMOS and MOS, combined with lactobacilli, like Lactobacillus rhamnosus (LPR, NCC4007), gave synergistic epithelial barrier protection, while no such effect was observed with Bifidobacterium longum (BL NCC3001), Escherichia coli (Nissle) or fructo-oligosaccharides. Mechanistically, for barrier protection with MOS, (i) viable LPR was required, (ii) acidification of growth medium was not enough, (iii) LPR did not directly neutralize toxin A, and (iv) physical proximity of LPR with the intestinal epithelial cells was necessary. This is the first study, highlighting the importance of structure–function specificity and the necessity of the simultaneous presence of prebiotic, probiotic and host cell interactions required for a biological effect.

Expression and Characterization of an Alginate Lyase and Its Thermostable Mutant in Pichia pastoris.

Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2–4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.

Preparation of alginate oligosaccharides and their biological activities in plants: A review

Alginate oligosaccharide (AOS) is the degradation product of alginates extracted from brown algae. As a multifunctional oligomer, it has attracted widespread attention in plant research. Different methods of preparation generate AOS possessing diverse structural properties, and result in differences in AOS activity. In this review, the methods of preparation and characterization of AOS are briefly summarized, followed by a systematic introduction to the activity and mechanisms of AOS in plants. AOS can act as a growth promoter at different growth stages of plants. AOS also enhances resistance to pathogens, drought, salt, heavy metals and other stressors by triggering plant immunity, exerting bioactivity just like a pathogen-associated molecular pattern. In addition, AOS can regulate ABA biosynthesis and metabolite to preserve fruit quality and enhance shelf life. This review provides a comprehensive summary of the biological activity of AOS in plants, which will support research and the application of AOS treatments for plants in the future.

In vitro digestibility of commercial and experimental isomalto-oligosaccharides

Isomalto-oligosaccharides (IMO) significantly contribute to the global oligosaccharide market. IMO are linear α-(1 → 6) linked oligosaccharides with isomaltotriose as the representative trisaccharide. Commercial IMO preparations ypically also contain panose-series oligosaccharides as a major component. In humans, IMO are partially digestible but the digestibility of specific components of commerical IMO preparations remains unknown. This study aimed to compare the in vitro digestibility of reference compounds, experimental α-gluco-oligosaccharides and commercial IMO. Experimental α-gluco-oligosaccharides were synthesized with the recombinant dextransucrase DsrM. Two in vitro digestion methods were used, a reference method matching the AOAC method for dietary fibre, and a protocol that uses brush border glycosyl hydrolases from the rat intestine. The α-gluco-oligosaccharides patterns after hydrolysis remain were analyzed by high performance anion exchange chromatography coupled to pulsed amperometric detection. Panose-series oligosaccharides were hydrolysed more rapidly by amylase and amyloglucosidase when compared to hydrolysis by rat intestinal enzymes. The rate of hydrolysis by rat intestinal enzymes decreased in the order panose > isomaltose, kojibiose or nigerose. Hydrolysis of panose-series oligosaccharides but not the hydrolysis of isomalto-oligosaccharides was dependent on the degree of polymerization. Qualitative analysis of oligosaccharides remaining after hydrolysis indicated that rat small intestinal enzymes hydrolyse their substrates from the non-reducing end. Taken together, results inform on the modification or optimization of current production processes for IMO to obtain tailored oligosaccharide preparations with reduced digestibility and an increased content of dietary fibre.

Cloning, Secretory Expression and Characterization of a Unique pH-Stable and Cold-Adapted Alginate Lyase.

Cold-adapted alginate lyases have unique advantages for alginate oligosaccharide (AOS) preparation and brown seaweed processing. Robust and cold-adapted alginate lyases are urgently needed for industrial applications. In this study, a cold-adapted alginate lyase-producing strain Vibrio sp. W2 was screened. Then, the gene ALYW201 was cloned from Vibrio sp. W2 and expressed in a food-grade host, Yarrowia lipolytica. The secreted Alyw201 showed the activity of 64.2 U/mL, with a molecular weight of approximate 38.0 kDa, and a specific activity of 876.4 U/mg. Alyw201 performed the highest activity at 30 °C, and more than 80% activity at 25–40 °C. Furthermore, more than 70% of the activity was obtained in a broad pH range of 5.0–10.0. Alyw201 was also NaCl-independent and salt-tolerant. The degraded product was that of the oligosaccharides of DP (Degree of polymerization) 2–6. Due to its robustness and its unique pH-stable property, Alyw201 can be an efficient tool for industrial production.

Research Article Product Composition Analysis and Process Research of Oligosaccharides Produced from Enzymatic Hydrolysis of High-Concentration Konjac Flour.

There is a huge variability in reducing sugars, viscosity, and composition of oligosaccharides in the hydrolyzed products of konjac flour with different concentrations. We analyzed the factors affecting reducing sugars, viscosity, and the average degree of polymerization (DP) during the preparation of oligosaccharides from konjac flour hydrolyzed by β-mannanase under the high-concentration solute hydrolysis model. Hydrolysate of konjac flour, using concentrations ranging from 50 to 200 g/L, was directly added into 20 U/mL of β-mannanase solution. The results showed that when the proportion of the water content in the solution decreased, the viscosity of the solution and the DP of polysaccharides changed significantly. When the viscosity of the hydrolysate was controlled within the range of 30–20 mPa·s, the concentration of the reducing sugars was maintained in the range of 9–13 g/L and the average DP of the polysaccharides was controlled in the range of 2.42–9.78. We also found that a high concentration of hydrolysate was beneficial for decreasing the production of reducing sugars, and the diversification of macromolecular glycan was beneficial to the preparation of functional sugars. Moreover, we observed that the proportion of reducing sugars with free water content was high and that the preparation of oligosaccharides via the high-concentration solid-state method increased product diversity.

Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides

Alginate lyases are important enzymes to prepare alginate oligosaccharides for industrial applications and elucidating the degradation pattern of alginate lyases is essential for expanding their applications. Herein, we cloned and expressed AlyPL6, a novel member of polysaccharide lyase family-6 (PL6) with high activity from Pedobacter hainanensis NJ-02. It was found that AlyPL6 could recognize tetrasaccharide as the minimal substrate to release oligosaccharides with low degrees of polymerization (DPs). As a result, it could be speculated that the cleavage site of substrate is located between −1 and +1 subsites. For further application, AlyPL6 was then immobilized onto mesoporous titanium oxide particles (MTOPs) with >55.4% of maximal activity retained at 45 °C after it was reused for 10 times. The research provided extended insights into the substrate recognition and degradation pattern of PL 6 alginate lyases, which may benefit the further application of alginate lyases a lot.

Cloning,Expression and Characterization of Recombinant β -mannanase from Bacillus subtilis

To achieve the heterologous expression of beta-mannanase which is highly specific to Konjac glucomannan,the beta-mannanase gene BsmanA was cloned from Bacillus subtilis G1,linked to the expression vector pACYCDuet-1 and transformed into E.coil BL21(DE3). The results showed that the full length of the beta-mannanase gene BsmanA was 1098 bp and encoded 366 amino acids. After purification by Ni affinity chromatography,the molecular weight of recombinant enzyme was determined to be 38 kD. The results of enzymatic properties of recombinant beta-mannanase showed that the optimum temperature and pH for the enzymatic reaction were 60℃ and pH6.5.The stability of recombinant beta-mannanase could be maintained in the range of pH4.5~7.0 and temperature 50~70℃. In this study,the heterologous expression of beta-mannanase was accomplished,which would provide a new choice for the industrialization of biocatalytic preparation of mannose oligosaccharides.

Elucidation of a Unique Pattern and the Role of Carbohydrate Binding Module of an Alginate Lyase.

Alginate oligosaccharides with different degrees of polymerization (DPs) possess diverse physiological activities. Therefore, in recent years, increasing attention has been drawn to the use of enzymes for the preparation of alginate oligosaccharides for food and industrial applications. Previously, we identified and characterized a novel bifunctional alginate lyase Aly7A, which can specifically release trisaccharide from three different substrate types with a unique degradation pattern. Herein, we investigated its degradation pattern by modular truncation and molecular docking. The results suggested that Aly7A adopted a unique action mode towards different substrates with the substrate chain sliding into the binding pocket of the catalytic domain to position the next trisaccharide for cleavage. Deletion of the Aly7A carbohydrate binding module (CBM) domain resulted in a complex distribution of degradation products and no preference for trisaccharide formation, indicating that the CBM may act as a “controller” during the trisaccharide release process. This study further testifies CBM as a regulator of product distribution and provides new insights into well-defined generation of alginate oligosaccharides with associated CBMs.

Preparation and antitumor activity of selenium-modified glucomannan oligosaccharides

Abstract Konjac glucomannan is safely and widely used in the functional food industry. Compared with glucomannan, glucomannan oligosaccharides have higher water solubility making them easier to absorb and utilize. These oligosaccharides are also associated with many biological activities. A novel glucomannan hydrolase PpGluA with high activity and substrate specificity which releases a series of konjac glucomannan oligosaccharides having DP 2–9 is described here. Selenium is one of the essential trace elements for humans. The selenium-modified glucomannan oligosaccharides were prepared by the sodium selenite-nitric acid method for the first time. Antitumor activity studies demonstrated that selenium-modified glucomannan oligosaccharides had better activity than inorganic sodium selenite and glucomannan oligosaccharides and inhibited tumor by inducing apoptosis through the mitochondrial pathway. This study suggests that selenium-modified glucomannan oligosaccharides may be have potential as a new functional food for inhibition of tumors.

Acceptor Specificity of β-N-Acetylhexosaminidase from Talaromyces flavus: A Rational Explanation.

Fungal β-N-acetylhexosaminidases, though hydrolytic enzymes in vivo, are useful tools in the preparation of oligosaccharides of biological interest. The β-N-acetylhexosaminidase from Talaromyces flavus is remarkable in terms of its synthetic potential, broad substrate specificity, and tolerance to substrate modifications. It can be heterologously produced in Pichia pastoris in a high yield. The mutation of the Tyr470 residue to histidine greatly enhances its transglycosylation capability. The aim of this work was to identify the structural requirements of this model β-N-acetylhexosaminidase for its transglycosylation acceptors and formulate a structure–activity relationship study. Enzymatic reactions were performed using an activated glycosyl donor, 4-nitrophenyl N-acetyl-β-d-glucosaminide or 4-nitrophenyl N-acetyl-β-d-galactosaminide, and a panel of glycosyl acceptors of varying structural features (N-acetylglucosamine, glucose, N-acetylgalactosamine, galactose, N-acetylmuramic acid, and glucuronic acid). The transglycosylation products were isolated and structurally characterized. The C-2 N-acetamido group in the acceptor molecule was found to be essential for recognition by the enzyme. The presence of the C-2 hydroxyl moiety strongly hindered the normal course of transglycosylation, yielding unique non-reducing disaccharides in a low yield. Moreover, whereas the gluco-configuration at C-4 steered the glycosylation into the β(1-4) position, the galacto-acceptor afforded a β(1-6) glycosidic linkage. The Y470H mutant enzyme was tested with acceptors based on β-glycosides of uronic acid and N-acetylmuramic acid. With the latter acceptor, we were able to isolate and characterize one glycosylation product in a low yield. To our knowledge, this is the first example of enzymatic glycosylation of an N-acetylmuramic acid derivative. In order to explain these findings and predict enzyme behavior, a modeling study was accomplished that correlated with the acquired experimental data.

Cloning, Expression, and Characterization of a New Glycosaminoglycan Lyase from Microbacterium sp. H14.

Glycosaminoglycan (GAG) lyase is an effective tool for the structural and functional studies of glycosaminoglycans and preparation of functional oligosaccharides. A new GAG lyase from Microbacterium sp. H14 was cloned, expressed, purified, and characterized, with a molecular weight of approximately 85.9 kDa. The deduced lyase HCLaseM belonged to the polysaccharide lyase (PL) family 8. Based on the phylogenetic tree, HCLaseM could not be classified into the existing three subfamilies of this family. HCLaseM showed almost the same enzyme activity towards hyaluronan (HA), chondroitin sulfate A (CS-A), CS-B, CS-C, and CS-D, which was different from reported GAG lyases. HCLaseM exhibited the highest activities to both HA and CS-A at its optimal temperature (35 °C) and pH (pH 7.0). HCLaseM was stable in the range of pH 5.0–8.0 and temperature below 30 °C. The enzyme activity was independent of divalent metal ions and was not obviously affected by most metal ions. HCLaseM is an endo-type enzyme yielding unsaturated disaccharides as the end products. The facilitated diffusion effect of HCLaseM is dose-dependent in animal experiments. These properties make it a candidate for further basic research and application.

Optimation for preparation of oligosaccharides from flaxseed gum and evaluation of antioxidant and antitumor activities in vitro

Flaxseed oligosaccharides (FGOS) were prepared by degradation of flaxseed gum (FG) using enzymatic method. Factors affecting the enzymatic hydrolysis of FG were investigated by single factor and orthogonal tests. In the optimum hydrolysis conditions (reaction time 12 h, temperature 50 °C, pH 4.5, cellulase concentration 100 U/mL), the reducing sugar ratio and extraction yield of FGOS were 33.6 ± 0.35% and 56.8 ± 0.41%, respectively. The average molecular weight of FGOS was about 1.6 kDa, which consists of mannose, galactose, glucose, arabinose, glucuronic acid, xylose, rhamnose, ribose, galacturonic acid. Fourier-transform infrared spectra and NMR indicated that FG was successfully degraded to FGOS. FGOS exhibited better antioxidant activities than FG on scavenging hydroxyl, ABTS and DPPH radicals. In vitro cytotoxicities experiments reveal FGOS acquire the ability of antiproliferation against HepG2 and Hela cells in a dose-dependent manner.

Convenient preparation of an antigenic oligosaccharide from white kidney bean powder: A useful plant oligosaccharide for synthesis of immunoactive glycopolymer

Plant glycoproteins, especially allergenic glycoproteins such as pollen allergens, often carry antigenic N-glycans with α1-3 fucose and/or β1-2 xylose residue(s) on the trimannosyl core structure. We previously reported that one of such antigenic free-form N-glycans, Man3Xyl1Fuc1GlcNAc2 (M3FX) suppressed IL-4 production from Th2 cells of pollinosis patients. For the molecular-level analysis of this immunoactivity, an effective and convenient procedure for large scale preparation of the immunoactive free-form N-glycan and a synthesis of glycopolymers bearing multivalent M3FX has been required. During the preparation of prebiotic oligosaccharides from several edible beans, we found that the free-form M3FX accumulates in relatively large amounts in white kidney beans. In this report, we describe a new procedure for preparation of M3FX from white kidney bean powders by a combination of ion-exchange method, gel-filtration, and hydrophilic partitioning. The high-purity of M3FX prepared by this procedure was confirmed by MS-analysis and 1H-NMR, suggesting that the free-form M3FX can be used for the synthesis of neoglycopolymer. Using this new procedure, the immunoactive oligosaccharide can be prepared without the chemical method such as hydrazinolysis and other purification steps required to exclude other type of N-glycans.

Biochemical characteristics and synergistic effect of two novel alginate lyases from Photobacterium sp. FC615

BackgroundMacroalgae and microalgae, as feedstocks for third-generation biofuel, possess competitive strengths in terms of cost, technology and economics. The most important compound in brown macroalgae is alginate, and the synergistic effect of endolytic and exolytic alginate lyases plays a crucial role in the saccharification process of transforming alginate into biofuel. However, there are few studies on the synergistic effect of endolytic and exolytic alginate lyases, especially those from the same bacterial strain.ResultsIn this study, the endolytic alginate lyase AlyPB1 and exolytic alginate lyase AlyPB2 were identified from the marine bacterium Photobacterium sp. FC615. These two enzymes showed quite different and novel enzymatic properties whereas behaved a strong synergistic effect on the saccharification of alginate. Compared to that when AlyPB2 was used alone, the conversion rate of alginate polysaccharides to unsaturated monosaccharides when AlyPB1 and AlyPB2 acted on alginate together was dramatically increased approximately sevenfold. Furthermore, we found that AlyPB1 and AlyPB2 acted the synergistic effect basing on the complementarity of their substrate degradation patterns, particularly due to their M-/G-preference and substrate-size dependence. In addition, a novel method for sequencing alginate oligosaccharides was developed for the first time by combining the 1H NMR spectroscopy and the enzymatic digestion with the exo-lyase AlyPB2, and this method is much simpler than traditional methods based on one- and two-dimensional NMR spectroscopy. Using this strategy, the sequences of the final tetrasaccharide and pentasaccharide product fractions produced by AlyPB1 were easily determined: the tetrasaccharide fractions contained two structures, ΔGMM and ΔMMM, at a molar ratio of 1:3.2, and the pentasaccharide fractions contained four structures, ΔMMMM, ΔMGMM, ΔGMMM, and ΔGGMM, at a molar ratio of ~ 1:1.5:3.5:5.25.ConclusionsThe identification of these two novel alginate lyases provides not only excellent candidate tool-type enzymes for oligosaccharide preparation but also a good model for studying the synergistic digestion and saccharification of alginate in biofuel production. The novel method for oligosaccharide sequencing described in this study will offer a very useful approach for structural and functional studies on alginate.

Preparation of gentiobiose-derived oligosaccharides by glucansucrase E81 and determination of prebiotic and immune-modulatory functions

Gentiobiose-derived oligosaccharides were synthesized by the acceptor reaction of glucansucrase E81 obtained from Lactobacillus reuteri E81 with sucrose and gentiobiose as donor-acceptor sugars, respectively. The reaction products were monitored by TLC analysis and gentiobiose-derived oligosaccharides up to DP 8 were formed during the acceptor reaction as determined by ESI-MS/MS analysis. The glycosylation of the gentiobiose with α-(1 → 6) linkages and α-(1 → 3) linkages was shown by 1H and 13C NMR analysis confirming the structure of these gentiobiose-derived oligosaccharides. The in vitro prebiotic function of the oligosaccharides was determined in which probiotic strains were stimulated whereas no growth was observed in pathogen strains. Gentiobiose-derived oligosaccharides showed immune-modulatory functions in vitro and triggered the production of IL-4, IL12 and TNF-α cytokines in HT29 cells in a dose dependent manner. This study showed the production and functional characterisation of gentiobiose-derived oligosaccharides establishing a promising avenue for future applications.

Cloning, Expression, and Characterization of a New PL25 Family Ulvan Lyase from Marine Bacterium Alteromonas sp. A321.

Ulvan lyases can degrade ulvan to oligosaccharides with potent biological activity. A new ulvan lyase gene, ALT3695, was identified in Alteromonas sp. A321. Soluble expression of ALT3695 was achieved in Escherichia coli BL21 (DE3). The 1314-bp gene encoded a protein with 437 amino acid residues. The amino acid sequence of ALT3695 exhibited low sequence identity with polysaccharide lyase family 25 (PL25) ulvan lyases from Pseudoalteromonas sp. PLSV (64.14% identity), Alteromonas sp. LOR (62.68% identity), and Nonlabens ulvanivorans PLR (57.37% identity). Recombinant ALT3695 was purified and the apparent molecular weight was about 53 kDa, which is different from that of other polysaccharide-degrading enzymes identified in Alteromonas sp. A321. ALT3695 exhibited maximal activity in 50 mM Tris-HCl buffer at pH 8.0 and 50 °C. ALT3695 was relatively thermostable, as 90% activity was observed after incubation at 40 °C for 3 h. The Km and Vmax values of ALT3695 towards ulvan were 0.43 mg·mL−1 and 0.11 μmol·min−1·mL−1, respectively. ESI-MS analysis showed that enzymatic products were mainly disaccharides and tetrasaccharides. This study reports a new PL25 family ulvan lyase, ALT3695, with properties that suggest its great potential for the preparation of ulvan oligosaccharides.