Preparation Of Oligosaccharides Research Articles

Biochemical Characterization and Substrate Degradation Mode of a Novel α-Agarase from Catenovulum agarivorans.

Agarose can be hydrolyzed into agarooligosaccharides (AOSs) by α-agarase, which is an important enzyme for efficient saccharification of agarose or preparation of bioactive oligosaccharides from agarose. Although many β-agarases have been reported and characterized, there are only a few studies on α-agarases. Here, we cloned a novel α-agarase named CaLJ96 with a molecular weight of approximately 200 kDa belonging to glycoside hydrolase family 96 from Catenovulum agarivorans. CaLJ96 has good pH stability and exhibits maximum activity at 37 °C and pH 7.0. The hydrolyzed products of agarose by CaLJ96 are analyzed as agarobiose (A2), agarotetraose (A4), and agarohexaose (A6), in which A4 is the dominant product. CaLJ96 can hydrolyze agaropentaose (A5) into A2 and agarotriose (A3) and A6 into A2 and A4 but cannot act on A2, A3, or A4. This is the first report to characterize the α-agarase action on AOSs in detail. Therefore, CaLJ96 has potential for the manufacture of bioactive AOSs.

Processivity and the Mechanisms of Processive Endoglucanases.

Cellulases, as environmentally appropriate catalysts specifically acting on cellulosic substrates, are important for the industrial conversion of lignocellulose and modification of cellulose products. After decades of research, a fundamental understanding of cellulase-mediated hydrolysis of cellulose is that its ability to processively act as a key for the complete enzymatic hydrolysis of natural crystalline cellulose. Two types of processive cellulases are known: exoglucanases and processive endoglucanases. Exoglucanases are typical processive enzymes, and they have been studied in detail so that their modes of action and mechanisms are reasonably well characterized. Conversely, endoglucanases are less well characterized because of the non-universality and structural diversity. However, processive endoglucanases have certain characteristics that exoglucanases lack such as hydrolysis product diversity and independent hydrolyze natural crystalline cellulose. Therefore, besides the conversion of cellulose to monosaccharide, they might be useful for modification of fibrous substrates and preparation of cellulose oligosaccharides. Herein, we review in detail the sources, hydrolysis products, application, and possible hydrolysis mechanisms of processive endoglucanases.

Secretory expression of biologically active chondroitinase ABC I for production of chondroitin sulfate oligosaccharides

Chondroitin sulfate ABC lyases (csABCs) have attracted intensive attention because of their wide potential applications in promoting tissue regeneration and generating oligosaccharides. In the present study, three csABC I encoding sequences were analyzed and site-directed mutagenesis results demonstrate that residues Leu125 and Leu322 are essential to activity and mutation of each leucine residue to proline dramatically decreased enzymatic activity. Additionally, our results showed that mutation of I309 V significantly increased the catalytic efficiency. By recruiting OmpA signal peptide and engineering the permeability of cell membrane with deletion of a lipoprotein encoding gene lpp, all recombinant enzymes were secreted and the extracellular activity was finally increased to 2.99 ± 0.1 U/mL in batch fermentation. More importantly, the engineered csABC I with high activity can rapidly degrade chondroitin sulfate to the end tetrasaccharides and disaccharides, demonstrating its applicability for preparation of chondroitin sulfate oligosaccharides.

Highly purified fucosylated chondroitin sulfate oligomers with selective intrinsic factor Xase complex inhibition

Fucosylated chondroitin sulfate (FCS) oligosaccharides of specific molecular weight have shown potent anticoagulant activities with selectivity towards intrinsic factor Xase complex. However, the preparation of FCS oligosaccharides by traditional methods requires multiple purification steps consuming large amounts of time and significant resources. The current study focuses on developing a method for the rapid preparation of FCS oligomers from sea cucumber Pearsonothuria graeffei having 6–18 saccharide residues. The key steps controlling molecular weight (Mw) and purity of these FCS oligomers were evaluated. Structural analysis showed the resulting FCS oligomers were primarily l-Fuc3,4diS-α1,3-d-GlcA-β1,3-(d-GalNAc4,6diS-β1,4-[l-Fuc3,4diS-α1,3-]d-GlcA-β1,3-)nd-anTal-ol4,6diS (n = 1˜5) accompanied by partial de-fucosylation and/or de-sulfation. In vitro and in vivo experiments demonstrate that these FCS oligomers selectively inhibit intrinsic factor Xase complex and exhibit remarkable antithrombotic activity without hemorrhagic and hypotension side effects. This method is suitable for large-scale preparation of FCS oligosaccharides as clinical anticoagulants.

Recent Developments in Stereoselective Chemical Glycosylation.

Because of their pivotal biological functions, attention to sugars and glycobiology has grown rapidly in recent decades, leading to increased demand for homogeneous oligosaccharides. The stereoselective preparation of oligosaccharides by chemical means remains challenging and continues to be a vivid research area for organic chemists. In the past decade, new approaches and reinvestigated traditional methods have transformed the field. These developments include novel catalyses, various types of glycosylation modulators and the use of photochemical energy to facilitate glycosylation. This Minireview presents a brief overview of the latest trends in chemical glycosylation, with emphasis on the stereoselective synthetic protocols developed in the past decade.

One-Step Process for Environment-Friendly Preparation of Agar Oligosaccharides From Gracilaria lemaneiformis by the Action of Flammeovirga sp. OC4.

Oligosaccharides extracted from agar Gracilaria lemaneiformis (G. lemaneiformis) have stronger physiological activities and a higher value than agar itself, but the pollution caused by the extraction process greatly restricts the sustainable use of agar. In this study, four bacterial strains with a high ability to degrade G. lemaneiformis were isolated from seawater by in situ enrichment in the deep sea. Among them, Flammeovirga sp. OC4, identified by morphological observation and its 16S rRNA sequencing (98.07% similarity to type strain JL-4 of Flammeovirga aprica), was selected. The optimum temperature and pH of crude enzyme produced by Flammeovirga sp. OC4 were 50°C and 8, respectively. More than 60% of the maximum enzyme activity remained after storage at pH 5.0–10.0 for 60 min. Both Mn2+ and Ba2+ could enhance the enzyme activity. A “one-step process” for preparation of oligosaccharides from G. lemaneiformis was established using Flammeovirga sp. OC4. After optimization of the Plackett–Burman (PB) design and response surface methodology (RSM), the yield of oligosaccharides was increased by 36.1% from 2.71 to 3.09 g L−1 in a 250-mL fermenter with optimized parameters: 30 g L−1 G. lemaneiformis powder, 4.84 g L−1 (NH4)2SO4, 44.8-mL working medium volume at 36.7°C, and a shaking speed of 200 × g for 42 h. The extracted oligosaccharides were identified by thin layer chromatography (TLC) and ion chromatography, which consisted of neoagarobiose, agarotriose, neoagarotetraose, agaropentaose, and neoagarohexaose. These results provided an alternative approach for environment-friendly and sustainable utilization of algae.

Structural insights into a novel Ca2+-independent PL-6 alginate lyase from Vibrio OU02 identify the possible subsites responsible for product distribution

Alginate lyases have a wide range of industrial applications, such as oligosaccharide preparation, medical treatment, and bioconversion. Therefore, the discovery and characterization of novel alginate lyases are extremely important. PL-6 alginate lyases are classified into two groups: those with a single domain or two domains. However, only one structure of a two-domain alginate lyase has been determined to date. In this study, we characterized a novel single-domain PL-6 alginate lyase (named AlyF). According to the biochemical analysis, AlyF possesses unique features compared with other PL-6 enzymes, including (1) a Ca2+-independent catalytic mechanism and (2) a PolyG-specific cleavage specificity that predominantly produces trisaccharides. The structures of AlyF and its complexes described here reveal the structural basis for these unique features and substrate binding mechanisms, which were further confirmed using mutagenesis. More importantly, we determined the possible subsites specifying the predominantly trisaccharide products of AlyF, which may facilitate the rational design of AlyF for potential applications in preparing a single alginate oligomer.

Production of a Thermostable Chitosanase from Shrimp Heads via Paenibacillus mucilaginosus TKU032 Conversion and its Application in the Preparation of Bioactive Chitosan Oligosaccharides.

Chitosanase has attracted great attention due to its potential applications in medicine, agriculture, and nutraceuticals. In this study, P. mucilaginosus TKU032, a bacterial strain isolated from Taiwanese soil, exhibited the highest chitosanase activity (0.53 U/mL) on medium containing shrimp heads as the sole carbon and nitrogen (C/N) source. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a chitosanase isolated from P. mucilaginosus TKU032 cultured on shrimp head medium was determined at approximately 59 kDa. The characterized chitosanase showed interesting properties with optimal temperature and thermal stability up to 70 °C. Three chitosan oligosaccharide (COS) fractions were isolated from hydrolyzed colloidal chitosan that was catalyzed by TKU032 chitosanase. Of these, fraction I showed the highest α-glucosidase inhibitor (aGI) activity (65.86% at 20 mg/mL); its inhibitory mechanism followed the mixed noncompetitive inhibition model. Fractions II and III exhibited strong 2,2-diphenyl1-picrylhydrazyl (DPPH) radical scavenging activity (79.00% at 12 mg/mL and 73.29% at 16 mg/mL, respectively). In summary, the COS fractions obtained by hydrolyzing colloidal chitosan with TKU032 chitosanase may have potential use in medical or nutraceutical fields due to their aGI and antioxidant activities.

Truncation of κ‑carrageenase for higher κ‑carrageenan oligosaccharides yield with improved enzymatic characteristics.

Carrageenase is useful for preparation of carrageenan oligosaccharides, which have significant bioactivity. We expressed a κ‑carrageenase gene from Zobellia sp. ZL-4 in full-length (κ-ZL-4) or after truncation of the carbohydrate binding module and the Type-IX secretion module (κ-ZL-4-GH16). κ-ZL-4-GH16 showed a specific activity (134.22 U/mg) 1.93 times higher than that of κ-ZL-4, and its thermal and pH stability also increased. The best activity of κ-ZL-4-GH16 was presented at pH 3.0-6.0, which was lower than the optimal pH of reported κ-carrageenases. The enzyme-substrate affinity of κ-ZL-4-GH16 was higher than that of κ-ZL-4, demonstrated by its lower Michaelis-Menten constant (0.704 mg/mL at pH 6.0). Importantly, κ-ZL-4-GH16 released 10-fold more κ-carrageenan disaccharides than κ-ZL-4. The κ-carrageenan tetrose and hexose produced by the two enzymes were purified and structurally identified. Molecular docking with κ-carrageenan hexose suggested that the efficiency improvement after truncation might be attributed to the conformation differences of the two enzymes.

Effects of Dietary FOS and CPP on growth performance and serum biochemical parameters for weaned piglets

The present experiment was conducted to study the effects of different levels of oligosaccharide(FOS) and complex CPP preparation(CPP) in diets on growth performance and serum biochemical parameters for weaned piglets. 240 crossbred DLY( Duroc× Landrace× Yorkshire) 21-day-old weaning piglets, about 7.2 kg in initial body weight were selected and randomly divided into 6 groups, with 2 repeats for each group, 20 each repeat(half male and half female.). Group 1, as the control group, was fed base diet(BD), group 2 was fed BD adding 0.10% aureomycin, group 3, 4, 5, 6, respectively, was fed BD adding 0.05% CPP, 0.05% FOS, 0.20% FOS and 0.10% FOS + 0.05% of CPP. Feeding trial was conducted for 35 days. Results: (1) The combination of 0.10%FOS and 0.05%CPP(group 6) were added, the average daily gain of weaned pigs increased by 13.71 % (P < 0.05) and the feed conversion ratio was reduced by 7.41 % (P < 0.05) compared with control. The diarrhea rate of piglets decreased by 94.63 % (P < 0.01) compared with the control (p < 0.05), and decreased by 62.86 % (P <0.05) compared with group 2.(2)There was no significant difference in the apparent digestibility of nutrient, such as ether extract and crude protein, by adding FOS and CPP individually or jointly in diet of weaned piglets, but apparent digestibility of crude fiber increased , to acertain extent, and there was no significant difference with diet adding 0.10 % of antibiotics (group 2)(P >0.05).(3) There was no significant difference in serum biochemical indexes, such as serum total protein, albumin, urea nitrogen, cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase and sugar by adding FOS and CPP individually or jointly in diet of weaned piglets (P > 0.05);there was no significant effect in serum immunoglobulin IgG and IgM (P> 0.05), but the serum IgE content was increased in different degrees. Conclusion: Adding FOS and CPP in diet for weaned piglets can improve the growth performance, reduce diarrhea rate of piglets, and promote digestion and absorption of feed nutrients, without significant difference between them and diet adding antibiotics. Among them, the effect of adding 0.10%FOS+0.05%CPP was the best.

Application of FOS and CPP in intestinal health of Weaned Piglets

The present experiment was conducted to study the effects of different levels of oligosaccharide(FOS) and complex CPP preparation(CPP) in diets on growth performance and serum biochemical parameters for weaned piglets. 240 crossbred DLY( Duroc× Landrace× Yorkshire) 21-day-old weaning piglets, about 7.2 kg in initial body weight were selected and randomly divided into 6 groups, with 2 repeats for each group, 20 each repeat(half male and half female.). Group 1, as the control group, fed base diet(BD) , group 2 fed BD adding 0.10% aureomycin, group 3, 4, 5, 6, respectively, fed BD adding 0.05% CPP, 0.05% FOS, 0.20% FOS and 0.10% FOS + 0.05% of CPP. Feeding trial was conducted for 35 days.Results: (1) Intestinal pH: Compared with the control group, the pH values of jejunum, ileum, cecum and colon in the 3, 4, 5 and 6 groups were significantly lower (P < 0.05), while there was no significant difference between the antibiotic group and the control group (P > 0.05).(2) Small intestinal villus morphology: Duodenal segment, villus length of 6 groups was significantly increased compared with the control group (P < 0.05), villus height to recess depth ratio (V/C) was significantly increased (P < 0.05). There was no significant difference in villus length between groups in jejunal segment (P > 0.05). Groups five V/C was significantly higher than that in the control group (P < 0.05). Compared with the control group and the antibiotic group, the ileal segment in the six groups significantly increased V/C (P < 0.05).(3) Intestinal flora Adding FOS, CPP and their combination could reduce the number of E. coli and Salmonella in different intestinal segments, and increase the number of Lactobacillus and Bifidobacteria. Among them, the effect of joint addition is the most obvious. Conclusion: Adding FOS and C PP to the diet of weaned piglets could effectively reduce the intestinal pH value of piglets, increase the length of intestinal villi and V/C, reduce the number of harmful bacteria and increase the number of beneficial bacteria. It is beneficial to maintain the balance of intestinal morphology and microflora and improve the intestinal health of piglets. Among them, the combination of 0.10% FOS and 0.05% CPP is the best, followed by 0.20% FOS.

Expression and Characterization of a New PolyG-Specific Alginate Lyase From Marine Bacterium Microbulbifer sp. Q7

Alginate lyases play an important role in preparation of alginate oligosaccharides. Although a large number of alginate lyases have been characterized, reports on directional preparation of alginate oligosaccharides by alginate lyases are still rather less. Here, a gene alyM encoding a new alginate lyase AlyM was cloned from Microbulbifer sp. Q7 and expressed in Escherichia coli. AlyM exhibited the maximumactivity at pH 7.0 and 55°C and showed special preference to poly-guluronic acid (polyG). Glycine promoted the extracellular secretion of AlyM by 3.6 times. PBS and glycerol significantly improved the thermal stability of AlyM, the enzyme activity remained 75 and 78% after heat-treatment at 45°C for 2 h, respectively. ESI-MS analysis suggested that AlyM mainly produced oligosaccharides with degrees of polymerization (DP) of 2–5. The results of 1H-NMR showed that guluronic acid (G) occupied the reducing end of the end products, indicating that AlyM preferred to degrade the glycosidic bond at the G-X linkage. HPLC analysis showed that the hydrolysis products with a lower degree of polymerization contained more G. Therefore, AlyM shows good potential to produce alginate oligosaccharides with specific M/G ratio and molecular weights.

Characterization of a thermostable κ-carrageenase from a hot spring bacterium and plant protection activity of the oligosaccharide enzymolysis product.

Seaweed oligosaccharides are environmentally-friendly natural products and their use for disease control in sustainable agriculture is extremely promising. Enzymatic digestion to prepare seaweed oligosaccharides has drawn considerable interest. However, the study of enzymatically degraded products of carrageenan is still in its infancy compared with that of other hydrocolloids such as agar and alginate. To prepare degraded carrageenan on a commercial scale, it is necessary to select superior producer bacterial strains to improve the yield and thermostability of carrageenases. The carrageenan-degrading bacterium Bacillus sp. HT19 was isolated from sediment of a hot spring in Indonesia, and a κ-carrageenase with high activity was purified from the culture supernatant. The purified enzyme, named Car19, had maximum activity (538 U mg-1 ) at 60 °C and pH 7.0. Notably, the enzyme retained >90% of its initial activity after incubation at 60 °C for 24 h. The Ca2+ obviously improved the thermostability of Car19 at 70 °C. The Km and Vmax values of purified Car19 were 0.061 mg mL-1 and 115.13 U mg-1 , respectively, with κ-carrageenan as substrate. Thin-layer chromatography and electrospray ionization mass-spectrometry analysis of hydrolysates indicated that the enzyme exolytically depolymerized κ-carrageenan to neo-carrabiose. The hydrolysate enhanced the resistance of cucumber to cucumber mosaic virus and increased the activity of antioxidant enzymes in infected plants. To our knowledge, Car19 is the most thermostable κ-carrageenase reported so far. Its high optimal reaction temperature and thermostability, and unitary hydrolysate constituent, makes Car19 a promising candidate for the preparation of carrageenan oligosaccharides with plant protection activity. © 2018 Society of Chemical Industry.

Front Cover: Preparation and identification of oligosaccharides in lotus seeds and determination of their distribution in different parts of lotus

Front Cover: Preparation and identification of oligosaccharides in lotus seeds and determination of their distribution in different parts of lotus

Synthesis of high-mannose oligosaccharides containing mannose-6-phosphate residues using regioselective glycosylation

Molecular recognition of mannose-6-phosphate (M6P)-modified oligosaccharides by transmembrane M6P receptors is a key signaling event in lysosomal protein trafficking in vivo. Access to M6P-containing high-mannose N-glycans is essential to achieving a thorough understanding of the M6P ligand–receptor recognition process. Herein we report the application of a versatile and reliable chemical strategy to prepare asymmetric di-antennary M6P-tagged high-mannose oligosaccharides in >20% overall yield and in high purity (>98%). Regioselective chemical glycosylation coupled with effective phosphorylation and product purification protocols were applied to rapidly assemble these oligosaccharides. The development of this synthetic strategy simplifies the preparation of M6P-tagged high-mannose oligosaccharides, which will improve access to these compounds to study their structures and biological functions.

The alginate lyase from Isoptericola halotolerans CGMCC 5336 as a new tool for the production of alginate oligosaccharides with guluronic acid as reducing end

Alginate oligosaccharides are depolymerization products of alginate by enzymatic degradation or physicochemical treatments. Alginate lyase has received great interests due to its use in oligosaccharide preparation. The substrate specificity of alginate lyase directly determines the type and degree of polymerization of alginate oligosaccharides. In this paper, the degradation products of Alginate lyase from Isoptericola halotolerans CGMCC 5336 were size-fractionated by gel filtration chromatography and the fractions were characterized by TLC, HPLC, MS and 1H NMR spectroscopy. The Mw values for these fractions were 352, 528, 704 Da which represented di-, tri-, and tetrasaccharide fragments, respectively. The 1H NMR revealed that the non-reducing ends of these oligosaccharides were 4-deoxy-L-erythro-hex-4-enepyranosyluronate and the reducing ends of these oligosaccharides were all guluronic acid. Therefore, it is believed that the alginate lyase produced by Isoptericola halotolerans CGMCC 5336 was capable of performing β elimination on guluronic acid residue so that low molecular weight oligosaccharides with guluronic acid on the reducing end could be obtained in this way.

Preparation and identification of oligosaccharides in lotus seeds and determination of their distribution in different parts of lotus.

Three fractions (I-III) were separated from crude oligosaccharides of lotus seeds by fast protein liquid chromatography with final purity of 97.6, 96.3, and 96.8%, respectively. The fractions were identified as sucrose, raffinose, and stachyose by using TLC, HPLC with charged aerosol detector (CAD), LC-MS, and methylation analysis. Subsequently sucrose and raffinose family oligosaccharides (RFOs) with degree of polymerization (DP) 3-5 (raffinose, stachyose, and verbascose) have been quantified by HPLC-CAD for the first time. All calibration curves for investigated analytes showed good linear regression (R2 >0.9952). Their limit of detection and limit of quantity were in the ranges 0.14-0.28 and 0.36-0.48μg/mL, respectively. The recoveries ranged from 96.6 to 103.4%. The contents of sucrose and RFOs DP3-DP5 were different in lotus seeds and other parts of lotus samples, but similar in their own variety. Additionally, the distribution of RFOs in different parts of lotus were also compared and the results indicated that RFOs might be mainly synthesized in lotus seeds. This work is helpful for understanding the way of biosynthesis of RFOs in lotus as well as quality control of plants containing RFOs.

Purification, characterization of Chondroitinase ABC from Sphingomonas paucimobilis and in vitro cardiocytoprotection of the enzymatically degraded CS-A

An extracellular chondroitinase ABC (ChSase ABC) produced by Sphingomonas paucimobilis was purified to homogeneity through ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephadex G-100 chromatography. The molecular weight was 82.3 kDa. It showed specific lyase activity toward chondroitin sulfate A (CS-A), CS-B, CS-C and hyaluronan (HA). Using CS-A as substrate, the specific activity was 98.04 U/mg, the maximal reaction rate (Vmax) and Michaelis-Menten constant (Km) were 0.49 μmol/min/ml and 0.79 mg/ml, respectively. Highest activity was obtained at pH 6.5 and 40 °C, and Hg2+ could strongly inhibit the enzyme activity. Mass spectrometry analysis indicated CS-A was degraded to unsaturated disaccharides by ChSase ABC. In vitro cytotoxic tests showed that CS-A oligosaccharide at the concentration of 50 and 100 μg/ml could promote the proliferation of normal H9c2 myocardial cells, decrease the damage induced by isoproterenol (ISO) and accelerate the recovery of cells injured by ISO. These findings suggested that ChSase ABC from Sphingomonas paucimobilis could be a promising tool for the structural analysis and bioactive oligosaccharide preparation of glucosaminoglycans.

Preparation of Oligosaccharides by Degradation of Polysaccharides from Chinese Jujube and Its Biological Activity

This study examined the degradation of polysaccharides to oligosaccharides in Chinese jujube fruits. Using a response surface model, the degradation conditions of polysaccharides under acid hydrolysis and enzymatic hydrolysis were optimized in laboratory conditions. A degradation rate of 66.9% was obtained under optimum acid hydrolysis conditions: 0.6 mol/L hydrochloric acid, 3% substrate concentration, and 1 h reaction time. A degradation rate of 41.4% was obtained under optimum enzyme hydrolysis conditions: 4.0 mL cellulose solution (10 mg/mL), 0.3 mL substrate solution (20 mg/mL), 0.7 mL citric acid buffer solution (pH 5), and 7.3 h reaction time. Using the stimulation effect for strain J-4 intestinal probiotic proliferation, the biological activity of oligosaccharides was determined. The results showed that the oligosaccharides from enzyme hydrolysis encouraged intestinal probiotic proliferation.

Biochemical Characterization and Degradation Pattern of a Unique pH-Stable PolyM-Specific Alginate Lyase from Newly Isolated Serratia marcescens NJ-07.

Enzymatic preparation of alginate oligosaccharides with versatile bioactivities by alginate lyases has attracted increasing attention due to its featured characteristics, such as wild condition and specific products. In this study, AlgNJ-07, a novel polyM-specific alginate lyase with high specific activity and pH stability, has been purified from the newly isolated marine bacterium Serratia marcescens NJ-07. It has a molecular weight of approximately 25 kDa and exhibits the maximal activity of 2742.5 U/mg towards sodium alginate under 40 °C at pH 9.0. Additionally, AlgNJ-07 could retain more than 95% of its activity at pH range of 8.0–10.0, indicating it possesses excellent pH-stability. Moreover, it shows high activity and affinity towards polyM block and no activity to polyG block, which suggests that it is a strict polyM-specific alginate lyase. The degradation pattern of AlgNJ-07 has also been explored. The activity of AlgNJ-07 could be activated by NaCl with a low concentration (100–300 mM). It can be observed that AlgNJ-07 can recognize the trisaccharide as the minimal substrate and hydrolyze the trisaccharide into monosaccharide and disaccharide. The TLC and ESI-MS analysis indicate that it can hydrolyze substrates in a unique endolytic manner, producing not only oligosaccharides with Dp of 2–5 but also a large fraction of monosaccharide. Therefore, it may be a potent tool to produce alginate oligosaccharides with lower Dps (degree of polymerization).