Biochemical Characterization of a Novel Endo-1,3-ß-Glucanase from the Scallop Chlamys farreri.

Endo-1,3-ß-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-ß-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-ß-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-ß-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-ß-glucanases, which facilitated development of a unique endo-1,3-ß-glucanase tool in the synthesis of novel glycosides.

Functional characterisation and product specificity of Endo-ß-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1.

Endo-ß-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast ß-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.

Identification and enzymatic characterization of an endo-1,3-ß-glucanase from Euglena gracilis.

Euglena produces paramylon as a storage polysaccharide, and is thought to require ß-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate ß-1,3-glucan degradation in Euglena, endo-1,3-ß-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by SDS-PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards ß-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0-5.5 and 60°C after 1h incubation or 50°C after 20h incubation. The enzyme had a Km of 0.21mg/ml and a Vmax of 40.5units/mg protein for laminarin degradation at pH 5.0 and 50°C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble ß-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-ß-glucanase to be identified from E. gracilis.

Catalytic properties and amino acid sequence of endo-1→3-ß-D-glucanase from the marine mollusk Tapes literata.

A specific 1→3-ß-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45°C. The 1→3-ß-D-glucanase catalyzes hydrolysis of ß-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K(m) for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-ß-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-ß-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-ß-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.

Molecular characterization of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans: effects of carbohydrate-binding module on enzymatic function and stability.

An endo-1,3-ß-glucanase was purified from Tunicase®, a crude enzyme preparation from Cellulosimicrobium cellulans DK-1, and determined to be a 383-residue protein (Ala1-Leu383), comprising a catalytic domain of the glycoside hydrolase family 16 and a C-terminal carbohydrate-binding module family 13. The Escherichia coli expression system of the catalytic domain (Ala1-Thr256) was constructed, and the protein with N-terminal polyhistidine tag was purified using a Ni-nitrilotriacetic acid column. We analyzed enzymatic properties of the recombinant catalytic domain, its variants, and the Tunicase®-derived full-length endo-1,3-ß-glucanase. Substitution of Glu119 with Ala and deletion of Met123, both of the residues are located in the catalytic motif, resulted in the loss of hydrolytic activity. In comparison between the full-length enzyme and isolated catalytic domain, their hydrolytic activities for soluble substrates such as laminarin and laminarioligosaccharides were similar. In contrast, the hydrolytic activity of the full-length enzyme for insoluble substrates such as curdlan and yeast-glucan was significantly higher than that of the catalytic domain. It should be noted that the acid stabilities for the hydrolysis of laminarin were clearly different. Secondary structure analysis using circular dichroism showed that the full-length enzyme was more acid stable than was the catalytic domain, possibly because of domain interactions between the catalytic domain and the carbohydrate-binding module.

Production, purification and characterization of a thermostable ß-1,3-glucanase produced by Moniliophthora perniciosa.

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L(-1) and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

Characterization of a new beta(1-3)-glucan branching activity of Aspergillus fumigatus.

A new HPLC method was developed to separate linear from beta(1-6)-branched beta(1-3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal beta(1-6)/beta(1-3)-glucan branching transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171-3180). Both enzymes release laminaribiose from the reducing end of a beta(1-3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked beta(1-3;1-6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a beta(1-3)-linked product with a beta(1-6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched beta(1-3)-glucans of the cell wall.

Co-purification of glucanase with acid trehalase-invertase aggregate in Saccharomyces cerevisiae.

An electrophoretically homogenous aggregate of acid trehalase, invertase and an unidentified 37-41 kDa protein was purified from Saccharomyces cerevisiae. N-terminal analysis of the protein revealed an amino acid sequence identical to that of Bgl2p (endo-beta-l,3-glucanase) of S. cerevisiae. Acid trehalase activity with co-eluted glucanase activity was observed from late growth phase through early stationary phase. Pools with high percentage of Bgl2p corresponded with high acid trehalase activity. A BGL2 deletion strain had lower acid trehalase activity. The 37-41 kDa protein represents Bgl2p which, besides imparting glucanase activity, could also be acting as a regulator for the acid trehalase activity by association in the enzyme aggregate.

Isolation of Aspergillus lentulus in Spain from a critically ill patient with chronic obstructive pulmonary disease

Aspergillus lentulus es un hongo de reciente descripción (año 2005) ypracticamente idéntico a Aspergillus fumigatus; posibles aislamientos deA. lentulus anteriores a 2005 eran identificados como A. fumigatus.Actualmente se han publicado muy pocos casos de infecciones invasorascausadas por A. lentulus, todos ellos con evolución fatal, en pacientesneutropénicos. Comunicamos el caso de un paciente crítico no neutropénicocon enfermedad pulmonar obstructiva crónica (EPOC) admitido en la unidadde cuidados intensivos médicos con una exacerbación de EPOC y quehabía sido sometido a un tratamiento prolongado con corticosteroides. Se aisló A. fumigatus en dos aspirados bronquiales, y A. lentulus y A. fumigatusde un tercero. En dos días consecutivos la detección de galactomanano ensuero fue negativa, mientras que la detección de (1-3) beta-D glucano fuepositiva (>518 pg/ml). Los valores de concentración mínima inhibitoria (CMI)obtenidos con el itraconazol, el voriconazol, la caspofungina y la anfotericinaB fueron altos para A. lentulus. Dado lo elevado de estos valores enA. lentulus, es necesaria la identificación precisa de esta nueva especie enaislamientos clínicos. A nuestro entender, este es el primer aislamiento de A.lentulus en un paciente crítico no-neutropénico, aunque como su aislamientose realizó una sola vez de secreciones respiratorias (dada la dificultad de laobtención de biopsias en este enfermo por su situación comprometida), suimplicación como agente etiológico de infección es dudosa en este enfermo(AU)

Aspergillus lentulus was first described in the year 2005, and since it cannotbe phenotypically distinguished from Aspergillus fumigatus, it is conceivablethat earlier descriptions (before 2005) could be attributed to this new species.Currently invasive infections caused by A. lentulus are rare and very few caseshave been previously published in neutropenic patients, all of them with fataloutcome. Here we report a critically ill non neutropenic patient with chronicobstructive pulmonary disease (COPD) who was admitted to the medicalintensive care unit with an exacerbation of COPD and who had been treatedwith long term corticosteroids. A. fumigatus was cultured from two bronchialaspirates and in a third bronchial aspirate both A. lentulus and A. fumigatuswere isolated. On two consecutive days detection of galactomannan in serumwas negative whilst detection of (1-3) beta-D glucan was positive(> 518 pg/ml). Minimal inhibitory concentrations (MIC) for itraconazole,voriconazole, caspofungin and amphotericin B were high for this strain ofA. lentulus. Given the high MIC values of A. lentulus to available antifungals,the accurate identification of this new species is warranted. To our knowledge,this is the first report of the isolation of A. lentulus in a non-neutropeniccritically ill patient, although we note that since it was isolated only once fromrespiratory specimens, its implication as an etiologic agent of infection for thispatient remains to be established(AU)

Crystallization and preliminary crystallographic analysis of endo-1,3-beta-glucanase from alkaliphilic Nocardiopsis sp. strain F96.

Endo-1,3-beta-glucanase, an enzyme that hydrolyzes the 1,3-beta-glycosyl linkages of beta-glucan, belongs to the family 16 glycosyl hydrolases, which are widely distributed among bacteria, fungi and higher plants. Crystals of a family 16 endo-1,3-beta-glucanase from the alkaliphilic Nocardiopsis sp. strain F96 were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 34.59, b = 71.84, c = 39.67 A, beta = 90.21 degrees, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and solvent content were 1.8 A3 Da(-1) and 31.8%, respectively. Diffraction data were collected to a resolution of 1.3 A and gave a data set with an overall Rmerge of 6.4% and a completeness of 99.3%.

Purification and characterization of laminaran hydrolases from Trichoderma viride.

At least three extracellular laminaran hydrolases which hydrolyzed laminaran (beta-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a beta-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for beta-1,3-linkages, but lam B was specific for beta-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7.

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase.

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).

Enzymatic properties and intracellular localization of the novel Trichoderma reesei beta-glucosidase BGLII (cel1A).

This paper describes the characterization of an intracellular beta-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific beta-glucosidase, having no beta-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this beta-glucosidase.

beta-1,3-Glucan binding by a thermostable carbohydrate-binding module from Thermotoga maritima.

The C-terminal 155 amino acids of the putative laminarinase, Lam16A, from T. maritima comprise a highly thermostable family 4 CBM that binds beta-1,3- and beta-(1,3)(1,4)-glucans. Laminarin, a beta-1,3-glucan, presented two classes of binding sites for TmCBM4-2, one with a very high affinity (3.5 x 10(7) M(-1)) and one with a 100-fold lower affinity (2.4 x 10(5) M(-1)). The affinities for laminarioligosaccharides and beta-(1,3)(1,4)-glucans ranged from approximately 2 x 10(5) to approximately 2.5 x 10(6) M(-1). Cellooligosaccharides and laminariobiose were bound only very weakly (K(a)s approximately 5 x 10(3) M(-1)). Spectroscopic and mutagenic studies implicated the involvement of three tryptophan residues (W28, W58, and W99) and one tyrosine residue (Y23) in ligand binding. Binding was enthalpically driven and associated with large negative changes in heat capacity. Temperature and osmotic conditions profoundly influenced binding. For the first time in solution, the direct uptake and release of water in CBM binding are demonstrated.

Polysaccharide hydrolases from leaves of Boscia senegalensis: properties of endo-(1-->3)-beta-D-glucanase.

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of alpha-amylase, beta-amylase, exo-(1-->3, 1-->4)-beta-D-glucanase, and endo-(1-->3)-beta-D-glucanase were detected in these leaves. The endo-(1-->3)-beta-D-glucanase (EC 3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI > or = 10.3. Its optimal activity occurred at pH 4.5 and 50 degrees C. Kinetic analysis gave Vmax, kcat, and Km values of 659 U/mg, 395 s(-1), and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1-->3)-beta-glucan chains in an endo fashion. This property is unusual for endo-acting (1-->3)-beta-D-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells.

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.

News & notes: production, purification, and properties of an endo-1, 3-beta-glucanase from the basidiomycete Agaricus bisporus.

Agaricus bisporus H 25 produced extracellular endo-1, 3-beta-glucanase when grown in a static culture at 25 degrees C in a minimal synthetic medium supplemented with A. bisporus cell walls plus fructose. Endo-1,3-beta-glucanase was purified 17.85-fold from 20-day-old culture filtrates by precipitation at 80% ammonium sulfate saturation, Sephadex G-75 gel filtration, and preparative PAGE followed by electroelution. The purified enzyme yielded a single band in both native and SDS-polyacrylamide gels with a molecular mass of 32 kDa (SDS-PAGE) and 33.7 kDa (MALDI-MS), showing an isoelectric point of 3.7. The enzyme was active against beta-1,3- linkages and, to a lesser extent, against beta-1,6-, exhibiting an endohydrolytic mode of action and a glycoprotein nature. Significant activities of the endo-glucanase against laminarin and pustulan were observed between pH 4 and 5.5, and between 40 degrees and 50 degrees C for laminarin, and between 30 degrees and 50 degrees C for pustulan. The optimum pH and temperature were 4.5 and 45 degrees C for both substrates.

A 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p.

A 1,3-beta-glucanosyltransferase which introduces intrachain 1,6-beta linkages into 1,3-beta-glucan was isolated from the cell wall of Aspergillus fumigatus. The biochemical and molecular characterization of the A. fumigatus transferase showed it was homologous to the Saccharomyces cerevisiae and Candida albicans transferase Bgl2p. A null mutant constructed in A. fumigatus by gene replacement did not show a distinct phenotype from the parental strain. The putative function of this major cell-wall-associated protein is discussed.

Purification and determination of the action pattern of Haliotis tuberculata laminarinase.

The major laminarinase activity (EC 3.2.1.39) from the gastropodean marine mollusc Haliotis tuberculata was purified to homogeneity by cation exchange chromatography and its action pattern was investigated by HPAEC-PAD analysis of the degradation of various laminarin samples. It consists of a 60 kDa protein capable of depolymerizing the unbranched portions of the beta-(1-->3), beta-(1-->6)-glucan, down to laminaritriose. The enzyme operates via a molecular mechanism retaining the anomeric configuration. As the purified protein does not cleave the beta-(1-->6) linkages, it can be used for the structural analysis of laminarins.