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.

Structural and thermodynamic characterization of endo-1,3-ß-glucanase: Insights into the substrate recognition mechanism.

Endo-1,3-ß-glucanase from Cellulosimicrobium cellulans is composed of a catalytic domain and a carbohydrate-binding module. We have determined the X-ray crystal structure of the catalytic domain at a high resolution of 1.66Å. The overall fold is a sandwich-like ß-jelly roll architecture like the enzymes in the glycoside hydrolase family 16. The substrate-binding cleft has a length and a width of ~28 and ~15Å, respectively, which is thought to be capable of accommodating at least six glucopyranose units. Laminarihexaose was placed into the substrate-binding cleft, namely at the subsites +2 to -4 from the reducing end, and the complex structure was analyzed using molecular dynamics simulations (MD) and using a rotamer search of the pocket. During the MD simulations, the substrate fluctuated more than the enzyme, where the residues at the subsites toward the non-reducing end fluctuated more than those toward the reducing end. Little conformational change of the protein was observed for the subsites +1 and +2, indicating that the glucose's position could be tightly restricted inside the pocket. Substrate binding experiments using isothermal titration calorimetry showed that the binding affinity of laminaritriose was higher than that of laminaribiose and similar to those of other longer laminarioligosaccharides. Taken together, the substrates mainly bind to the subsites -1 to -3 with the highest affinity, while the part bound to the reducing end would be hydrolyzed.

C-Terminal sequence is involved in the incorporation of Bgl2p glucanosyltransglycosylase in the cell wall of Saccharomyces cerevisiae.

A cell wall (CW) provides a protective barrier for a yeast cell and is a firm structure that nevertheless dynamically changes during cell's growth. Bgl2p is a non-covalently anchored glucanosyltransglycosylase in the CW of the yeast Saccharomyces cerevisiae. The mode of its anchorage is poorly understood, while its association with CW components is tight and resistant to 1-h treatment with 1% SDS at 37°C. In order to demarcate the potential structural block responsible for incorporation of Bgl2p into the CW, bioinformatics analysis of its sequence was performed, and a conservative structural region was identified in the C-terminal region of Bgl2p, which was absent in its homologues in S. cerevisiae, the Scw4p and Scw10p. Deletion of this region disrupted the incorporation of Bgl2p into the CW and led to release of this protein through the CW into the culture medium. Two left-handed polyproline-II helices were identified in the C-terminal region of the structure model of a wild-type Bgl2p. These helices potentially formed binding sites, which were absent in the truncated protein. Using immune fluorescence microscopy, we demonstrated that C-truncated Bgl2p was exported into culture medium and lost its ability to form fibrils described earlier. It was also shown that the C-terminal truncation of Bgl2p led to a more severe decrease of cell survivability in extreme conditions than BGL2 deletion.

Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p-glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils.

We performed a comparative study of the process of amyloid formation by short homologous peptides with a substitution of aspartate for glutamate in position 2 - VDSWNVLVAG (AspNB) and VESWNVLVAG (GluNB) - with unblocked termini. Peptide AspNB (residues 166-175) corresponded to the predicted amyloidogenic region of the protein glucantransferase Bgl2 from the Saccharomyces cerevisiae cell wall. The process of amyloid formation was monitored by fluorescence spectroscopy (FS), electron microscopy (EM), tandem mass spectrometry (TMS), and X-ray diffraction (XD) methods. The experimental study at pH3.0 revealed formation of amyloid fibrils with similar morphology for both peptides. Moreover, we found that the morphology of fibrils made of untreated ammonia peptide is not mentioned in the literature. This morphology resembles snakes lying side by side in the form of a wave without intertwining. Irrespective of the way of the peptide preparation, the rate of fibril formation is higher for AspNB than for GluNB. However, preliminary treatment with ammonia highly affected fibril morphology especially for AspNB. Such treatment allowed us to obtain a lag period during the process of amyloid formation. It showed that the process was nucleation-dependent. With or without treatment, amyloid fibrils consisted of ring-like oligomers with the diameter of about 6nm packed either directly ring-to-ring or ring-on-ring with a slight shift. We also proposed the molecular structure of amyloid fibrils for two studied peptides.

Tryptophan introduction can change ß-glucan binding ability of the carbohydrate-binding module of endo-1,3-ß-glucanase.

Endo-1,3-ß-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, ß-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to ß-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in ß- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in ß- or γ-repeat can substitute the α-repeat by primarily contributing to ß-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.

The role of Bgl2p in the transition to filamentous cells during biofilm formation by Candida albicans.

The fungal pathogen Candida albicans undergoes a transition from yeast cells to filamentous cells that is related to its pathogenicity. The complex multicellular processes involved in biofilm formation by this fungus also include this transition. In this work, we investigated the morphological role of the Bgl2 protein (Bgl2p) in the transition to filamentous cells during biofilm formation by C. albicans. Bgl2p has been identified as a ß-1, 3-glucosyltransferase, and transcription of the CaBGL2 gene is upregulated during biofilm formation. We used scanning electron microscopy to observe the microstructure of a bgl2 null mutant during biofilm formation and found a delay in the transition to filamentous cells in the premature phase (24 hours) of biofilm formation. Deletion of the CaBGL2 gene led to a decrease in the expression of CPH2 and TEC1, which encode transcription factors required for the transition to the filamentous form. These findings indicate that Bgl2p plays a role in the transition to filamentous cells during biofilm formation by C. albicans.

Modulating the function of a ß-1,3-glucanosyltransferase to that of an endo-ß-1,3-glucanase by structure-based protein engineering.

A glycoside hydrolase (GH) family 17 ß-1,3-glucanosyltransferase (RmBgt17A) from Rhizomucor miehei CAU432 (CGMCC No. 4967) shared very low sequence homology (∼20 % identity) with that of other ß-1,3-glucanases,despite their similar structural folds. Structural comparison and sequence alignment between RmBgt17A and GH family 17 ß-1,3-glucanases suggested important roles for three residues (Tyr102, Trp157, and Glu158) located in the substrate-binding cleft of RmBgt17A in transglycosylation activity. A series of site-directed mutagenesis studies indicated that a single Glu-to-Ala mutation (E158A) modulates the function of RmBgt17A to that of a ß-1,3-glucanase. Mutant E158A exhibited high hydrolytic activity (39.95 U/mg) toward reduced laminarin, 348.5-fold higher than the wild type. Optimal pH and temperature of the purified RmBgt17A-E158A were 4.5 and 55 °C, respectively. TLC analysis suggested that RmBgt17A-E158A is an endo-ß-1,3-glucanase. Our study provides novel insight into protein engineering of the substrate-binding cleft of glycoside hydrolases to modulate the function of transglycosylation and hydrolysis.

The first crystal structure of a glycoside hydrolase family 17 ß-1,3-glucanosyltransferase displays a unique catalytic cleft.

ß-1,3-Glucanosyltransferase (EC 2.4.1.-) plays an important role in the formation of branched glucans, as well as in cell-wall assembly and rearrangement in fungi and yeasts. The crystal structures of a novel glycoside hydrolase (GH) family 17 ß-1,3-glucanosyltransferase from Rhizomucor miehei (RmBgt17A) and the complexes of its active-site mutant (E189A) with two substrates were solved at resolutions of 1.30, 2.30 and 2.27 Å, respectively. The overall structure of RmBgt17A had the characteristic (ß/α)8 TIM-barrel fold. The structures of RmBgt17A and other GH family 17 members were compared: it was found that a conserved subdomain located in the region near helix α6 and part of the catalytic cleft in other GH family 17 members was absent in RmBgt17A. Instead, four amino-acid residues exposed to the surface of the enzyme (Tyr135, Tyr136, Glu158 and His172) were found in the reducing terminus of subsite +2 of RmBgt17A, hindering access to the catalytic cleft. This distinct region of RmBgt17A makes its catalytic cleft shorter than those of other reported GH family 17 enzymes. The complex structures also illustrated that RmBgt17A can only provide subsites -3 to +2. This structural evidence provides a clear explanation of the catalytic mode of RmBgt17A, in which laminaribiose is released from the reducing end of linear ß-1,3-glucan and the remaining glucan is transferred to the end of another ß-1,3-glucan acceptor. The first crystal structure of a GH family 17 ß-1,3-glucanosyltransferase may be useful in studies of the catalytic mechanism of GH family 17 proteins, and provides a basis for further enzymatic engineering or antifungal drug screening.

Using dielectrophoresis to study the dynamic response of single budding yeast cells to Lyticase.

Budding yeast cells are quick and easy to grow and represent a versatile model of eukaryotic cells for a variety of cellular studies, largely because their genome has been widely studied and links can be drawn with higher eukaryotes. Therefore, the efficient separation, immobilization, and conversion of budding yeasts into spheroplast or protoplast can provide valuable insight for many fundamentals investigations in cell biology at a single cell level. Dielectrophoresis, the induced motion of particles in non-uniform electric fields, possesses a great versatility for manipulation of cells in microfluidic platforms. Despite this, dielectrophoresis has been largely utilized for studying of non-budding yeast cells and has rarely been used for manipulation of budding cells. Here, we utilize dielectrophoresis for studying the dynamic response of budding cells to different concentrations of Lyticase. This involves separation of the budding yeasts from a background of non-budding cells and their subsequent immobilization onto the microelectrodes at desired densities down to single cell level. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts, in a highly dynamic process that depends on the concentration of Lyticase. We also introduce a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes.

Bifunctional immobilization of a hyperthermostable endo-ß-1,3-glucanase.

Laminarinase A (LamA) from Pyrococcus furiosus is a hyperthermostable endo-ß-1,3-glucanase (EC 3.2.1.39) belonging to the glycosyl hydrolase family GH16. Here, we report the two-step immobilization of LamA on macroporous acrylic epoxy beads, extra-functionalized with disulfide groups. To facilitate initial immobilization via thiol-disulfide exchange, we introduced, by site-directed mutagenesis, a superficial cysteine residue near the protein C-terminal end. The thus-obtained S296C variant showed similar catalytic properties as native LamA. The activity of immobilized S296C displayed an inverse relationship with particle size. Use of conventional beads (150-300 µm in diameter) obstructed the catalytic efficiency due to pore diffusion limitation of the polysaccharide substrate. Bifunctional attachment to milled beads (20-40 µm) resulted in high enzyme load and outstanding catalytic features. Bifunctional immobilized S296C showed extreme pH stability and could be repeatedly used at 60 °C without significant activity loss.

The structure of a glycoside hydrolase family 81 endo-ß-1,3-glucanase.

Endo-ß-1,3-glucanases catalyze the hydrolysis of ß-1,3-glycosidic linkages in glucans. They are also responsible for rather diverse physiological functions such as carbon utilization, cell-wall organization and pathogen defence. Glycoside hydrolase (GH) family 81 mainly consists of ß-1,3-glucanases from fungi, higher plants and bacteria. A novel GH family 81 ß-1,3-glucanase gene (RmLam81A) from Rhizomucor miehei was expressed in Escherichia coli. Purified RmLam81A was crystallized and the structure was determined in two crystal forms (form I-free and form II-Se) at 2.3 and 2.0 Šresolution, respectively. Here, the crystal structure of a member of GH family 81 is reported for the first time. The structure of RmLam81A is greatly different from all endo-ß-1,3-glucanase structures available in the Protein Data Bank. The overall structure of the RmLam81A monomer consists of an N-terminal ß-sandwich domain, a C-terminal (α/α)6 domain and an additional domain between them. Glu553 and Glu557 are proposed to serve as the proton donor and basic catalyst, respectively, in a single-displacement mechanism. In addition, Tyr386, Tyr482 and Ser554 possibly contribute to both the position or the ionization state of the basic catalyst Glu557. The first crystal structure of a GH family 81 member will be helpful in the study of the GH family 81 proteins and endo-ß-1,3-glucanases.

Glycosidases of marine organisms.

This review discusses the catalytic properties, activity regulation, structure, and functions of O-glycoside hydrolases from marine organisms exemplified by endo-1→3-ß-D-glucanases of marine invertebrates.

Presence of a large ß(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control.

Previous results suggested that the chitin ring present at the yeast mother-bud neck, which is linked specifically to the nonreducing ends of ß(1-3)glucan, may help to suppress cell wall growth at the neck by competing with ß(1-6)glucan and thereby with mannoproteins for their attachment to the same sites. Here we explored whether the linkage of chitin to ß(1-3)glucan may also prevent the remodeling of this polysaccharide that would be necessary for cell wall growth. By a novel mild procedure, ß(1-3)glucan was isolated from cell walls, solubilized by carboxymethylation, and fractionated by size exclusion chromatography, giving rise to a very high-molecular-weight peak and to highly polydisperse material. The latter material, soluble in alkali, may correspond to glucan being remodeled, whereas the large-size fraction would be the final cross-linked structural product. In fact, the ß(1-3)glucan of buds, where growth occurs, is solubilized by alkali. A gas1 mutant with an expected defect in glucan elongation showed a large increase in the polydisperse fraction. By a procedure involving sodium hydroxide treatment, carboxymethylation, fractionation by affinity chromatography on wheat germ agglutinin-agarose, and fractionation by size chromatography on Sephacryl columns, it was shown that the ß(1-3)glucan attached to chitin consists mostly of high-molecular-weight material. Therefore, it appears that linkage to chitin results in a polysaccharide that cannot be further remodeled and does not contribute to growth at the neck. In the course of these experiments, the new finding was made that part of the chitin forms a noncovalent complex with ß(1-3)glucan.

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.

Two high-resolution structures of potato endo-1,3-ß-glucanase reveal subdomain flexibility with implications for substrate binding.

Endo-1,3-ß-glucanases are widely distributed among bacteria, fungi and higher plants. They are responsible for hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted ß-1,3-linked glucosyl residues. The plant enzymes belong to glycoside hydrolase family 17 (GH17) and are also members of class 2 of pathogenesis-related (PR) proteins. X-ray diffraction data were collected to 1.40 and 1.26 Å resolution from two crystals of endo-1,3-ß-glucanase from Solanum tuberosum (potato, cultivar Désirée) which, despite having a similar packing framework, represented two separate crystal forms. In particular, they differed in the Matthews coefficient and are consequently referred to as higher density (HD; 1.40 Å resolution) and lower density (LD; 1.26 Å resolution) forms. The general fold of the protein resembles that of other known plant endo-1,3-ß-glucanases and is defined by a (ß/α)(8)-barrel with an additional subdomain built around the C-terminal half of the barrel. The structures revealed high flexibility of the subdomain, which forms part of the catalytic cleft. Comparison with structures of other GH17 endo-1,3-ß-glucanases revealed differences in the arrangement of the secondary-structure elements in this region, which can be correlated with sequence variability and may suggest distinct substrate-binding patterns. The crystal structures revealed an unusual packing mode, clearly visible in the LD structure, caused by the presence of the C-terminal His(6) tag, which extends from the compact fold of the enzyme molecule and docks in the catalytic cleft of a neighbouring molecule. In this way, an infinite chain of His-tag-linked protein molecules is formed along the c direction.

Critical roles of Asp270 and Trp273 in the α-repeat of the carbohydrate-binding module of endo-1,3-ß-glucanase for laminarin-binding avidity.

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with ß-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10(6) M(-1) and 10(4) M(-1), respectively, indicating that 2 or 3 binding sites in the α-, ß-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to ß-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to ß-glucan binding. The α-repeat bound ß-glucan with an affinity of approximately 10(4) M(-1), and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.

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.

Hydrolytic Enzymes as Potentiators of Antimicrobials against an Inter-Kingdom Biofilm Model.

Biofilms are recalcitrant to antimicrobials, partly due to the barrier effect of their matrix. The use of hydrolytic enzymes capable to degrade matrix constituents has been proposed as an alternative strategy against biofilm-related infections. This study aimed to determine whether hydrolytic enzymes could potentiate the activity of antimicrobials against hard-to-treat interkingdom biofilms comprising two bacteria and one fungus. We studied the activity of a series of enzymes alone or in combination, followed or not by antimicrobial treatment, against single-, dual- or three-species biofilms of Staphylococcus aureus, Escherichia coli, and Candida albicans, by measuring their residual biomass or culturable cells. Two hydrolytic enzymes, subtilisin A and lyticase, were identified as the most effective to reduce the biomass of C. albicans biofilm. When targeting interkingdom biofilms, subtilisin A alone was the most effective enzyme to reduce biomass of all biofilms, followed by lyticase combined with an enzymatic cocktail composed of cellulase, denarase, and dispersin B that proved previously active against bacterial biofilms. The subsequent incubation with antimicrobials further reduced the biomass. Enzymes alone did not reduce culturable cells in most cases and did not interfere with the cidal effects of antimicrobials. Therefore, this work highlights the potential interest of pre-exposing interkingdom biofilms to hydrolytic enzymes to reduce their biomass besides the number of culturable cells, which was not achieved when using antimicrobials alone. IMPORTANCE Biofilms are recalcitrant to antimicrobial treatments. This problem is even more critical when dealing with polymicrobial, interkingdom biofilms, including both bacteria and fungi, as these microorganisms cooperate to strengthen the biofilm and produce a complex matrix. Here, we demonstrate that the protease subtilisin A used alone, or a cocktail containing lyticase, cellulase, denarase, and dispersin B markedly reduce the biomass of interkingdom biofilms and cooperate with antimicrobials to act upon these recalcitrant forms of infection. This work may open perspectives for the development of novel adjuvant therapies against biofilm-related infections.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-ß-1,3-glucanase from Thermotoga petrophila.

1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal ß-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

Characterization of the GPI-anchored endo ß-1,3-glucanase Eng2 of Aspergillus fumigatus.

A GPI-anchored endo ß-1,3-glucanase of Aspergillus fumigatus was characterized. The enzyme encoded by ENG2 (AFUA_2g14360) belongs to the glycoside hydrolase family 16 (GH16). The activity was characterized using a recombinant protein produced by Pichiapastoris. The recombinant enzyme preferentially acts on soluble ß-1,3-glucans. Enzymatic analysis of the endoglucanase activity using Carboxymethyl-Curdlan-Remazol Brilliant Blue (CM-Curdlan-RBB) as a substrate revealed a wide temperature optimum of 24-40°C, a pH optimum of 5.0-6.5 and a K(m) of 0.8 mg ml(-1). HPAEC analysis of the products formed by Eng2 when acting on different oligo-ß-1,3-glucans confirmed the predicted endoglucanase activity and also revealed a transferase activity for oligosaccharides of a low degree of polymerization. The growth phenotype of the Afeng2 mutant was identical to that of the wt strain.