Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

Heterologous expression of α-1,3-glucanase Agn1p from Schizosaccharomyces pombe, and efficient production of nigero-oligosaccharides by enzymatic hydrolysis from solubilized α-1,3;1,6-glucan.

The glycoside hydrolase family 71 α-1,3-glucanase (Agn1p) of Schizosaccharomyces pombe was expressed in Escherichia coli Rosetta-gami B (DE3). Agn1p (0.5 nmol/mL) hydrolyzed insoluble α-1,3-glucan (1%), and about 3.3 mm reducing sugars were released after 1440 min of reaction. The analysis of reaction products by high-performance liquid chromatography revealed that pentasaccharides accumulated in the reaction mixture as the main products, along with a small amount of mono-, di-, tri-, tetra-, and hexasaccharides. Soluble glucan was prepared from insoluble α-1,3;1,6-glucan by alkaline and sonication treatment to improve the hydrolytic efficiency. As a result, this solubilized α-1,3;1,6-glucan maintained a solubilized state for at least 6 h. Agn1p (0.5 nmol/mL) hydrolyzed the solubilized α-1,3;1,6-glucan (1%), and about 8.2 mm reducing sugars were released after 240 min of reaction. Moreover, Agn1p released about 12.3 mm reducing sugars from 2% of the solubilized α-1,3;1,6-glucan.

Design, synthesis, and evaluation of the self-assembled antimicrobial peptides based on the ovalbumin-derived peptide TK913.

The preparation, self-assembly, and antimicrobial activity of peptides based on TK913 is described. TK9Z4 incorporating a Pro-Pro motif exhibited self-assembly but no cytotoxicity. However, peptide TKZ3 (obtained by changing the amino acid sequence of TK9Z4) showed morphological changes at different concentrations, potent antimicrobial activity, low cytotoxicity, and trypsin resistance. Accordingly, TKZ3 is proposed as new AMP derived from ovalbumin-derived peptides.

The Hydrophobicity and Antifungal Potentiation of Burkholdine Analogues.

The burkholdines are a family of cyclic lipopeptides reported to exhibit antifungal activity. We synthesized a series of 18 burkholdine analogues in good yield by conventional Fmoc-SPPS followed by cyclization with DIPCI/HOBt in the solution phase. Although none of the synthesized peptides exhibited antifungal activity, several did potentiate the antibiotic effect of the antibiotic G418, including the Thr-bearing Bk analogue (4b) and the tartaramide-bearing Bk analogue (5b). This work exemplifies the potential of burkholdine analogues as potentiating agents.

Mixed Polyplex Micelles with Thermoresponsive and Lysine-Based Zwitterionic Shells Derived from Two Poly(vinyl amine)-Based Block Copolymers.

Two series of poly(vinyl amine) (PVAm)-based block copolymers with zwitterionic and thermoresponsive segments were synthesized by the reversible addition-fragmentation chain transfer polymerization. A mixture of the two copolymers, poly(N-acryloyl-l-lysine) (PALysOH) and poly(N-isopropylacrylamide) (PNIPAM), which have the same cationic PVAm chain but different shell-forming segments, were used to prepare mixed polyplex micelles with DNA. Both PVAm-b-PALysOH and PVAm-b-PNIPAM showed low cytotoxicity, with characteristic assembled structures and stimuli-responsive properties. The cationic PVAm segment in both block copolymers showed site-specific interactions with DNA, which were evaluated by dynamic light scattering, zeta potential, circular dichroism, agarose gel electrophoresis, atomic force microscopy, and transmission electron microscopy measurements. The PVAm-b-PNIPAM/DNA polyplexes showed the characteristic temperature-induced formation of assembled structures in which the polyplex size, surface charge, chiroptical property of DNA, and polymer-DNA binding were governed by the nitrogen/phosphate (N/P) ratio. The DNA binding strength and colloidal stability of the PVAm-b-PALysOH/DNA polyplexes could be tuned by introducing an appropriate amount of zwitterionic PALysOH functionality, while maintaining the polyplex size, surface charge, and chiroptical property, regardless of the N/P ratio. The mixed polyplex micelles showed temperature-induced stability originating from the hydrophobic (dehydrated) PNIPAM chains upon heating, and remarkable stability under salty conditions owing to the presence of the zwitterionic PALysOH chain on the polyplex surface.

Crystal structure of the catalytic unit of thermostable GH87 α-1,3-glucanase from Streptomyces thermodiastaticus strain HF3-3.

α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 Å resolution using X-ray crystallography. The catalytic unit is composed of two modules, a ß-sandwich fold module, and a right-handed ß-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the ß-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST.

Antimicrobial activity and secondary structure of a novel peptide derived from ovalbumin.

A novel antimicrobial peptide derived from ovalbumin has been discovered. First, the peptide fragment RKIKVYLPRMK (TK9.1) was identified based on computerized predictions of the secondary structure of peptides in a protein data bank. Using HeliQuest, the sequence was developed into RKIKRYLRRMI (TK9.1.3), which was synthesized using Fmoc-solid phase peptide synthesis, and found to have strongly antimicrobial activity against Gram-positive and Gram-negative bacteria, and fungi but not cytotoxic to HeLa cells and hemolysis in mouse red blood cells. Although ovalbumin itself does not have an antibacterial activity, our results suggest that it may supply the organisms that consume it with antimicrobial peptides, in support of their immunodefence.

Synthesis, Assembled Structures, and DNA Complexation of Thermoresponsive Lysine-Based Zwitterionic and Cationic Block Copolymers.

A series of anionic, zwitterionic, and cationic lysine-based block copolymers with a thermoresponsive segment were synthesized by the reversible addition-fragmentation chain transfer (RAFT) polymerization of N-acryloyl- N-carbobenzoxy-l-lysine [A-Lys(Cbz)-OH], which contains a carboxylic acid and a protected amine-functionality in the monomer unit. Carboxylic acid-containing homopolymers, poly(A-Lys(Cbz)-OH), with predetermined molecular weights with relatively low polydispersities were initially synthesized by RAFT polymerization of A-Lys(Cbz)-OH. The chain extension of the dithiocarbamate-terminated poly(A-Lys(Cbz)-OH) to N-isopropylacrylamide (NIPAM) via the RAFT process and subsequent deprotection afforded the zwitterionic block copolymer composed of thermoresponsive poly(NIPAM) and poly(A-Lys-OH), which exhibited switchability among the zwitterionic, anionic, and cationic states by pH change. The assembled structures and thermoresponsive and chiroptical properties of these block copolymers were evaluated by dynamic light scattering, circular dichroism, and turbidity measurements. Finally, the cationic block copolymer, poly(A-Lys-OMe)- b-poly(NIPAM), was obtained by the methylation of the carboxylic acid group in the zwitterionic poly(A-Lys-OH) segment. Selective interactions of DNA with the cationic poly(A-Lys-OMe) segment in the lysine-based block copolymer were further evaluated by agarose gel electrophoresis and atomic force microscopy measurements, which revealed characteristic assembled structures and temperature-responsive properties of the polyplexes.

Synthesis of a burkholdine analogue containing ß-hydroxytyrosine.

Synthesis of a ß-OHTyr-containing Bk analogue, a cyclic octalipopeptide with antifungal activities, is described. Since ß-OHTyr-containing peptides generally are unstable in strong acidic conditions, synthesis of ß-HOTyr-containing peptides by SPPS have rarely been reported. To overcome this problem, we found that using distilled TFA removed the protecting groups of side chains of ß-OHTyr-containing Bk analogue, which was prepared by Fmoc-SPPS. Abbreviations: ß-OHTyr: ß-hydroxytyrosine; ß-OHAsn: ß-hydroxyasparagine; Bk: burkholdine; FAA: fatty acyl amino acid; ß-MeOTyr: ß-methoxytyrosine; SPPS: solid phase peptide synthesis; MIC: minimun inhibitory concentration; DMF: dimethyl formamide; DIPEA: diisopropylethylamine; DIPC: diisopropylcarbodiimide; HOBt: 1-hydroxybenzotriazole; Fmoc: 9-fluorenylmethyloxycarbonyl; HFIP: 1,1,1,3,3,3-hexafluoropropan-2-ol; TFA: trifluoroacetic acid; LAP: N-lauryl -3-amino-4-carbamolypropanoic acid; HPLC: high performance liquid chromatography; ESI-TOFMS: electrospray ionization-time of flight mass spectrometry; Bn: benzyl; Boc: t-butyloxycatbonyl; 2-CTC: 2-chlorotritylchloride.

Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor.

Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives gave SK80 with an IC50 value of 43µM against SARS CoV 3CL R188I mutant protease.

α-1,3-Glucanase: present situation and prospect of research.

α-1,3-Glucanases hydrolyze α-1,3-glucan which is an insoluble linear α-1,3-linked homopolymer of glucose and these enzymes are classified into two families of glycoside hydrolases on the basis of amino acid sequence similarity; type-71 α-1,3-glucanases found in fungi and type-87 enzymes in bacteria. α-1,3-Glucan (also called 'mutan') is a major component of dental plaque formed by oral Streptococci and has important physiological roles in various fungal species, including as a component of cell walls, an endogenous carbon source for sexual development, and a virulent factor. Considering these backgrounds, α-1,3-glucanases have been investigated from the perspectives of applications to dental care and development of cell-wall lytic enzymes. Compared with information regarding other glycoside hydrolases such as amylases, cellulases, chitinases, and ß-glucanases, there is limited biochemical and structural information available regarding α-1,3-glucanase. Further research on α-1,3-glucanases on enzyme application to dental care and biological control of pathogenic fungi is expected. In this mini-review, we briefly describe how α-1,3-glucanases are categorized and characterized and present our study findings regarding α-1,3-glucanase from Bacillus circulans KA-304. Furthermore, we briefly discuss potential future applications of α-1,3-glucanases.

Structure activity relationship study of burkholdine analogues toward simple antifungal agents.

Cyclic and linear lipopeptides, burkholdine analogues, were synthesized by conventional Fmoc-SPPS and cyclisation with DIPC/HOBt in the solution phase. Synthesized peptides were evaluated for antifungal activities with MIC values against Saccharomyces cerevisiae and Aspergillus oryzae. As a result, the stereochemistry of the amino acid residues and sequences of burkholdine analogues exerted a significant influence on antifungal activities. In addition, we found a linear burkholdine analogue with moderate antifungal activities.

Surface α-1,3-glucan facilitates fungal stealth infection by interfering with innate immunity in plants.

Plants evoke innate immunity against microbial challenges upon recognition of pathogen-associated molecular patterns (PAMPs), such as fungal cell wall chitin. Nevertheless, pathogens may circumvent the host PAMP-triggered immunity. We previously reported that the ascomycete Magnaporthe oryzae, a famine-causing rice pathogen, masks cell wall surfaces with α-1,3-glucan during invasion. Here, we show that the surface α-1,3-glucan is indispensable for the successful infection of the fungus by interfering with the plant's defense mechanisms. The α-1,3-glucan synthase gene MgAGS1 was not essential for infectious structure development but was required for infection in M. oryzae. Lack or degradation of surface α-1,3-glucan increased fungal susceptibility towards chitinase, suggesting the protective role of α-1,3-glucan against plants' antifungal enzymes during infection. Furthermore, rice plants secreting bacterial α-1,3-glucanase (AGL-rice) showed strong resistance not only to M. oryzae but also to the phylogenetically distant ascomycete Cochlioborus miyabeanus and the polyphagous basidiomycete Rhizoctonia solani; the histocytochemical analysis of the latter two revealed that α-1,3-glucan also concealed cell wall chitin in an infection-specific manner. Treatment with α-1,3-glucanase in vitro caused fragmentation of infectious hyphae in R. solani but not in M. oryzae or C. miyabeanus, indicating that α-1,3-glucan is also involved in maintaining infectious structures in some fungi. Importantly, rapid defense responses were evoked (a few hours after inoculation) in the AGL-rice inoculated with M. oryzae, C. miyabeanus and R. solani as well as in non-transgenic rice inoculated with the ags1 mutant. Taken together, our results suggest that α-1,3-glucan protected the fungal cell wall from degradative enzymes secreted by plants even from the pre-penetration stage and interfered with the release of PAMPs to delay innate immune defense responses. Because α-1,3-glucan is nondegradable in plants, it is reasonable that many fungal plant pathogens utilize α-1,3-glucan in the innate immune evasion mechanism and some in maintaining the structures.

Enzyme immobilization on α-1,3-glucan: development of flow reactor with fusion protein of α-1,3-glucan binding domains and histamine dehydrogenase.

α-1,3-Glucanase Agl-KA from Bacillus circulans KA-304 consists of a discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), a threonine-proline-rich-linker (TP linker), a discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. The binding of DS1, CBM6, and DS2 to α-1,3-glucan can be improved in the presence of two of these three domains. In this study, DS1, CBM6, and TP linker were genetically fused to histamine dehydrogenase (HmDH) from Nocardioides simplex NBRC 12069. The fusion enzyme, AGBDs-HmDH, was expressed in Escherichia coli Rosetta 2 (DE3) and purified from the cell-free extract. AGBDs-HmDH bound to 1% micro-particle of α-1,3-glucan (diameter: less than 1 µm) and 7.5% coarse-particle of α-1,3-glucan (less than 200 µm) at about 97 % and 70% of the initial amounts of the enzyme, respectively. A reactor for flow injection analysis filled with AGBDs-HmDH immobilized on the coarse-particle of α-1,3-glucan was successfully applied to determine histamine. A linear calibration curve was observed in the range for about 0.1 to 3.0 mM histamine. These findings suggest that the combination of α-1,3-glucan and α-1,3-glucan binding domains is a candidate for novel enzyme immobilization.

Domain structure and function of α-1,3-glucanase Agl-EK14 from the gram-negative bacterium Flavobacterium sp. EK-14.

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the Km values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the Km value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself.

Addition of α-1,3-glucan-binding domains to α-1,3-glucanase Agn1p from　 Schizosaccharomyces pombe enhances hydrolytic activity of insoluble α-1,3-glucan.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 ß-1,3-glucanase.

Domain structure and function of α-1,3-glucanase from Bacillus circulans KA-304, an enzyme essential for degrading basidiomycete cell walls.

Bacillus circulans KA-304 α-1,3-glucanase (Agl-KA) includes an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CB6), threonine and proline repeats (TPs), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Domain deletion enzymes lacking DS1, CB6, and DS2 exhibited lower α-1,3-glucan-hydrolyzing and -binding activities than the wild type, Agl-KA. An α-1,3-glucan binding assay with fluorescent protein fusion proteins indicated that DS1, CB6, and DS2 bound to α-1,3-glucan and fungal cell walls, and that binding efficiency was increased by their combined action. In contrast, UCD did not exhibit any α-1,3-glucan-binding activity. A dramatic decrease in protoplast formation in the Schizophyllum commune mycelium was observed given only a DS1 deletion. An Agl-KA with deletion DS1, CB6, and DS2 produced no protoplasts. These results indicate that the combined actions of DS1, CB6, and DS2 contributed to increased cell-wall binding and were indispensable for efficient Agl-KA cell-wall degradation.

Nigero-oligosaccharide production by enzymatic hydrolysis from alkaline-pretreated α-1,3-glucan.

Nigero-oligosaccharides are α-1,3-linked oligomers of glucose. Glycoside hydrolase 87 type α-1,3-glucanase Agl-KA from Bacillus circulans KA304 is an endo-lytic enzyme that releases nigero-oligosaccharides (tetra-, tri-, and di-saccharide) from α-1,3-glucan. α-1,3-Glucan is insoluble under natural conditions, thus the efficiency of enzymatic hydrolysis is low and only 5 mM of reducing sugars were released from 1% glucan by Agl-KA. To improve hydrolytic efficiency, α-1,3-glucan was solubilized by 1 M NaOH and alkaline-solubilized glucan was adjusted to approximately pH 8. As a result, glucan maintained a solubilized state. This alkaline-pretreated α-1,3-glucan (1%) was hydrolyzed by Agl-KA (0.64 nmol/mL) and approximately 11.6 mM of reducing sugars were released at 240 min of reaction. When 0.016, 0.032, and 0.13 nmol/mL enzyme were added, reducing sugar reached approximately 5.1, 7.5, and 9.8 mM, respectively, and reaction mixtures containing 0.016 and 0.032 nmol/mL enzyme gradually became cloudy. Our findings suggest α-1,3-glucan cannot maintain its solubilized state and gradually becomes insoluble. For deletion enzyme of α-1,3-glucan binding domains from Agl-KA (AglΔDCD-UCD) on glucan hydrolysis (2%), reducing sugar concentrations released by AglΔDCD-UCD were almost the same as Agl-KA. These findings suggest that alkaline-pretreated α-1,3-glucan maintains a soluble state during a short time period and that glucan is efficiently hydrolyzed even by α-1,3-glucanase without α-1,3-glucan binding domains.

α-1,3-Glucanase from the gram-negative bacterium Flavobacterium sp. EK-14 hydrolyzes fungal cell wall α-1,3-glucan.

The glycoside hydrolase (GH) 87 α-1,3-glucanase (Agl-EK14) gene was cloned from the genomic DNA of the gram-negative bacterium Flavobacterium sp. EK14. The gene consisted of 2940 nucleotides and encoded 980 amino acid residues. The deduced amino acid sequence of Agl-EK14 included a signal peptide, a catalytic domain, a first immunoglobulin-like domain, a second immunoglobulin-like domain, a ricin B-like lectin domain, and a carboxyl-terminal domain (CTD) involved in extracellular secretion. Phylogenetic analysis of the catalytic domain of GH87 enzymes suggested that Agl-EK14 is distinct from known clusters, such as clusters composed of α-1,3-glucanases from bacilli and mycodextranases from actinomycetes. Agl-EK14 without the signal peptide and CTD hydrolyzed α-1,3-glucan, and the reaction residues from 1 and 2% substrates were almost negligible after 1440 min reaction. Agl-EK14 hydrolyzed the cell wall preparation of Aspergillus oryzae and released glucose, nigerose, and nigero-triose from the cell wall preparation. After treatment of A. oryzae live mycelia with Agl-EK14 (at least 0.5 nmol/ml), mycelia were no longer stained by red fluorescent protein-fused α-1,3-glucan binding domains of α-1,3-glucanase Agl-KA from Bacillus circulans KA-304. Results suggested that Agl-EK14 can be applied to a fungal cell wall lytic enzyme.

Construction of a fusion protein consisting of α-1,3-glucan-binding domains and tetrameric red fluorescent protein, which is involved in the aggregation of α-1,3-glucan and inhibition of fungal biofilm formation.

Agl-KA, an α-1,3-glucan-hydrolyzing enzyme from Bacillus circulans KA-304, has three α-1,3-glucan-binding domains DS1, CB6, and DS2 (DCD). While their individual binding activities toward insoluble α-1,3-glucan and fungal cell-wall are weak, the three domains in combination bind strongly to the α-1,3-glucan and the cell-wall. In this study, we constructed DCD-tetraRFP by fusing DCD with DsRed-Express2, a tetrameric red fluorescent protein. DCD-tetraRFP forms a tetramer in an aqueous solution and contains twelve substrate-binding domains in one complex. We also constructed DCD-monoGFP by fusing DCD with AcGFP1, a monomeric green fluorescent protein. The molecular weight of DCD-tetraRFP and DCD-monoGFP were compared. The results of gel filtration chromatography and dynamic light scattering indicated that DCD-tetraRFP was larger than DCD-monoGFP, suggesting that DCD-tetraRFP had a tetrameric structure. In addition, DCD-tetraRFP bound to insoluble α-1,3-glucan strongly, and the amount of DCD-tetraRFP binding to 0.01% α-1,3-glucan was about twice of DCD-monoGFP. The Kd values of DCD-tetraRFP (measurements per subunit) and DCD-monoGFP were 0.16 and 0.84 µM, respectively. Adding DCD-tetraRFP to a suspension of α-1,3-glucan caused glucan aggregation; however, adding DCD-monoGFP did not. These data suggested that DCD-tetraRFP had four DCDs sterically arranged in different directions so that DCD-tetraRFP cross-linked with the substrate, causing aggregation. Lastly, the aggregates of DCD-tetraRFP and α-1,3-glucan captured Aspergillus oryzae conidia and decreased their biofilm formation by 80% in a 24-well dish.