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.

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.

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.

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.

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.

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.

GH-16 Type ß-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall.

The GH-16 type ß-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type ß-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with ß-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using ß-glucans.

Antimicrobial Activity of Polysorbate 80-iodine Complex: Polysorbate 80 Firmly Retains Iodine.

Considering that iodine is highly volatile and has low solubility in water, it is utilized as an antiseptic in its complex form (iodophor) with a carrier material. Herein, we prepared the polysorbate 80-iodine complex and investigated its properties. In the presence of 0%, 0.01%, 0.1%, and 1% polysorbate, Pseudomonas putida NBRC 100650 growth was inhibited at 75, 75, 50, and 25 ppm iodine, respectively, indicating that high concentrations of polysorbate 80 enhanced the antibacterial activity of iodine. Absorption spectra of the mixtures of polysorbate 80 and iodine were analyzed; we observed that two peaks at 287 and 350 nm, derived from triiodide ions, shifted to the longer wavelength side in the presence of 0.1% and 1% polysorbate 80. Further, when 1% polysorbate 80 was added to the mixture of soluble starch and iodine, the peak around 580 nm arising from the amylose-iodine complex disappeared, indicating that polysorbate 80 captured iodine from the starch-iodine complex. We also found that polysorbate 80 retained iodine for approximately 4 months and prevented its volatilization; moreover, the mixture did not lose its growth inhibitory activity upon storage for approximately 4 months. Collectively, our data indicated that polysorbate 80 firmly retains low concentrations of iodine and that the polysorbate 80-iodine complex can serve as an antiseptic that can be stably stored for a long time.

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.

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.

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae.

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

Corrigendum: A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans.

[This corrects the article DOI: 10.3389/ffunb.2021.821946.].

Optimization of protease production by Bacillus cereus HMRSC30 for simultaneous extraction of chitin from shrimp shell with value-added recovered products.

Chitin extraction from shrimp shell powder (SSP) using protease-producing microbes is an attractive approach for valorizing shrimp shell waste because it is simple and environmentally friendly. In this study, the protease production and chitin extraction from SSP by Bacillus cereus HMRSC30 were simultaneously optimized using statistical approaches. As a result, fermentation in medium composed of 30 g/L SSP, 0.2 g/L MgSO4 · 7H2O, 3 g/L (NH4)2SO4, 0.5 g/L K2HPO4, and 1.5 g/L KH2PO4 (pH 6.5) for 7 days maximized protease production (197.75 ± 0.33 U/mL) to approximately 1.64-fold compared to unoptimized condition (126.8 ± 0.047 U/mL). This level of enzyme production was enough to achieve 97.42 ± 0.28% deproteinization (DP) but low demineralization (DM) of 53.76 ± 0.21%. The high DM of 90% could be easily accomplished with the post-treatment using 0.4 M HCl and acetic acid. In addition, the study evaluated the possible roadmap to maximize the value of generated products and obtain additional profits from this microbial process. The observation showed the possibility of serving crude chitin as a bio-adsorbent with the highest removal capacity against Coomassie brilliant blue (97.99%), followed by methylene blue (74.42%). The recovered protease exhibited the function to remove egg yolk stain, indicating its potential for use as a detergent in de-staining. The results corroborated the benefits of microbial fermentation by B. cereus HMRSC30 as green process for comprehensive utilization of shrimp shell waste as well as minimizing waste generation along the established process.

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 Selective Antibacterial Activity of the Mixed Systems Containing Myristic Acid against Staph ylococci.

Fatty acids and their derivatives are interesting cosmetic ingredients because they show the selective antibacterial activity against Staphylococcus aureus (S. aureus). However, the antibacterial activity in mixed systems containing several active ingredients is unclear because previous studies focused antibacterial systems containing one kind of fatty acid. In the present study, the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC) were evaluated for myristic acid/lauric acid, myristic acid/palmitoleic acid, and myristic acid/lactic acid mixed systems to show the effect of the coexisting components on the selective antibacterial activity of myristic acid. In the myristic acid/palmitoleic acid mixed system, the antibacterial activity against S. aureus was enhanced by additive effect, whereas the antibacterial activity was not observed against S. epidermidis. On the other hand, the myristic acid/lauric acid mixed system showed antibacterial activity against S. epidermidis: Lauric acid impaired the selectivity of antibacterial activity of myristic acid. These results suggest that the selective activity of myristic acid varies with the additives. The present findings are useful for designing formulations of cosmetics and body cleansers containing myristic acid.

Antibacterial Activity of the Mixed Systems Containing 1,2-Dodecanediol against Staphylococcus aureus and Staphylococcus epidermidis.

1,2-Alkanediols are characteristic cosmetic ingredients because these moisturizers exhibit the antibacterial activity against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). However, the antimicrobial behavior in mixed systems containing several active ingredients is unclear because previous reports focus on an antibacterial system containing only 1,2-alkanediol. In this study, the minimal inhibitory concentration (MIC) and the fractional inhibitory concentration (FIC) were evaluated for 1,2-dodecanediol/lactic acid, 1,2-dodecanediol/myristic acid, 1,2-dodecanediol/methylparaben, and 1,2-dodecanediol/isopropyl methylphenol mixed systems to show the effect of the addition of other antimicrobial components to 1,2-dodecanediol. The antibacterial property of 1,2-dodecanediol/lactic acid mixed system was almost similar compared to 1,2-dodecanediol monomeric system. On the other hand, the antimicrobial activity of 1,2-dodecanediol against S. epidermidis was inhibited in the 1,2-dodecanediol/myristic acid mixed system. Because the selective antimicrobial activity of myristic acid against S. aureus was demonstrated in the mixed system. The present findings are useful for designing formulations of cosmetics and body cleansers containing 1,2-dodecanediol.

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.

Functional analysis of α-1,3-glucanase domain structure from Streptomyces thermodiastaticus HF3-3.

α-1,3-Glucanase from Streptomyces thermodiastaticus HF3-3 (Agl-ST) has been classified in the glycoside hydrolase (GH) family 87. Agl-ST is a multi-modular domain consisting of an N-terminal ß-sandwich domain (ß-SW), a catalytic domain, an uncharacterized domain (UC), and a C-terminal discoidin domain (DS). Although Agl-ST did not hydrolyze α-1,4-glycosidic bonds, its amino acid sequence is more similar to GH87 mycodextranase than to α-1,3-glucanase. It might be categorized into a new subfamily of GH87. In this study, we investigated the function of the domains. Several fusion proteins of domains with green fluorescence protein (GFP) were constructed to clarify the function of each domain. The results showed that ß-SW and DS domains played a role in binding α-1,3-glucan and enhancing the hydrolysis of α-1,3-glucan. The binding domains, ß-SW and DS, also showed binding activity toward xylan, although it was lower than that for α-1,3-glucan. The combination of ß-SW and DS domains demonstrated high binding and hydrolysis activities of Agl-ST toward α-1,3-glucan, whereas the catalytic domain showed only a catalytic function. The binding domains also achieved effective binding and hydrolysis of α-1,3-glucan in the cell wall complex of Schizophyllum commune.

A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans.

α-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsAOE) strain was larger than that in an agsB-overexpressing (agsBOE) strain. The mechanism that regulates its MM is poorly understood. Although the gene amyD, which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD (ΔamyD) or overexpressed amyD (amyDOE) in the genetic background of the ABPU1 (wild-type), agsAOE, or agsBOE strain, and characterized the chemical structure of α-1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of α-1,3-glucan from the agsBOE amyDOE strain was smaller than that in the parental agsBOE strain. In addition, the MM of α-1,3-glucan from the agsAOE ΔamyD strain was greater than that in the agsAOE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.

Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain. The catalytic domain of Chi19MK is approximately 60% similar to those of ChiB from Burkholderia gladioli CHB101, chitinase N (ChiN) from Chitiniphilus shinanonensis SAY3T, ChiF from Streptomyces coelicolor A3(2), Chi30 from Streptomyces olivaceoviridisis, ChiA from Streptomyces cyaneus SP-27, and ChiC from Streptomyces griseus HUT6037. Chi19MK lacking the signal and uncharacterized sequences (Chi19MKΔNTerm) was expressed in Escherichia coli Rosetta-gami B(DE3), resulting in significant chitinase activity in the soluble fraction. Purified Chi19MKΔNTerm hydrolyzed colloidal chitin and released disaccharide. Furthermore, Chi19MKΔNTerm inhibited hyphal extension in Trichoderma reesei and Schizophyllum commune. Based on quantitative antifungal activity assays, Chi19MKΔNTerm inhibits the growth of Trichoderma viride with an IC50 value of 0.81 µM.