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.

This study investigated the cloning and expression of the gene encoding α-1,3-glucanase (Agl-EK14) in Flavobacterium sp.EK-14.In 1975, Flavobacterium sp.EK-14 was isolated from soil on a medium containing insoluble sticky glucan produced by Streptococcus mutans OMZ 176 27 .α-1,3-Glucanase (EK-14 enzyme) was purified from a culture filtrate of strain EK-14, and various properties of the enzyme were investigated.Furthermore, the EK-14 enzyme hydrolyzed α-1,3-glucans synthesized by oral Streptococcus as well as the glucan in fungal cell walls.These findings suggest that Flavobacterium is a potential source of α-1,3-glucanase genes.Here, an α-1,3-glucanase gene was cloned from strain EK-14 based on putative GH87 enzyme α-1,3-glucanase genes of Flavobacterium registered in the database.Agl-EK14 was also expressed in Escherichia coli, and the purified enzyme strongly hydrolyzed the α-1,3-glucan prepared by GTF-I of S. mutans.Agl-EK14 also hydrolyzed the cell wall α-1,3-glucan of Aspergillus oryzae, suggesting that GH87 α-1,3-glucanase from gram-negative bacteria is a useful enzyme for fungal control, as are GH87 enzymes from gram-positive bacteria and GH71 enzymes from fungi.

Sequence analysis of the α-1,3-glucanase gene of Flavobacterium sp. EK-14
Flavobacterium is a gram-negative bacterium belonging to Bacteroidetes and is often isolated from soil and environmental water.α-1,3-Glucanase genes have never been cloned from Flavobacterium species.Therefore, this study cloned α-1,3-glucanase from strain EK-14 based on putative α-1,3-glucanases from the CAZy database.To amplify the gene via polymerase chain reaction (PCR), sense and antisense primers were designed based on this putative enzyme's conserved region, such as Flavobacterium anhuiense M168 and Flavobacterium johnsoniae UW101.PCR product, approximately 2700-bp long, showed a high sequence similar to the α-1,3-glucanase encoding gene from F. anhuiense M168.The DNA fragment containing upstream and downstream regions of the partial Agl-EK14 gene was amplified by PCR, and the primers were designed based on the upstream and downstream regions of the open reading frame (ORF) of the F. anhuiense gene.As a result, an approximately 3000-bp PCR product was obtained.The product contains an ORF corresponding to about 980 amino acid residues (Supplementary Fig. S1), with a sequence highly similar to those of the putative enzymes described above (Supplementary Fig. S2), indicating that a complete α-1,3-glucanase gene was obtained.
A phylogenetic tree was constructed based on the amino acid sequences of the catalytic domains of GH87 α-1,3-glucanases and mycodextranases.The phylogenetic tree revealed that Agl-EK14 was distinct from several clusters containing enzymes that have already been cloned and characterized (Supplementary Fig. S4).

Expression and purification of recombinant Agl-EK14
To clarify enzymatic properties, the Agl-EK14 gene excluding the signal peptide and CTD, was expressed in E. coli (Fig. 1A).The expression plasmid pET-aglEK14 was introduced into E. coli Rosetta-gami B (DE3) cells.The α-1,3-glucanase activity of the cell-free extract of E. coli harboring the plasmid was 29.6 units/mg.The recombinant enzyme was purified from cell-free extracts using anion exchange and hydrophobic interaction column.Figure 1B shows the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Agl-EK14.The predicted molecular mass of Agl-EK14 was 92,970, and its molecular mass, as estimated by SDS-PAGE, was consistent with the predicted values.The purified Agl-EK14 exhibited a specific activity of 134 units/mg (Supplementary Table S2).

α-1,3-Glucan hydrolytic activity
Figure 4A shows the effect of substrate concentration on the glucan hydrolysis of Agl-EK14.The concentration of reducing sugars increased with increasing α-1,3-glucan, and approximately 12.0, 20.4,29.5, 38.6, and 42.2 mM reducing sugars were released from 1%, 2%, 3%, 4%, and 5% α-1,3-glucan, respectively, after 1440 min of reaction.Agl-KA from B. circulans KA-304 is one of the most investigated α-1,3-glucanases, and its various properties have been well characterized.Compared to the hydrolytic activity of Agl-KA on α-1,3-glucan, the amounts of reducing sugars released by Agl-EK14 were almost 1.5-fold higher than those by Agl-KA after 24 h of reaction (Supplementary Fig. S5).After the reaction, the residues were collected by centrifugation (Fig. 4B).The residues of the reaction mixture with 1% and 2% substrates were almost negligible after 1440 min.Reaction products released from 1% α-1,3-glucan were analyzed by high-performance liquid chromatography (HPLC; Fig. 4C), and four peaks were detected.Because the retention time of peak 1 coincided with glucose, electrospray ionizationmass spectrometry (ESI-MS) analysis was performed against peaks 2-4 (Supplementary Fig. S6).Results revealed that peaks 2-4 were assigned to the [M + Na] + ions of di-, tri-, and tetra-saccharide at 365.0, 527.0, and 689.1 m/z, respectively.These results suggested that Agl-EK14 released di-to tetra-saccharides after a 15-min reaction.Tetra-saccharides gradually decreased, but glucose, di-, and tri-saccharides increased during incubation.The cell wall of A. oryzae is composed of cross-linked polysaccharides and glycoproteins.Chitin and β-glucan are the main skeletal polysaccharides, and α-1,3-glucan is mainly localized on the cell wall surface 8 .The cell wall preparation of A. oryzae included polysaccharide complexes containing α-1,3-glucan, and the hydrolytic activity of Agl-EK14 was investigated against the cell wall preparation.Figure 5A shows the time course of hydrolysis of the cell wall preparation of Agl-EK14.Agl-EK14 released approximately 2.0 mM reducing sugars after a 24 h hydrolysis reaction.The reaction products were analyzed by thin-layer chromatography (TLC; Fig. 5B), and mono-, di-, and tri-saccharides were detected.These reaction products were consistent with hydrolytic products from insoluble α-1,3-glucan synthesized by GTF-I of S. mutans.
GH19 chitinase (Chi19MK) from Lysobacter sp.MK9-1 inhibited the growth of fungi such as Trichoderma reesei, Trichoderma viride, and Shizophyllum commune.GH16 β-1,3-glucanase (BgluC16MK) of Lysobacter sp.MK9-1 enhanced the growth-inhibitory activity of Chi19MK against T. reesei 38,40 .This study investigated the growth inhibition assay of Agl-EK14 with Chi19MK and BgluC16MK against A. oryzae (Fig. 7).As a result, a mixture of Chi19MK and BgluC16MK did not inhibit the growth of A. oryzae, but the addition of Agl-EK14 Figure 5. Hydrolysis of the cell wall preparation of A. oryzae (A) and TLC analysis of reaction products from the cell wall preparation (B).The reaction mixture contained 2% cell wall preparation of A. oryzae, 50 mM potassium phosphate (pH 6.5), and 1.0 nmol/ml Agl-EK14.The mixture was incubated at 37 °C.(A) Each data point represents the mean ± standard deviation from triplicate experiments.(B) The 2-24h reaction products were spotted at 2 μl on TLC.TLC was performed as described in Methods.Glucose (Glc), nigerose (NG2), nigerotriose (NG3), and nigerotetraose (NG4) were used as markers.Lane M, markers; lane G, α-1,3-glucan hydrolytic products of Agl-EK14 after 24 h. to the mixture inhibited the mycelial growth of A. oryzae.In the case of the mixture containing Agl-EK14, Chi19MK, and BgluC16MK, the colony diameter was 380-820 μm, and the colonies existed individually (Supplementary Fig. S8).In contrast, under other conditions, colonies were extended and connected, and individual colony diameters could not be measured.
Furthermore, Agl-EK14 hydrolyzed the cell surface α-1,3-glucan of A. oryzae (Fig. 6).After treatment of the live mycelia of A. oryzae with Agl-EK14, BGBD-GFP bound to the cell wall.In some of the mycelial tips of A. oryzae, BGBD-GFP bound firmly.At the mycelial tip, the synthesis and degradation of cell wall polysaccharides proceed simultaneously 47 .The mycelial tip was probably in the process of becoming a complex structure, such as a multi-layered structure of glycans and cross-linked structures between glycans.Thus, BGBD-GFP could readily bind.Although AGBD-RFP bound strongly to the cell surface before Agl-EK14 treatment, AGBD-RFP hardly bound after the treatment.Because α-1,3-glucan was located on the cell surface and not the main skeletal polysaccharide, only α-1,3-glucanase treatment hardly influenced the growth and fungi form.The combination of Agl-EK14 with chitinase Chi19MK and β-glucanase BgluC16MK inhibited the growth of A. oryzae (Fig. 7), suggesting that their combination provides a potent antifungal enzyme preparation.
As mentioned in the Introduction, α-1,3-glucanase, an EK-14 enzyme, was purified from a culture filtrate of Flavobacterium sp.EK-14, and some properties of the EK-14 enzyme were reported 27 .Some enzymatic properties of the EK-14 enzyme, such as optimum pH, optimal temperature, and substrate specificity, were almost similar to those of Agl-EK14 obtained in this study.However, there were some inconsistencies between the EK-14 enzyme and Agl-EK14.The molecular weight of the EK-14 enzyme was about 65,000, and the specific activity of the EK-14 enzyme was 0.813 units/mg.In contrast, the molecular weight of Agl-EK14 was 92,970, and the specific activity was 134 units/mg, implying that Agl-EK14 is an isozyme of the EK-14 enzyme.
Although the relation between the previously purified EK-14 enzyme and cloned Agl-EK14 based on the DNA sequence is yet to be clarified, this study revealed that Agl-EK14 has excellent α-1,3-glucan-degrading activity and forms a cluster independent of known α-1,3-glucanases from gram-positive bacteria.Hence, Agl-EK14 is a valuable enzyme to be studied for industrial applications.Functional and structural analyses are in progress for further improvement of this enzyme.

Microorganisms and culture
Flavobacterium sp.EK-14 was grown at 22 °C in trypticase soy broth.A. oryzae NBRC 100959 was cultured at 28 °C in a potato dextrose agar (PDA) medium.The A. oryzae conidial solution was prepared by suspending conidia formed in the PDA medium in a suspension buffer containing 0.01% Tween 20 and 100 mM NaCl.The conidial solution was inoculated into a Czapek-Dox (CD) medium for the cell wall binding assay and incubated at 28 °C.

Cell wall preparation of A. oryzae
A. oryzae live mycelia (from a 5-day culture with 2 L CD medium) were suspended in 100 ml acetone.Mycelia were collected by filtration and air-dried.Acetone-dried mycelia were suspended in water and heated at 120 °C for 20 min in an autoclave.Autoclaved mycelia were washed with distilled water and lyophilized.Lyophilized mycelia were used as cell wall preparation of A. oryzae.

Cloning of the α-1,3-glucanase gene
All primers used in cloning the Agl-EK14 gene are listed in Supplementary Table S1.PCR amplified the partial Agl-EK14 gene from the genomic DNA of Flavobacterium sp.EK-14.The sense primer, Flavo-for, was designed from a conserved region of putative GH 87 α-1,3-glucanase genes from F. anhuiense M168 (accession no.CP023642.1;region: 32,333,180-2,336,068) and F. johnsoniae UW101 (accession no.CP000685.1;region: 3,818,600-3,821,545).The antisense primer, Flavo-rev, was designed from their conserved regions.The DNA fragment containing upstream and downstream regions of the partial Agl-EK14 gene was amplified by PCR using the primer FlavoEK-f4 and FlavoEK-r4.The FlavoEK-f4 was designed based on the upstream region (position at − 190 to − 156) of the ORF of the F. anhuiense strain M168 α-1,3-glucanase gene.The FlavoEK-r4 was designed based on the downstream region (position at 3081 to 3115) of the ORF of the F. anhuiense gene.The nucleotide sequence of the Agl-EK14 gene from Flavobacterium sp.EK-14 was assigned to the DNA Data Bank of Japan (DDBJ; https:// www.ddbj.nig.ac.jp/ index.html), Accession No. LC735653.

Expression of the Agl-EK14 gene
The ORF, excluding the signal peptide, was amplified by PCR from the genomic DNA of Flavobacterium sp.EK-14 using primers the Flavof1RE and Flavo5rRE.The PCR fragment was digested with Nde I and BamH I; the resulting fragment was cloned into pET22b (+); and the vector was designated as pET-aglEK14CTD.Subsequently, the gene, excluding CTD, was amplified from pET-aglEK14CTD using primers the T7 promoter and Cbam4.The PCR fragment was digested with Nde I and BamH I, and the resulting fragment was cloned into pET22b (+).The plasmid expressing Agl-EK14 was designated as pET-aglEK14.The constructed plasmid was transformed into E. coli Rosetta-gami B (DE3) cells, and the resulting transformant was inoculated into a 2 l flask containing 1 L LB medium with ampicillin, chloramphenicol, and kanamycin incubated at 30 °C on a reciprocal shaker (100 strokes/min).After incubation, until the optical density at 600 nm was about 0.6, isopropyl-β-d-thiogalactopyranoside was added to the culture medium at a final concentration of 0.4 mM.The culture was incubated at 16 °C for an additional 18 h.
To purify Agl-EK14, the dialysate (cell-free extract) was fractionated with ammonium sulfate, and the fraction of 25-70% saturation was dialyzed against 10 mM potassium phosphate (pH 7.0).The dialysate was applied to a Cellufine A-500 column (2.0 × 4.5 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.0).After washing with the same buffer, Agl-EK14 was found in the unabsorbed fractions.The activity fraction was mixed with ammonium sulfate at 25% saturation and applied to a HiTrap Butyl 650M column (1.5 × 2.5 cm) equilibrated with 10 mM potassium phosphate buffer (pH 7.0) containing 25% ammonium sulfate.The column was washed with the buffer containing 25% ammonium sulfate, and Agl-EK14 was eluted by linearly decreasing the ammonium sulfate concentration from 25 to 0%.The fraction containing Agl-EK14 was dialyzed against 10 mM potassium phosphate buffer (pH 7.0).

Assay of α-1,3-glucanase activity
α-1,3-Glucanase activity was measured as described previously 48 .The reaction mixture contained 50 mM potassium phosphate buffer (pH 6.5), 1% α-1,3-glucan, and appropriate amounts of enzyme.The reaction mixture was incubated at 37 °C.One unit of enzyme is defined as the amount of enzyme that releases 1 μmol of reducing sugar per minute.The optimal temperature for Agl-EK14 (0.005 nmol/ml) activity was determined by monitoring the amount of reducing sugar produced between 0 and 100 °C for 30 min in 50 mM potassium phosphate buffer (pH 6.5).To determine thermal stability, Agl-EK14 (0.1 nmol/ml) in 50 mM potassium phosphate buffer (pH 6.5) was treated at various temperatures ranging from 0 to 100 °C for 30 min.After temperature treatment, α-1,3-glucanase activity was measured using 0.005 nmol/ml of the treated enzyme in 50 mM potassium phosphate buffer (pH 6.5) at 37 °C.

Analytical methods
Protein concentrations were measured during enzyme purification using Lowry's method 50 , with bovine serum albumin as the standard.Otherwise, the protein concentration of purified Agl-EK14 was estimated by measuring the absorbance at 280 nm with a molar absorption coefficient of 165,880 M -1 cm -151 .AGBD-RFP concentration was estimated by measuring the absorbance at 554 nm with a molar absorption coefficient of 35,600 M −1 cm −1 .The BGBD-GFP concentration was estimated by measuring the absorbance at 475 nm with a molar absorption coefficient of 32,500 M −1 cm −1 .SDS-PAGE was performed using Laemmli's method 52 .PM1500 Excel Band All Blue Regular Range Plus Protein Marker (purchased from SMOBIO, Taiwan) was used as a molecular marker.Reducing sugars were quantified with dinitrosalicylic acid reagent using the Miller method, and glucose (0-4 mM) was used as the standard 53 .HPLC was used to analyze the products of hydrolysis.The reaction products were applied onto a column of Inertsil amide (4.6 × 250 mm; GL Sciences, Inc., Tokyo, Japan), and the products were monitored using an RID-10A refractive index detector (Shimadzu Co., Kyoto, Japan).The elution solvent was 70% acetonitrile at a flow rate of 0.5 ml/min with LC-10AT pumps (Shimadzu).The hydrolytic products from the cell wall preparation of A. oryzae were analyzed by TLC as described previously 49 .

Figure 1 .Figure 2 .
Figure 1.Schematic design of the domain of Agl-EK14 (A) and SDS-PAGE analysis of the purified proteins (B).(A) SP signal peptide, Ig1 first immunoglobulin-like domain, Ig2 second immunoglobulin-like domain, Ricin B Ricin B-like lectin domain, CTD carboxy-terminal domain.(B) The 10% SDS-PAGE gel was stained with Coomassie brilliant blue G250.Lane M, molecular weight markers; lane 1, Agl-EK14.The original image is presented in Supplementary Fig. S9.

Figure 3 .
Figure 3. Optimum pH (A), optimal temperature (B), pH stability (C), and thermal stability (D) of Agl-EK14.The activity values are the mean ± standard deviation of three independent experiments.Symbols: closed triangles, sodium acetate buffer; closed circles, potassium phosphate buffer; closed squares, Tris-HCl buffer; and closed diamonds, borate buffer.