Action of an endo-β-1,3(4)-glucanase on cellobiosyl unit structure in barley β-1,3:1,4-glucan

β-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single β-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-β-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley β-1,3:1,4-glucan. To find the minor structure on which the endo-β-1,3(4)-glucanase acts, we prepared oligosaccharides from barley β-1,3:1,4-glucan by endo-β-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-β-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as β-Glc-1,3-β-Glc-1,4-β-Glc-1,3-β-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-β-1,3(4)-glucanase acts on the cellobiosyl units of barley β-1,3:1,4-glucan in an endo-manner.

Measurement of enzyme activity by reducing sugar assay.
The activities of enzymes were measured using reaction mixtures (0.1 mL) consisting of the enzyme, 0.1% (w/v) polysaccharide, and 200 mM acetate buffer, pH 5.0. After incubation at 37°C for the appropriate reaction time, the liberated sugars were determined reductometrically by the method of Nelson 23) and Somogyi. 24) One unit of enzyme activity liberates 1 μmol of reducing sugar per min. The concentration of protein was determined by the method of Bradford 25) using bovine serum albumin as the standard.
Preparation of C5 oligosaccharides. One gram of barley β-1,3:1,4-glucan, E70-S, was digested with endoβ-1,4-glucanase from A. niger in 10 mM sodium acetate buffer (pH 4.5) at 37°C for 24 h. The hydrolysate was lyophilized by freeze-dry and dissolved into 4 mL of water. Oligosaccharides released from the β-glucan were separated by gel permeation chromatography on a Bio-Gel P-2 column (26 mm × 925 mm, Bio-Rad). The V 0 and V i of the column were determined with dextran (Sigma) and Glc. Oligosaccharides were fractionated into C3, C4, and C5 fractions in order of increasing degree of polymerization (DP), which was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) with a Bruker AutoflexIII (Bruker Daltonics, Bremen, Germany). C5 fraction was further fractionated into C5-a, -b, and -c by paper chromatography using Whatman 3MM filter paper with 6:4: Fig. 2). The sugar content of the fractions was determined by the phenol-sulfuric acid method using Glc as the standard. 26) Methylation analysis.
For the analysis of sugar linkage, the oligosaccharide (approximately 100 μg) was subjected to the methylation analysis. Methylation was performed by the Hakomori method, 27) and the products were analyzed by gas liquid chromatography (GLC). GLC of neutral sugars as their alditol acetate derivatives was done with a Shimadzu gas chromatograph GC-6A equipped with a column (0.28 mm × 50 m) of Silar-10C, according to the method of Albersheim et al. 28) Action of enzymes on oligosaccharides. The action of the Trichoderma enzyme, rGI, and rGII on C4 and C5-b was analyzed using a reaction mixture (total volume, 20 μL) containing the enzyme, 0.1 mM oligosaccharide, and 50 mM sodium acetate buffer (pH 5.0). After incubation at 37°C for 24 h, the sample was inactivated by heating. The reducing sugars liberated were coupled at their reducing terminals with p-aminobenzoic acid ethyl ester (ABEE) by the method of Matsuura and Imaoka. 29) The ABEE-derivatized sugars were analyzed on an HPLC system equipped with a TSKgel Amide-80 column (4.6 mm × 250 mm; Tosoh). The column was eluted with a linear gradient of CH 3 CN:water from 74:26 to 58:42 (v/v), for 40 min at a flow rate of 1 mL/min and 40°C. ABEE sugars were monitored by a fluorescence detector model RF-10A XL at 305 nm (excitation) and 360 nm (emission).

Structural analysis of oligosaccharide.
For MALDI-ToF/ToF-MS/MS, per-methylation of glycans was performed using the NaOH slurry method described by Ciucanu and Kerek 30) using 1 mL of methyl iodide (Fluka, Buchs, Switzerland). Dry samples were resuspended in 100 μL of methanol and were kept at room temperature for MALDI-ToF/ToF-MS/MS analysis. Per-methylated methanol dissolved samples (5 μL) were mixed with 5 μL of 2,5-dihydroxybenzoic acid matrix {10 mg/mL dissolved in 50% (v/v) metha-nol} and 1 μL of the mixture was spotted on a MALDI target plate and analyzed by MALDI-ToF/ToF-MS/MS (4700 proteomics analyzer, Applied Biosystems, Foster City, CA, USA) as previously described. 31) Highenergy MALDI collision-induced dissociation (CID) spectra were acquired with an average 10,000 laser shots/spectrum, using a high collision energy (1 kV). The oligosaccharide ions were allowed to collide in the CID cell with argon at a pressure of 2 × 10 −6 Torr.
Polysaccharide analysis using carbohydrate gel electrophoresis. Products from the C5-b oligosaccharide by the Trichoderma enzyme were analyzed by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). The derivatization of carbohydrates was performed according to previously developed protocols. 32) Carbohydrate electrophoresis and PACE gel scanning were performed as described by Goubet et al. 32)

Linkage analysis of oligosaccharides
To determine the structure, C5-b oligosaccharide was subjected to methylation analysis for glucosidic linkages together with C3 and C4 oligosaccharides. In the analysis, sugars at reducing end were converted to their respective alditols before methylation of free OH groups. C4 oligosaccharide appeared to have nearly equal molecular ratio of terminal Glc (t-Glc), 3-linked Glc (3-Glc), 4-linked Glc (4-Glc), and 4-linked reducing-end Glc (4-Glcol), which coincides with the ratio obtained from G3G4G4G (Table 2). Similarly, C3 was identified as G3G4G. Compared with C3 and C4, C5-b had roughly two units of 3-Glc, indicating that C5-b is derived from a cellobiosyl unit or continuous β-1,3glucosyl residues in β-1,3:1,4-glucan.

Structure of C5-b oligosaccharide
For further structural analysis, C5-b oligosaccharide was also per-methylated and analyzed by high-energy MALDI-CID (Fig. 4, Supplemental Fig. 4). 33) However, because the direct annotation of the fragmentation spectrum was very ambiguous, we followed a comparative approach where the CID spectra of per-methylated cellopentaose, laminaripentaose, and C5-b oligosaccharide were simultaneously analyzed. In particular, we compared the relative proportions of various molecular ions in the corresponding spectra in order to decipher the structure of C5-b oligosaccharide. Comparing the CID spectra of cellopentaose and laminaripentaose, it becomes apparent that the intensity of the D 1 "elimina-  Notes: A-D, β-1,3:1,4-Glucan, E70-S, and the β-glucans with high, medium, and low viscosity were digested with the Trichoderma endoβ-1,3(4)-glucanase, respectively. E and F, E70-S was treated with barley rGI and rGII, respectively. As the products were monitored by refractive index, smaller products eluted with salts at V i could not be detected. tion ion" (m/z 227.3) 34) is higher than the neighboring E 1 or G 1 "elimination ions" (m/z 211.3) 35,36) when the nonreducing-end Glc is 1,3-linked to the second Glc residue (Fig. 4, panel I). In fact, the relative intensity of the D 1 ion in the C5-b CID spectrum is higher than the G 1 or E 1 ion intensity, suggesting that the nonreducing-end Glc is linked via a 1,3-linkage to the second Glc residue. This is further supported by the absence of the 3,5 A 2 cross-ring fragment ion (m/z 329.4) 33) in the C5-b CID spectrum (Fig. 4, panel II) and the absence of the V 4 "elimination ion" (m/z 809.4) (Fig. 4, panel VII). A strong 3,5 A 3 cross-ring fragment ion (m/z 533.4) over a 0,2 X 2 cross-ring fragment ion (m/z 519.4) 33) indicates that the second Glc is 1,4-linked to the middle Glc residue (Fig. 4, panel IV). This is further supported by the presence of a strong V 3 "elimination ion" (m/z 605.4) (Fig. 4, panel V). From the comparison of the CID cellopentaose spectrum with the corresponding laminaripentaose spectrum, it becomes apparent that a weak V 2 "elimination ion" (m/z 401.4) is indicative of a 1,3-linkage between the middle and penultimate from the reducing-end Glc residues (Fig. 4, panel III). Since the C5-b pre-methylated spectrum has a weak V 2 ion, we conclude that in this oligosaccharide the middle Glc residue is 1,3linked to the penultimate Glc residue. This is further supported by the absence of the 3,5 A 4 cross-ring fragment ion (m/z 737.4) (Fig. 4, panel VI) and the absence of the D 4 "elimination ion" (m/z 839.4) (Fig. 4, panel VII). In Fig. 4, panel VIII, it is shown that a strong 3,5 A 5 cross-ring fragment ion (m/z 941.4) is indicative of a 1,4-linkage between the penultimate and the reducing-end Glc residues. Taken together, these data allow the C5-b oligosaccharide to be identified as β-Glcp-1,3β-Glcp-1,4-β-Glcp-1,3-β-Glcp-1,4-Glcp (G3G4G3G4G).
Products released from C5-b by Trichoderma enzyme As shown in Fig. 3, Trichoderma enzyme properly hydrolyzed C5-b into smaller saccharides; however, the oligosaccharide could not be identified on HPLC. Therefore, the products released from C5-b were also subjected to PACE. As shown in Fig. 5, the smaller saccharides in the products were identified as laminaribiose and Glc. The result suggests that the enzyme acted on both β-1,4-linkages in G3G4G3G4G.

Discussion
Poaceae β-1,3:1,4-glucan is mainly consisted of cellotriosyl and cellotetraosyl units linked through single β-1,3-glucosidic linkages, but it has also been shown to possess cellobiosyl units as the minor structure. 4) Through the structural analysis of unexpected oligosaccharides released by endo-β-1,3:1,4-glucanase, the cellobiosyl units appeared to locate at the nonreducing side of cellotriosyl units in barley, lichen, and horsetail. 5) Based on the proportion of the released oligosaccharides, the frequency of cellobiosyl unit was estimated less than 2% in barley β-1,3:1,4-glucan. 5) In this study, based on sugar content, C5 fraction obtained by digestion with A. niger endo-β-1,4-glucanase belonging to GH12 was less than 1.5% of total sugar released from barley β-1,3:1,4-glucan, confirming that the cellobiosyl units exist as the minor structure in barley β-1,3:1,4-glucan. Notes: A, the action of the Trichoderma enzyme on C4; B, the action of the Trichoderma enzyme on C5-b; C, the action of rGI on C5-b; and D, the action of rGII on C5-b were analyzed. Reducing sugars before and after enzyme reaction were derivatized with ABEE and analyzed on HPLC, respectively. Together with cellobiosyl unit and long stretch of β-1,4-glucosidic linkage, continuous β-1,3-glucosidic linkages have also been presumed in maize β-1,3:1,4glucan. 4) However, we could not detect any hydrolysis of barley β-1,3:1,4-glucan by barley endo-β-1,3-glu-canases belonging to GH17, rGI, and rGII. The fact that laminaritriose is the smallest substrate for GII 22,37) suggests that barley β-glucan does not have three continuous β-1,3-glucosyl residues. Hence, the hydrolysis of β-1   Notes: The signals used for characterization were boxed numbered from I to VIII. Glucosidic and cross-ring fragments are identified according to the nomenclature of Domon and Costello. 33) not likely occur in barley. On the other hand, we cannot exclude the possibility that barley β-1,3:1,4-glucan has two continuous β-1,3-glucosyl residues that can be hydrolyzed by distinct endo-β-1,3-glucanases secreted by fungi and bacteria.
In the analysis of 3-D structure of PcLam16A, 20,21) two Trp residues have been shown to be involved in the specific recognition of β-1,3-glucosidic linkage between the subsites −1 and −2. In the enzyme, the substrate-binding cleft has a narrow and straight canyon structure along which a linear oligosaccharide such as G4G3G can lay. The Trichoderma enzyme hydrolyzed C5-b oligosaccharide, G3G4G3G4G, into laminaribiose and Glc as the final products. This result suggests that the Trichoderma enzyme either first hydrolyzed G3G4G3G4G into G3G4G3G and Glc, and then into two laminaribioses and Glc, or the enzyme first hydrolyzed G3G4G3G4G into G3G4G and laminaribiose, and then into two laminaribioses and Glc. Because the Trichoderma enzyme did not act on C4 oligosaccharide (G3G4G4G), the former case is more probable. These facts also suggest that the smallest substrate for the Trichoderma enzyme is G3G4G3G.