Effects of artificial cell walls on syringyl-lignin growth reaction using recombinant cationic cell wall peroxidase

Artificial lignin called dehydrogenative polymers (DHPs) has been used to develop models for investigating lignin characteristics and structures. However, these models are inadequate because they have lower molecular weight and less content of β-O-4 than natural lignin. The conditions required for dehydrogenative polymerization, particularly the reaction within the polysaccharide matrix, are believed to be critical for facilitating lignin polymer growth reaction. This study investigated the effects of the polysaccharide matrix on syringyl-DHPs properties following dehydrogenative polymerization reaction on artificial cell walls. Specifically, artificial cell walls were prepared by esterifying ferulic acid to xyloglucan, which was then adsorbed on nematic ordered cellulose film. Sinapyl alcohol or coniferyl alcohol was polymerized on the film using recombinant cationic cell wall peroxidase. Syringyl-DHP and guaiacyl-DHP were prepared in the buffer and served as comparative subjects. DHPs formed on artificial cell walls had lower molecular weights and less content of β-O-4 than DHPs prepared in the buffer. These results suggest that binding DHPs to a polysaccharide matrix may limit their mobility and inhibit their growth reaction.

The plant secondary cell wall, which is located on the inner side of the primary cell wall, contributes to the overall strength and hydrophobic properties of the plant [1, 2]. The secondary cell wall is composed of cellulose microfibrils (CMF) formed from accumulated cellulose, with hemicellulose adhering to CMF. Lignin, which fills the spaces between cellulose and hemicellulose components, is one of the main cell wall components in vascular plants. The principal precursors of lignin include coniferyl alcohol (CA), sinapyl alcohol (SA), and p-coumaryl alcohol, which are polymerized via a random radical coupling reaction [3]. Because of the complex structure of lignin, its natural form has not been fully elucidated.

Artificial lignin (dehydrogenative polymers; DHPs) has been used as a model to investigate lignin characteristics and structures. However, DHPs have less content of β-O-4 and lower molecular weight than natural lignin [4,5,6]. Efforts have been made to prepare DHPs under various polymerization conditions in vitro to mimic natural lignin. Compared with Zulaufverfahren (ZL) polymerization, during which all reactants are introduced simultaneously, Zutropfverfahren (ZT) polymerization, which involves the gradual addition of substrates and hydrogen peroxide in a buffer containing peroxidase, results in minor increases in the molecular weight and the number of β-O-4 bonds [7, 8]. DHPs prepared by the dialysis membrane method (a process where enzymes are introduced inside a cellulose dialysis membrane within a buffer solution, and the addition of hydrogen peroxide and CA outside it) exhibited higher molecular weights compared to DHPs prepared through the ZT method [9]. A relatively small increase in the number of β-O-4 bonds in DHPs may occur if the pH is adjusted to make the buffer solution more acidic [10, 11]. Notably, this change in polymerization conditions results in only a slight improvement. Lignification, which involves the radical coupling of monolignols in plant cell walls, occurs within the polysaccharide matrix made up of cellulose and hemicellulose [12, 13]. Therefore, the polysaccharide matrix is thought to play a crucial role during lignin formation. In earlier studies, DHPs prepared in buffers containing xylans [14] or pectins [15] had higher molecular weight and content of β-O-4 than those prepared in buffers without hemicellulose. However, these conditions did not substantially improve the molecular weight and content of β-O-4, possibly because dehydrogenative polymerization in a buffer containing dissolved polysaccharides differs considerably from that occurring during the natural formation of lignin in plant cell walls. Recently, artificial cell walls have been used to investigate the influence of the polysaccharide matrix on lignin structures and properties within the plant cell wall [16, 17]. The primary benefit of using artificial cell walls lies in the ability to easily separate the generated lignin from artificial cell walls, thereby allowing for a detailed examination of the effects of each polysaccharide matrix component on lignin polymerization. Lyu et al. [18] demonstrated that some hemicelluloses were adsorbed on bacterial cellulose films, after which CA may be polymerized using horseradish peroxidase (HRP) to assess the influence of each hemicellulose on DHP structures. Their findings showed that the content of β-O-4 in DHPs is higher when xylan is present than when xyloglucan (XG) and cellulose are present. Elschner et al. [19] prepared DHPs on ferulic acid (FA) esterified to cellulose films and then analyzed the types of linkages in DHPs. They revealed a higher ratio of β-β bond pattern to DHPs prepared, consistent with the structural features typically associated with the ZL method. Prior research has mainly concentrated on investigating the effect of artificial cell walls on the growth reactions of guaiacyl-DHPs derived from CA using HRP. Moreover, even in the documented instances, only the quantity of syringyl-DHPs deposited on the artificial polysaccharide matrix was assessed [16], leaving the effects on the chemical structure. In this study, dehydrogenative polymerization reaction, followed by the growth of syringyl-DHPs polymers, was conducted on an artificial polysaccharide matrix to explore the effects of artificial cell walls on the properties of DHPs. To generate artificial cell walls, nematic ordered cellulose (NOC) film was prepared and used as a cellulose layer. The cellulose was a highly oriented and non-crystalline material created by uniaxially stretching cellulose hydrogel from a LiCl/DMAc solution containing cellulose, with an amphiphilic structure containing alternating hydrophilic regions of hydroxyl groups and hydrophobic regions from glucose rings [20, 21]. CMF in the plant secondary cell wall is oriented in one direction. Researchers examined simulated plant cell walls and proposed that lignin is adsorbed in the same orientation as cellulose [22,23,24]. Therefore, NOC was assumed to be an appropriate cellulose layer in artificial secondary cell walls. XG [25, 26] was selected as hemicellulose because of its strong affinity for cellulose. It was esterified with FA and then adsorbed on NOC. FA, which has been detected in grass family plants, forms a polysaccharide–FA–lignin structure after an esterification with hemicellulose [27, 28]. Furthermore, FA has been hypothesized to be the starting point of lignin polymerization [29]. Therefore, FA was used as an anchor for lignin polymerization. Finally, SA or CA was polymerized on the film using recombinant cationic cell wall peroxidase (rCWPO-C) (Fig. 1). CWPO-C was initially identified in Populus alba L. [30, 31] and can oxidize CA as well as SA and lignin polymers that are hardly oxidized by HRP. Next, DHPs of these artificial cell walls were analyzed.

Synthesis of FA esterified to XG

XG was selected because of its favorable interaction with cellulose [25]. FA, which served as an anchor for DHPs on the polysaccharide matrix, was esterified to XG. FA esterified to XG (FA-XG) with different degrees of FA substitution (DS_FA) was synthesized by altering the composition ratio and reaction time. Two specific types of FA-XG, designated as high ferulic acid xyloglucan (HFXG) and low ferulic acid xyloglucan (LFXG), with DS_FA values of 1.72 and 0.72, respectively (Table 1), were used for subsequent analyses. According to a Fourier transform infrared spectroscopy (FT-IR) analysis (Fig. S1), –C=O signal corresponding to the ester group in HFXG and LFXG was detected at 1,724 cm^–1; this signal was undetectable for samples containing only XG and FA. Additionally, aromatic group (–C=C) stretching was detected in HFXG and LFXG as signals at 1,594 and 1,510 cm^–1, whereas carboxylic group (HO–C = O) stretching was not detected in HFXG and LFXG (i.e., no signal at 1,690 cm^–1) [32]. These findings confirm that XG was successfully esterified with FA, resulting in the synthesis of FA-XG with varying degrees of FA substitution.

Adsorption of FA-XG on NOC

FA is hydrophilic due to its carboxyl group and phenolic hydroxyl group [33]. XG is also hydrophilic. However, the ester of FA and XG (FA-XG) was insoluble in a single solvent. An oil-in-water emulsion was formulated using FA-XG as a surfactant and dodecane, with XG and FA positioned externally and internally, respectively. The emulsion was adsorbed on NOC. The generated FA-XG film was analyzed using a confocal laser scanning microscope (Fig. S2), which was calibrated to detect fluorescence only when both polysaccharide-derived wavelengths (495–545 nm) and FA-derived wavelengths (562–700 nm) were present. Fluorescence was not detected for NOC (Fig. S2-A), but was observed for both HFXG-film and LFXG-film (Fig. S2-B, C). Thus, FA-XG was indeed adsorbed on NOC. The fluorescence intensity was stronger for HFXG-film than for LFXG-film. The FA contents in HFXG-film and LFXG-film were quantified by saponification followed by analyses via gas chromatography with a flame ionization detector (GC-FID). The FA content was determined after a normalization against the film weight (Table S1). In terms of density, the FA content was 6 times higher in HFXG-film than in LFXG-film, suggesting that HFXG-film was a more effective anchor for DHPs. Therefore, HFXG-film was used for the subsequent polymerization of monolignols.

Preparation of syringyl-lignin cellulose complex film (SLCF) and guaiacyl-lignin cellulose complex film (GLCF)

HFXG-film was incubated for 30 min in 50 mM Tris–HCl buffer (pH 7.5) containing SA or CA, rCWPO-C, and H2O2. The films were then washed with 50 mM Tris–HCl buffer (pH 7.5). This process was repeated for 3, 6, and 12 cycles. The resulting samples were designated as SLCF3, SLCF6, SLCF12, GLCF3, GLCF6, and GLCF12. Both SLCF12 and GLCF12 were analyzed by confocal laser scanning microscopy (Fig. S2-D, E). Compared with HFXG-film, SLCF12 and GLCF12 had a more intense red fluorescence, indicating the presence of DHPs on these artificial cell walls.

Bromide (Br)-stained SLCF and GLCF were further analyzed by scanning electron microscopy (SEM) (Fig. 2). Aggregates were detected in SLCF12 and GLCF12 that were absent in HFXG-film (Fig. 2-D, E, H). In GLCF12, spherical DHP aggregates were observed, in contrast to the linear DHP aggregates (like stalactites in a cave) in SLCF12. The width and height of linear aggregates in SLCF increased as the number of polymerization cycles increased (Fig. 2-B, C, D). Spherical DHP aggregates were detected in GLCF3 (Fig. 2-F), but were rare in GLCF6, which had DHPs spread across the artificial cell wall surface (Fig. 2-G). Following 12 polymerization cycles, spherical DHPs were observed in GLCF12 (Fig. 2-H). These findings reflected the differences in DHPs aggregation and the underlying processes between DHPs of SLCF and GLCF.

Characterization of SLCF and GLCF

DHPs content and the proportion of FA used as an anchor for DHPs were determined for both SLCF and GLCF. The DHPs content increased for SLCF and GLCF as the number of polymerization cycles increased. However, the DHP content was consistently higher for SLCF than for GLCF at each polymerization cycle. The proportion of FA used as an anchor for DHPs increased as the number of polymerization cycles increased. At the same polymerization cycle, syringyl-DHPs in SLCF used a higher proportion of FA monomers than guaiacyl-DHPs in GLCF (Fig. 3). The slope of the approximation curve was steeper for SLCF than for GLCF, suggesting that SLCF had a higher DHPs content per FA anchor than GLCF. Thus, DHPs of SLCF may have a higher molecular weight than that of GLCF.

Figure 4 presents gel permeation chromatography (GPC) chromatograms of DHPs of SLCF and GLCF for various polymerization cycles. The absorbance wavelengths were 280 and 321 nm for DHPs and FA residues, respectively. Both DHPs and FA were monitored at 280 nm, while the wavelength at 321 nm specifically monitored FA residues in DHPs. As the number of polymerization cycles increased, the molecular weight distribution curve at 280 nm for DHPs of SLCF shifted toward a higher molecular weight. The molecular weight distribution curve at 321 nm for DHPs of SLCF also shifted toward a higher molecular weight. The molecular weight distribution curves at 280 and 321 nm for the DHPs of SLCF were similar in shape. For DHPs of GLCF, as the number of polymerization cycles increased, the molecular weight distribution curves at 280 and 321 nm shifted toward a higher molecular weight. These results reflected the growth reaction of DHPs bound to the FA anchor.

GPC chromatograms (Fig. 5) for DHPs of SLCF12 and GLCF12 were compared with GPC chromatograms for the corresponding DHPs prepared in buffer without HFXG-film [designated as syringyl-DHP (S-DHP) and guaiacyl-DHP (G-DHP), respectively]. DHPs of SLCF12 had a higher molecular weight than those of GLCF12, indicating that SLCF12 facilitated the accumulation of DHPs anchored by FA better than GLCF12. A comparison between DHPs of SLCF12 and S-DHP indicated that SLCF12 had a lower molecular weight than S-DHP. A similar trend was observed when DHPs of GLCF12 were compared with G-DHP.

To investigate the effects of artificial cell walls on DHPs linkage types, SLCF12 and GLCF12 were subjected to a derivatization followed by reductive cleavage (DFRC) analysis. This analytical technique is useful for determining the content of β-O-4 in lignin on the basis of acetylated monolignols released through the selective cleavage of β-O-4 bonds, which are the main bonds in natural lignin [34]. The findings indicated that the content of β-O-4 was less in DHPs of SLCF12 than in S-DHP (Table 2). Similarly, the content of β-O-4 was less in DHPs of GLCF12 than in G-DHP. Accordingly, the linkage type of DHPs formed by an FA anchor on the polysaccharide matrix differed from that of natural lignin.

Although we used the ZL-based polymerization method in our previous studies, we used the ZT method for polymerization in the current study. Additionally, prior to the polymerization reaction, rCWPO-C was adsorbed on HFXG-film, after which monolignols and hydrogen peroxide were added for polymerization. Because peroxidase is localized in plant cell walls during plant cell wall formation, the above-mentioned method was considered to simulate the conditions found in plant systems relatively closely [12]. Furthermore, the effect of FA densities on DHP formation was investigated using LFXG (DS_FA = 0.72). DHPs of these films were then analyzed by GPC. The data indicated that all polymerization conditions resulted in molecular weights that were lower than that of S-DHP (Fig. S3).

This study was conducted to clarify the effects of artificial cell walls on DHPs growth reaction. SEM images indicated that SLCF12 and GLCF12 differ in terms of DHP aggregation. Lignin creates spherical aggregates through hydrogen bonding and van der Waals forces [35]. In previous studies, spherical DHP aggregates were detected on an artificial polysaccharide matrix [16, 19]. However, DHPs of SLCF12 were observed as linear aggregates aligned in the same orientation as cellulose (Fig. 2). To explore the mechanism underlying the formation of these linear aggregates, SA was polymerized on NOC or on films in which XG was adsorbed on NOC (SL-NOC or SL-XG-NOC). SEM images of SL-NOC and SL-XG-NOC revealed a lack of these linear aggregates of DHPs (Fig. S4-C, D). An earlier study demonstrated the polymerization of SA on acetyl xylans adsorbed on cellulose films using rCWPO-C formed spherical aggregates of DHPs [16].

HFXG was adsorbed on non-ordered cellulose film, after which Br plots were analyzed by SEM–EDX (Fig. S5). The results detected the irregular distribution of FA on cellulose without a uniform orientation (Fig. S5-B1), whereas FA was oriented in the same direction as cellulose in HFXG-NOC (Fig. S5-A1). SA was polymerized on HFXG-non-ordered cellulose film and analyzed by SEM, revealing that DHP aggregates were not consistently orientated (Fig. S4-E). Furthermore, SA was polymerized on NOC film with adsorbed LFXG. An SEM analysis confirmed that the DHPs were aligned in the same orientation as cellulose; however, linear DHP aggregates were undetectable (Fig. S4-B). SLCF contained more DHPs and had a higher molecular weight than GLCF, indicating that DHPs density was higher for SLCF than for GLCF. Additionally, β-O-4 bonds in DHPs were more abundant in SLCF than in GLCF, implying that the DHPs adopted a linear configuration. As a result, the chains likely interacted with each other and formed linear aggregates rather than spheres (Fig. 6-A).

According to the GPC chromatogram of DHPs (Fig. 4-A), DHPs of SLCF12 had similar profiles at detection wavelengths of 280 and 321 nm. By contrast, DHPs of GLCF12 had different profiles at these two wavelengths. Specifically, for DHPs of GLCF12, a peak at Rt. 18.5 was observed if only the wavelength at 280 nm was monitored (Fig. 4-D). This observation implies that GLCF12 contains low molecular weight DHPs that are not covalently bound to FA. An examination of GLCF revealed that DHPs formed in a buffer solution were deposited on artificial cell walls. Consequently, spherical aggregates of DHPs in GLCF were probably detected by SEM, unlike the linear DHPs of SLCF (Fig. 6B).

In a previous investigation, DHPs growth reaction in a buffer containing polysaccharides was attributed to the formation of complexes between polysaccharides and DHPs, which hindered precipitation by small angle X-ray scattering [36, 37]. Lignin carbohydrate complexes formed by covalent bonds between lignin and polysaccharides might modulate lignin structures [38]; however, their exact role remains unclear [39, 40]. Therefore, DHPs of SLCF12 and GLCF12 were compared with S-DHP and G-DHP in terms of their molecular weight and content of β-O-4 in DHPs. The findings indicated that DHPs of SLCF and GLCF had lower molecular weight and less content of β-O-4 than S-DHP and G-DHP (Fig. 5 and Table 2). These results showed that DHPs growth reaction was suppressed on artificial cell walls. Elschner et al. [19] reported that DHPs polymerized on films containing FA esterified to cellulose have a low molecular weight and high β-β content, which is consistent with the structural features typically associated with the ZL method. In the current study, S-DHP and G-DHP were prepared on artificial cell walls using rCWPO-C. Additionally, we expected that DHPs growth reaction would be promoted. However, we unexpectedly did not observe enhanced DHPs growth reaction. In this study, the binding of DHPs to HFXG-films may have restricted their free movement and suppressed their growth reaction.

We determined that DHPs bound to a polysaccharide matrix film have a lower molecular weight and less content of β-O-4 than DHPs prepared in a buffer. Furthermore, DHPs bound to HFXG-film may exhibit limited mobility and their growth reaction.

NOC film was prepared following the method outlined by Kondo et al. [20]. Simply put, bleached cotton linters were mechanically disintegrated into smaller fragments and subsequently dispersed in water, methanol, and N, N-dimethylacetamide (DMAc) in that order. Cellulose was then dissolved in a LiCl/DMAc solution while being stirred continuously at room temperature. Gel films were formed from this cellulose solution in a water vapor environment. These gel films were subjected to uniaxial elongation using a manual stretching device and then dried under ambient air conditions. Gel films were not elongated in the manual stretching device and were dried at room temperature for the non-ordered cellulose film.

XG was extracted from tamarind seeds sourced from Megazyme (Ireland) by a previous study [41]. The monosaccharide composition was determined to be xylose:glucose:galactose = 5.6:7.9:1.4. Sinapyl alcohol and coniferyl alcohol were synthesized using the method described in a previous study [42] and were purified through silica gel column chromatography. The preparation of rCWPO-C was conducted as per the procedures established in a previous study [43]. rCWPO-C exhibited an RZ value of 2.1, with an enzyme activity of 481 U/mg (using 2.6-DMP as the substrate). All other reagents, of extra pure grade, were procured from Sigma-Aldrich, TCI, and Fuji Film Wako and were utilized without any further purification.

Synthesis of FA esterified to XG

The synthesis of FA-XG was conducted utilizing Steglich’s esterification method [44]. 150 mg of XG was dissolved in DMSO containing 5% LiCl. FA was incorporated in quantities equivalent to either 10 or 4 times the weight of XG. Following this, DCC was added in an amount corresponding to 1 mol of FA, while DMAP was included in a quantity of 1/100 mol of FA. The resulting mixture was stirred at 65 °C for either 18 h or 3 h for esterification. Following the reaction, the sample was washed with water once and methanol four times, then subjected to vacuum drying. Once dried, the samples were ground using a mortar, and the resulting FA-XG was analyzed by FT-IR. Spectra were collected using 670 FT-IR (JASCO Japan) with ATR system. Spectra were recorded in absorbance mode from 4000 to 550 cm⁻¹ at 4 cm⁻¹ resolution with 32 scans per spectrum, and background spectra were collected in air. FA-XG was then hydrolyzed in 1 M NaOH for 20 h at room temperature. Post-reaction, the mixture was acidified with 1 M HCl to a pH of 3.0 and subsequently extracted with diethyl ether (15 mL ×ばつ 3). An internal standard of 2 mL of a 60 μmol/mL acetosyringone-methanol solution was added. The combined organic layers were washed with saturated salt water and dried over anhydrous Na₂SO₄. A volume of 3 mL of the solution was transferred to a vial, dried under nitrogen gas, and 1 mL of pyridine was introduced. After thorough shaking, 50 μL of the solution was transferred to another vial, to which 50 μL of BSTFA was added for trimethylsilylation at 65 °C for 30 min, followed by analysis using GC-FID. GC-FID analysis was performed using GC-2014 (SHIMADZU Co., Ltd., Kyoto, Japan) equipped with an InertCap column (0.25 mm ×ばつ 30 m; GL Sciences Inc., Tokyo, Japan). The temperature was programmed at a rate of 10 °C/min from 140 to 240 °C and at 30 °C/min from 240 to 310 °C. The final temperature (310 °C) was held for 12 min. The content of FA in the esterified compound was determined, and this value was subtracted from the total to ascertain the amount of XG present in the esterified compound. The average molecular weight per monosaccharide was calculated based on the ratio of the constituent monosaccharides, and DS_FA was derived from the molar mass ratio of each component.

Preparation FA-XG emulsion and adsorption of FA-XG on NOC

FA-XG was added into a mixed solution consisting of RO-water and dodecane in a volume ratio of 6:4, achieving a concentration of 5.0 g/L. An oil-in-water emulsion was prepared using an ultrasonic homogenizer operating at 2.8 W for 7 min. Subsequently, the mixed solution was diluted to a concentration of 1.0 g/L and underwent further emulsification through ultrasonic treatment for 5 min. The resulting emulsified solution, with 100 μm, was collected using a pipette and adsorbed on NOC. The film formed was then dried in a desiccator for 24 h. Following this drying process, the film was incubated in 50 mM Tris–HCl buffer (pH 7.5) for 24 h and dried. The product was designated as FA-XG-film, which was subsequently analyzed using confocal laser scanning microscopy Leica TSC SP8 (Leica, Tokyo, Japan) analysis conditions: excitation wavelength was 488 nm. Light intensity was 15%. Detection wavelengths were 495–545 nm (green) and 562–700 nm (red).

Preparation SLCF and GLCF

FA-XG film was incubated in 2 mL of 50 mM Tris–HCl buffer (pH 7.5) and subsequently combined with a solution of SA or CA (25 μmol/mL) that had been dissolved in a minimal volume of methanol and the buffer (400 μL), along with rCWPO-C (3.8 U/mL) and 200 μL of 100 mM H₂O₂ [45]. After 30 min, the polymerized FA-XG film was washed with 50 mM Tris–HCl buffer (pH 7.5). This process was conducted for 3, 6, and 12 cycles, respectively. Following this, the samples were washed with dioxane/water, 7:3 (v/v) for an hour. Artificial cell walls resulting from the polymerization of SA or CA were designated as SLCF and GLCF, respectively. S-DHP and G-DHP were prepared under identical conditions without FA-XG films, then collected by centrifugation (5000 G for 10 min), washed twice with distilled water, and subsequently freeze-dried.

SLCF and GLCF bromination and SEM–EDX analysis

SLCF and GLCF underwent bromination following the method outlined in a previous study [46]. Artificial cell walls were incubated in a solution of Br₂ and chloroform, 3:97 (v/v). After a duration of an hour, the films were sequentially washed with chloroform and ethanol. Subsequently, these films were analyzed by using SEM–EDX SU3500 (Hitachi, Tokyo, Japan), with scanning electron microscopy conducted at an accelerating voltage of 5 kV.

Proportion of FA monomers as an anchor on SLCF and GLCF

Each SLCF and GLCF was incubated in 1 M NaOH solution for 24 h at room temperature, after which the solution was neutralized using 1 M HCl and freeze-drying. Subsequently, 1 mL of 30% methanol was introduced to the dried sample, and the resulting dissolved samples were analyzed by high-performance liquid chromatography (HPLC) to determine the free FA monomers that are unbound to DHPs.

The analysis conditions were inert sustain C18 5 μm by column. The mobile phase was 30% MeOH in H₂O. The injection volume was 20 μL; the flow rate was 1.0 mL/min. The detection wavelength was 321 nm.

The all FA on FA-XG was determined by analyzing the amount of FA monomer on the FA-XG adsorbent film before polymerization and dividing by the weight of the film.

Determination of acetyl bromide-soluble lignin (ABSL)

DHPs content in SLCF and GLCF was analyzed by the method of acetic bromide [47]. SLCF and GLCF were dried at 70 °C for 3 days. SLCF and GLCF (5 mg) were digested with 1 mL of 25% acetyl bromide at 70 °C for 3 h in a glass vial, immediately cooled in ice, and then 2 mL glacial acetic acid was added to complete the reaction. The diluted sample was mixed with 900 μL of 2 M NaOH and 100 μL of 7.5 M hydroxylamine hydrochloride, and the volume was adjusted to 5 mL with acetic acid. The absorbance at 280 nm of the mixture was recorded using a spectrophotometer (Shimadzu UV-1850, Japan). The absorptivity of acetylated S-DHP and G-DHP at 280 nm was found to be 15.6 and 16.5 L/g cm, respectively. The absorption coefficient of the acetylated FA monomer was determined using commercially available FA. The absorbance of the free FA monomer contained in SLCF and GLCF, analyzed by the aforementioned method, was calculated and subtracted to ascertain DHPs content formed on artificial cell walls:

As is the absorbance of sample, αf is the absorbance of free FA monomers, V is the volume of final solution (L), α is molar extinction coefficient (L/g cm), W is the weight of the sample (g), and L is the cell thickness (cm).

Derivatization followed by reductive cleavage (DFRC)

The content of β-O-4 was analyzed by DFRC according to the previous study [48]. SLCF and GLCF (15 mg) were added into 3 mL acetyl bromide:acetic acid (20:80 v/v) at 70 °C for 30 min in a glass vial. The evaporation residue was resuspended in 3 mL acidic reduction solvent (dioxane/acetic acid/water, 5:4:1, v/v/v). Following the addition of 50 mg zinc dust, the mixture was transferred into a separating funnel with 10 mL dichloromethane and saturated ammonium chloride. The internal standard, 50 μL of tetracosane (1 mg/mL), was added, and the aqueous phase was adjusted to between pH 2 and 3 using 3% HCl. After vigorous mixing, the organic layer was collected, and the extraction was repeated three times with 10 mL dichloromethane. The organic layer was dehydrated with magnesium sulfate, redissolved in 1.5 mL dichloromethane, and acetylated overnight with 0.5 mL acetic anhydride and 0.5 mL pyridine. Once acetylation reagents were removed, the samples were dissolved in 50 μL dichloromethane and subjected to gas chromatography with GC–FID. GC–FID analysis was performed using a Shimadzu GC-2014 (SHIMADZU Co., Ltd., Kyoto, Japan) equipped with an InertCap column (0.25 mm ×ばつ 30 m; GL Sciences Inc., Tokyo, Japan). The temperature was programmed at a rate of 3 °C/min from 140 to 240 °C and at 30 °C/min from 240 to 310 °C. The final temperature (310 °C) was held for 12 min. The amounts of coniferyl alcohol and sinapyl alcohol were determined using calibration curves derived from acetylated pure standards.

GPC analysis

Artificial cell wall was incubated in 0.5 M NaOH solution (2 mL) at room temperature for 24 h, after which the alkaline solution was neutralized using 1 M HCl. Following the drying process, the resulting residues were dissolved in a mixture of dioxane and water (4:1 v/v) containing 40 mM LiCl, and the molecular weight was analyzed by GPC. The analysis was conducted with TSKgel α-3000 (TOSOH, Tokyo, Japan, exclusion limit: 90,000 for PEG in water, 100,000 for polystyrene in THF) GPC column. Dioxane and water (4:1, v/v) containing 40 mM LiCl served as the mobile phase with a flow rate of 0.5 mL/min and at 25 °C. The absorbance of the eluted samples was detected at 280, 270, 260, and 321 nm.

Examination of polymerization conditions of DHPs

HFXG-film was incubated in 50 mM Tris–HCl buffer (pH 7.5) containing rCWPO-C at a concentration of 30 U/mL for an hour at room temperature. Subsequently, HFXG-film was washed in the buffer without rCWPO-C for 2 h. This film was designated as HFXG-film-AD. HFXG-film-AD was then incubated in 2 mL of 50 mM Tris–HCl buffer (pH 7.5) and combined with a solution of SA (5.0 mg/mL) dissolved in a minimal volume of methanol and RO-water. Additionally, 4 μL of 100 mM hydroxide peroxide was introduced at 2-h intervals for a total of three additions, after which the mixture was allowed to stand for an additional 18 h. The film prepared by this process was named SLCF-AD.

Three distinct solutions were prepared. Solution I consisted of 25 mg of SA dissolved in 5 mL of 50 mM Tris–HCl buffer (pH 7.5). Solution II contained 12 U/mL of rCWPO-C dissolved in 5 mL of 50 mM Tris–HCl buffer (pH 7.5) and was combined with HFXG-films. Solution III was created by mixing 50 mM hydrogen peroxide with 5 mL of 50 mM Tris–HCl buffer (pH 7.5). Solutions I and III were introduced dropwise into Solution [8] II using two separate syringes connected to SJ-1211 II-L syringe pump (ATTO, Tokyo, Japan) at a flow rate of 0.5 mL/h for 10 h, after which the mixture was allowed to react for an additional 16 h. Following the reaction, the films were washed in 70% dioxane for one hour and were designated as SLCF-ZT. LFXG (DS_FA = 0.72) was utilized, and SLCF-LFXG was prepared as described. The DHPs of all artificial cell walls were extracted as outlined earlier and subsequently analyzed by GPC.

Data sharing does not apply to this article as no data sets were generated or analyzed during the current study.

ABSL:

Acetyl bromide-soluble lignin

Br:

Bromide

CMF:

Cellulose microfibrils

CA:

Coniferyl alcohol

DFRC:

Derivatization followed by reductive cleavage

DHPs:

Dehydrohydrogenative polymers

DMAc:

Dimethylacetamide

FA:

Ferulic acid

FA-XG:

Ferulic acid-xyloglucan

GC-FID:

Gas chromatography with a flame ionization detector

G-DHP:

guaiacyl-dehydrogenative polymers

GLCF:

Guaiacyl-lignin cellulose complex film

GPC:

Gel permeation chromatography

HFXG:

High ferulic acid xyloglucan

HPLC:

High performance liquid chromatography

HRP:

Horseradish peroxidase

LFXG:

Low ferulic acid xyloglucan

NOC:

nematic ordered cellulose

rCWPO-C:

recombinant cationic cell wall peroxidase

SA:

Sinapyl alcohol

S-DHP:

syringyl-dehydrogenative polymers

SEM:

Scanning electron microscopy

SLCF:

Syringyl-lignin cellulose complex film

XG:

Xyloglucan

ZT:

Zutropfverfahren

ZL:

Zulaufverfahren

The suggestions from Professor Yasumitsu Uraki are acknowledged. We thank Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

This work was supported by JSPS Grants-in-Aid for Scientific Research (KAKENHI) (Grant Number JP21H04730).

Authors and Affiliations

1. Department of Agro-Environmental Sciences, Graduate School of Agriculture, Kyushu University, 744 Motooka Nishi-Ku, Fukuoka, 819-0395, Japan

   Naofumi Matsuhisa, Go Tamura & Natsumi Kawaguchi

2. Faculty of Agriculture, Kyushu University, 744 Motooka Nishi-Ku, Fukuoka, 819-0395, Japan

   Koki Fujita & Yuji Tsutsumi

3. Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo, 060-8589, Japan

   Naofumi Matsuhisa

4. Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo, 060-8589, Japan

   Kengo Shigetomi

Contributions

YT was the principal investigator of this project. YT and NM designed the experiments. NM, GT, and NK performed the experiments and analyzed the data. KF and KS participated in analyses and discussions of the results. NM wrote the manuscript. YT revised the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Yuji Tsutsumi.

Competing interests

The authors declare that they have no competing interests.

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