Polysaccharide Degrading Biocatalysts

The transformation of polysaccharides into valuable compounds for health and industry requires the careful application of enzyme protocols and controlled biocatalysis.

Polysaccharide-Degrading Biocatalysts provides a thorough grounding in these biocatalytic processes and their growing role in the depolymerization of polysaccharides, empowering researchers to discover and develop new enzyme-based approaches across pharmaceuticals, fuels, and food engineering. Here, over a dozen leading experts offer a close examination of structural polysaccharides, genetic modification of polysaccharides, polysaccharide degradation routes, pretreatments for enzymatic hydrolysis, hemicellulose-degrading enzymes, biomass valorization processes, oligosaccharide production, and enzyme immobilization for the hydrolysis of polysaccharides, among other topics and related research protocols. A final chapter considers perspectives and challenges in an evolving, carbohydrate-based economy.

• Describes the role of enzymes in the degradation of polysaccharides to obtain building blocks for biochemical processes
• Covers new tools for enzymatic evolution, research protocols, and process strategies contributing to large-scale applications
• Explores the use of polysaccharide hydrolysis products in the areas of pharmaceuticals, fuels, and food engineering
• Features chapter contributions from international experts

• Language: English
• Publisher: Academic Press
• Release date: Feb 15, 2023
• ISBN: 9780323983150

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Chapter 1: Plant cell wall polysaccharides: Methodologies for compositional, structural, and physicochemical characterization

Ingrid Santos Migueza,b; Fernanda Thimoteo Azevedo Jorgea; Roberta Pereira Espinheiraa,b; Ronaldo Rodrigues de Sousaa,b; Viridiana Santana Ferreira Leitãoa,b; Ricardo Sposina Sobral Teixeirab; Carmen Lucia de Oliveira Petkowiczc; Ayla Sant’Ana da Silvaa,b    a Division of Catalysis, Biocatalysis and Chemical Processes, Ministry of Science, Technology, and Innovations, Instituto Nacional de Tecnologia, Rio de Janeiro, RJ, Brazil

b Department of Biochemistry, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

c Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba, PR, Brazil

Abstract

Plant wastes are the prime choice of feedstock for producing new chemicals and materials required for the transition toward a biobased economy. Plant biomass is mainly constituted by plant cell walls (PCWs), primarily composed of polysaccharides presenting a wide diversity of glycosyl residues content and linkages, leading to different structures and physicochemical properties. Thus, the effective use of plant biomass requires a comprehensive understanding of PCW polysaccharides through characterizing methods that can guide the selection of suitable industrial applications well-matched with each biomass source's properties. In this context, this chapter discusses consolidated analytical methodologies for PCW polysaccharides characterization grouped into three categories: (i) glycosyl residue composition analysis, (ii) structural analysis, and (iii) complementary analysis. Furthermore, a critical discussion of their main advantages and limitations is provided to aid the reader in identifying the most appropriate methods for different biomass samples.

Keywords

Plant cell wall; Structural polysaccharide; Sugar compositional analysis; Glycosidic linkage analysis; Polysaccharide structural analysis; Polysaccharide physicochemical properties

1: Introduction to the analysis of plant cell wall polysaccharides

The plant cell wall (PCW) has mechanical properties related to its components and their interactions. As the main PCW components, polysaccharides assume a structural role in providing mechanical stability for the cell [1]. Arranged in the primary and secondary walls, the wall layers of this extracellular matrix consist of polymer networks based on covalent and noncovalent bonds [2].

In the primary cell wall, 90% of the dry weight corresponds to polysaccharides, among which cellulose microfibrils and hemicelluloses are embedded in a matrix of pectin. Proteins and phenolics are also typical components of primary PCWs of dicots and grass, respectively. On the other hand, in the secondary cell wall, pectin and proteins are low or absent, and 60% of the dry weight is constituted of cellulose and hemicelluloses, enclosed by lignin, composing a recalcitrant structure whose polymers are harder to isolate compared with the primary wall components [3,4]. Fig. 1 illustrates a general representation of the layered and heterogeneous PCW.

Fig. 1

Fig. 1 Representation of the major components from the primary and secondary plant cell walls.

Despite the similarities, the composition and organization of the PCWs vary depending on the biomass species and other biological characteristics, such as the cell type, function of the tissue, and growth conditions [1]. Moreover, botanical studies indicate that polysaccharides change their structure during plant cell development, impacting the overall composition [5].

The cell wall polysaccharides are highly heterogeneous due to the diversity of glycosyl residues with different configurations (D/L and α/β), glycosidic linkages, degree and pattern of branching, and presence of substituents such as methyl and acetyl groups [6], which render them as challenging analytical targets. Increased complexity of cell wall polymers arises from inter- and intrachain interactions between polysaccharides and the cross-linking between structural carbohydrates by lignin. Consequently, these carbohydrates can exhibit many physicochemical properties, biological functions, and potential industrial applications that significantly depend on their structural and molecular features [7].

Nowadays, lignocellulosic biomass (i.e., PCWs) is the primary choice of feedstock for producing alternative fuels and chemicals to meet the needs of the transition to a biobased economy as an action to address the climate change [8]. Given that PCWs are primarily composed of polysaccharides, methods for characterizing the PCW carbohydrates become fundamental for determining target applications well-matched with each biomass source's properties to reach economic and reproducible processes. For this, the accurate determination of glycosyl residues composition, structures of polysaccharides, and properties is essential for understanding the structure-activity relationship and calculating conversion yields and process economics [9].

Due to the complexity of those macromolecules, a complete characterization of PCW polysaccharides requires time-consuming and often complex and expensive analytical methods. Thus, depending on the scientific question behind the characterization effort, different strategies can be traced and combined as analytical approaches based on the sample's conditions and biological characteristics. In this chapter, we will discuss techniques to analyze different aspects of the chemical and physical nature of polysaccharides from the PCWs (Fig. 2), aiming to aid the reader in identifying the most appropriate methods for different biomass samples. Still, we do not intend to deliver an exhaustive review of each technique.

Fig. 2

Fig. 2 Analytical tools to characterize structural polysaccharides from the plant cell wall.

The chapter initiates by presenting the most common carbohydrates’ isolation and purification methods that can be necessary before multiple characterization techniques. Then, the discussion of consolidated methodologies was grouped into three categories: (i) glycosyl residues composition analysis, (ii) structural analysis, and (iii) complementary methodologies. Nevertheless, it is necessary to emphasize that we do not intend to be comprehensive but to give an overview of the most commonly reported techniques, in view of the vast amount of work related to this topic and other numerous valuable characterizing methods not included here.

Considering the importance of PCW characterization to the economic competitiveness of the production of sugar syrups derived from lignocellulosic biomass, this chapter has gathered relevant information on this subject, aiming to provide a critical discussion on the limitations and advantages of the primary methodologies applied today by most research groups in this area.

2: Sample preparation for the polysaccharides’ analysis

The analysis of PCW polysaccharides can range from total sugar composition identification techniques to advanced methods for detailed structural features. Depending on the chosen methodology, the purpose of the analysis, and the samples’ nature, sample preparation steps may be required.

The main objective of sample preparation before polysaccharide analysis is to fractionate (partially or totally) the PCW components to eliminate or reduce as much as possible interferences in the analysis, which can occur by the presence of proteins, lipids, phenols, pigments, free sugars, or other polysaccharides [10,11]. However, separating polysaccharides from PCW can be laborious due to the heterogeneous nature of the samples [7]. Thus, critical analysis regarding the stability and representativeness of the obtained samples after preparation should always be carried out for a more accurate determination of carbohydrates in the PCWs [12].

Typically, before polysaccharides’ analysis, several steps can be applied to PCW samples, starting from routine procedures for size reduction and drying, passing through solvent extraction, and, depending on the study's objective, going through a series of fractionation steps for isolating an enriched fraction of specific polysaccharides (Fig. 3).

Fig. 3

Fig. 3 Fractionation steps for isolating enriched fractions of specific polysaccharides from plant cell walls. AIR , alcohol-insoluble residue.

As starting points for preparing the samples, a myriad of processes may include cut operations, drying by air, heating or lyophilization, freezing, grinding, and sieving. Grinding operations are of utmost importance to obtain relatively homogeneous powder suitable for subsequent chemical treatment steps and can be applied to different tissues [13]. In some cases, it is recommended to freeze the sample by using liquid nitrogen as quickly as possible to suppress enzymatic activities in vegetal tissues, which could result in partial degradation and/or modification of polymers [14].

After grinding, the most widely adopted step is the samples’ solvent extraction to prepare an alcohol-insoluble residue (AIR). The AIR preparation method consists of alcohol-based solid-liquid extractions, in which nonstructural materials are separated from plant biomass, such as inorganic salts, low-molecular-weight metabolites, and free saccharides. At the same time, the alcohol treatment provides the inactivation of endogenous enzymes. For this process, methanol, ethanol, or isopropanol can be used in concentrations that vary from 10% to 96% in aqueous solution in combination or not with other additional organic compounds and heating, although ethanol is ubiquitously adopted [12,15]. Lipids, chlorophyll, and some pigments can also be removed, depending on the solvent used [12]. Considering that there is a significant variation in AIR preparation protocols, one must evaluate the methodology most suitable for the samples’ characteristics, as different AIR preparation parameters might impact on extraction, detection, and degradation of certain PCW components [12,13]. For more detailed information, refer to the study of Fangel et al. [12], which compared 10 AIR preparation protocols.

The obtained AIR by those procedures enables further analysis of PCW polysaccharides by many methodologies, such as the ones aiming at the total glycosyl residues composition; however, AIR is not effective in fractionating polymeric carbohydrates, which requires several additional steps for a more comprehensive analysis of individual polysaccharides [16] (Fig. 3). Also, other macromolecules that are not related to structural polysaccharides may remain insoluble in alcohol, such as starch, RNA, lipids, polyphenolic compounds, and proteins from the cytoplasm, demanding additional extraction steps of the obtained AIR, depending on the accuracy required for polysaccharide characterization and the type of vegetal tissue [16,17]. For example, if necessary, AIR may be defatted by a proper organic solvent (as petroleum ether) and deproteinated by using Sevag reagent, a mixture of chloroform and butanol (4:1 v/v) able to denature the free proteins present in PCW [18].

A detailed fractionation protocol usually starts with removing starch from the AIR. The presence of starch (a nonstructural carbohydrate) may lead to an inaccurate interpretation of the glycosyl compositional analysis of structural polysaccharides and complicate the interpretation of linkage data [13]. Starch can be removed by enzymatic extractions using α-amylases in a neutral phosphate buffer or by extraction with 90% dimethyl sulfoxide (DMSO). Mammalian pancreatic amylases are preferable, as microbial amylases might contain hydrolytic enzymes that attack other structural polysaccharides. In contrast, the use of DMSO needs to be carefully evaluated as it may lead to the removal of other cell wall components [13].

The destarched AIR samples then undergo a series of sequential extraction with increasingly harsh reagents, aiming to obtain fractions enriched in different polysaccharides. Hot water, ammonium oxalate, and diluted sodium carbonate are respectively used to remove pectins that are weakly bounded, calcium-associated, and covalently attached. Ethylenediaminetetraacetic acid (EDTA) and 1,2-cyclohexylenedinitrilotetraacetic acid (CDTA) are alternative chelators used in this step that can affect the calcium bridges in which pectins are held. Still, their use has the drawback of difficult removal by further dialysis steps [19].

The sequential fractioning steps involve using aqueous alkaline solutions (NaOH or KOH) in different concentrations and treatment conditions. Alkaline solutions enable the removal of hemicelluloses, including lignin-bounded polysaccharides, by affecting the hydrogen bonds and promoting hydrolysis of ester linkages between polysaccharides and lignin. However, in the alkaline environment, terminal reducing units of polysaccharides may be attacked in a chain-reaction mechanism leading to an intense carbohydrates degradation. Thus, borohydride salts (as NaBH4) are used in association with alkaline solutions to promote a reduction of end units and avoid the depolymerization of carbohydrates [20].

After alkaline extraction, lignin can be removed from the recovered solid by sodium chlorite [21]. For samples undergoing sodium chlorite treatment, after removing chlorite gas, the washed pellet obtained after lignin extraction can undergo a postchlorite extraction with an alkaline solution to extract lignin-bound polysaccharides. However, this step can be omitted for samples that are not lignified or do not contain significant levels of lignin [16].

Finally, the residue obtained will contain the typically insoluble polysaccharides, such as cellulose and linear mannan. In addition, all the extracts obtained in each of the mentioned steps can be dialyzed and lyophilized for storage until undergoing detailed characterization analysis [16]. Alternatively, the polysaccharides can be recovered from the dialyzed extract by precipitation using ethanol or isopropanol (2–3 volumes), followed by drying under mild temperatures.

After AIR preparation or for polysaccharides derived from each fractionation step, the determination of the monosaccharide composition requires breaking the polysaccharides into their monomeric units [22,23]. In these cases, total acid hydrolysis should be applied to conciliate effective depolymerization of polysaccharides without noticeable degradation of resultant monosaccharides, which means that the concentration of reagents, time, and temperature should be carefully adjusted [15]. The use of trifluoroacetic acid (TFA), proposed by Albersheim in 1967 [24], is wide disseminated due to the easy separation by evaporation posthydrolysis. However, TFA hydrolysis cannot depolymerize all the cellulose content in crystalline form, being more suitable for analyzing other components of PCW [13]. A typical alternative is carried out with mineral acids (HCl or H2SO4), as the method proposed by Saeman et al. and optimized by Sluiter and coworkers [25,26]. While TFA is volatile and easily removed from the sample (which is a requirement for some analysis), it will only hydrolyze the noncellulosic component of PCW. Thus, for some samples, the use of sulfuric acid will be necessary, and its removal (if needed) will require precipitation with barium hydroxide or neutralization with N,N-dioctylamine in chloroform followed by successive washes [13]. However, constraints related to toxicity and safety issues, reaction time, and separation posthydrolysis led researchers to find alternatives, such as protocols based on enzymatic hydrolysis [27].

It should be noted that, in this section, we intended to bring some standard preparation methods before general procedures of PCW polysaccharide analysis. Still, specific studies may require additional preparation steps not included here.

3: Chemical analysis of plant cell wall polysaccharides—Glycosyl residues composition

Usually, an initial assessment of the polysaccharide content of biomass samples is performed by analyzing the glycosyl residue composition. The qualitative and quantitative information obtained with these analyses is essential to have clues on polysaccharides’ content, which will help select other overlapping and complementary characterizing methodologies aiming to reconstitute the original macromolecular structure that they are derived from. After preparing the AIR from the target sample or isolating the carbohydrate fraction, it is possible to go on with the glycosyl residues composition determination and quantification [28].

In these analyses, the polysaccharides are hydrolyzed into monosaccharides (as commented in the Sample Preparation section) that are usually detected by chromatographic methods that can further require an additional derivatization step. The accuracy of the quantitative data obtained by these approaches will depend on the efficiency of the depolymerizing method for the cleavage of glycosidic linkages, the stability of released monosaccharides, and the effectiveness of derivatizing methods if required [28,29]. Despite the constraints of chromatographic glycosyl residues analyses aiming to build up the PCW polysaccharide content quantitatively, those techniques are preferable and more specific than the routine detergent fiber gravimetric methods used to predict total cellulose and hemicellulose content in forage crops, such as the Van Soest method [30,31]. However, it is important to mention that the glycosidic linkages between acidic monosaccharides and acidic or neutral monosaccharides are notoriously stable [32]. Thus, irrespective of the monosaccharide analysis method, it is difficult to determine the quantitative monosaccharide composition of polysaccharides rich in uronic acids based on the monomers released by acid hydrolysis.

In this section, three common quantitative methods used for monosaccharide composition analysis, high-performance liquid chromatography, high-performance anion-exchange chromatography, and gas chromatography, will be discussed. These analytical tools are summarized in Fig. 4.

Fig. 4

Fig. 4 Analytical methods for identifying and quantifying glycosyl residues constituents of PCW polysaccharides; (A) HPLC-RID/UV and HPAEC-PAD; (B) alditol acetate (AA) and trimethylsilyl (TMS) derivatives analysis by GC-FID/MS.

3.1: High-performance liquid chromatography (HPLC)

HPLC is a common tool available in many research laboratories for quantitative analysis of sugars derived from the hydrolysis of PCW polysaccharides. Combining the appropriate stationary phase and detection system can give rapid and specific responses with versatility [28]. The choice of the stationary and mobile phase type varies according to the sugar's nature that will be separated. The separation mechanisms include ion-exchange, ion-exclusion, ion-pair, hydrophilic interaction, or reverse-phase [33]. The detection systems for sugar analysis by HPLC include the refractive index detector (RID), ultraviolet and visible light absorption (UV-Vis), photodiode array detector (PDA), fluorescence, evaporative light scattering detector (ELSD), charged aerosol detector (CAD), or mass spectrometry (MS), for example [34].

Among the detectors, HPLC-RID has become one of the most common methods for a direct quantitative determination of five neutral monosaccharides derived from structural polysaccharides (glucose, xylose, galactose, mannose, and arabinose) since intact carbohydrates do not have UV chromophores. RID is a universal detector that responds to all solutes as long as the refractive index of analytes is distinguished from the eluent [35]. Much of the preference for this methodology comes from the simplicity of the technique, mainly concerning the sample preparation (no derivatization required) [36]. In fact, one of the most well-established and cited modern protocols for biomass composition analysis, developed by the National Renewable Energy Laboratory (NREL), United States, uses HPLC-RID as a default method to quantify monosaccharides in biomass hydrolysates [37]. This applicability of HPLC-RID for reliable routine quantification of glycosyl residues from PCW was reported decades ago [38] and has been used to characterize a variety of woody and herbaceous biomasses. For monosaccharide determination, the protocol recommends using lead cation (Pb²+) exchange columns and a simple isocratic HPLC separation with water as a mobile phase, which gives a near-baseline resolution for the common biomass neutral sugars [39].

However, the NREL methodology does not detect deoxy (rhamnose and fucose) and acidic sugars (glucuronic and galacturonic acids), preventing the correct analysis of pectins and some hemicelluloses such as xyloglucans and glucuronoarabinoxylans [25]. Another inconvenience of the method is that RID detection is based on differential measurements; therefore, it is influenced by temperature, composition, and the flow rate variation of the mobile phase, which must be expertly controlled, without using gradient elution. Also, this method is not sensitive enough for trace sugar analysis and has poor selectivity [40]. In fact, the quantification of glucose and xylose, the major biomass sugars, by HPLC-RID using a Pb²+ column has been demonstrated to be more accurate when compared to the analysis of less abundant sugars, such as mannose, galactose, and arabinose [41].

As an alternative to RID, photometers detectors based on UV-Vis or PDA are widely used in reversed-phase (RP) HPLC analysis of carbohydrates [42]. UV-Vis detectors quantify the analyte concentration according to the absorption light between 190 and 600 nm wavelengths, have relatively high sensitivity, but are nonselective, and usually depend on the previous derivatization of the sugars since most carbohydrates absorb light at a UV range below 200 nm [33,35]. There are precolumn and postcolumn reported methods to derivatize the samples, where the most popular and facile method applied is the precolumn derivatization using 3-methyl-1-phenyl-2-pyrazoline-5-one (PMP), which reacts with reducing carbohydrates under mild conditions [43]. This method has been successfully applied in various studies employing carbohydrate analysis to determine monosaccharides, including its validation using an HPLC-DAD with precolumn PMP derivatization for simultaneous estimation of seven neutral (mannose, rhamnose, glucose, galactose, xylose, arabinose, and fucose) and two acidic sugars (glucuronic and galacturonic acids) with baseline separation aiming to analyze cell wall polysaccharides from agro-industrial wastes [42,44]. Other precolumn methods applied to RP-HPLC-UV-Vis detection of PCW sugars report the derivatization by reductive amination using 2-aminobenzamide or 3-amino-9-ethylcarbazole [45,46]. Despite the advantageous increase in detection levels with the UV-Vis detectors, the derivatization techniques are a laborious step that makes the method less attractive for routine analysis than HPLC-RID. Some studies evaluated the UV-based identification of unlabeled sugars using both isocratic and gradient methods, providing comparable results to RID in terms of the limits of detection and quantification, but its application to the variety of PCW sugars needs to be demonstrated [35].

Many other HPLC-based methods have also been proposed to analyze PCW sugars. For example, to improve the selectivity and sensitivity of the technique, online derivatization through a postcolumn reaction of reducing sugars with Cu(II)-neocuproine reagent has been reported [47]. Moreover, to better detect hemicellulose and cellulose monosaccharides, different detectors have been successfully applied, such as HPLC-CAD, which increased the specificity of the analysis compared with the NREL method [48].

HPLC-based analyses can range from simple and rapid assays to more complex procedures to accurately determine PCW sugar composition. The HPLC-RID has excellent versatility as a universal detector in routine analysis of PCW sugars. However, it has low stability and low sensibility, and the isocratic elution does not enable the separation of all monomers from PCW polysaccharides. The UV-Vis solves the problem with sensibility; nevertheless, it requires laborious derivatization steps. Therefore, the appropriate technique can be chosen by balancing the importance of accuracy versus the time consumed for the analysis and the available resources [34].

3.2: High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

Among liquid chromatography techniques, HPAEC-PAD is suitable for the simultaneous identification of neutral and acidic sugars from PCW polysaccharides with high selectivity and sensitivity (low-picomole quantities), also enabling the detection of oligosaccharides derived from partial hydrolysis of PCW, having the benefit of not requiring derivatization steps [49,50].

HPAEC-PAD analysis for mono- and oligosaccharides was developed in the 1980s due to the development and combination of two analytical technologies (HPAEC and PAD). The methodology was based on the fortuitous observation of borate exclusion from a NaOH-borate eluent, which led to the separation of reducing sugars as oxyanions by anion-exchange chromatography at high pH values [51,52]. In parallel, the development of a cyclic, pulsed amperometric technique with cleaning potentials applied to an electrode that is regenerated in every cycle allowed the stable and reproducible detection of carbohydrates, which circumvented the problem of electrode fouling when using constant-potential amperometric detection [52].

The HPAEC carbohydrate separation lies in the weakly acidic properties of sugar molecules and their dissociation constants, in high pH (>12), sugars show different net acidity due to differences in their hydroxyl groups [53], resulting in different pKa values and retention capacities. For example, mannose, glucose, and galactose, three hexoses, have pKa values of 12.08, 12.28, and 12.39, respectively [54].

The eluents used for HPAEC analysis are generally composed of NaOH or other hydroxides, and pusher anions with higher affinity to the stationary phase than hydroxide (such as acetate) and gradients can be used to increase the quality of the results. The standard stationary phase used for HPAEC carbohydrate analysis is the quaternary-ammonium-bonded pellicular anion-exchange resins that, in alkaline solutions, interact with neutral sugars. The alkaline environment is crucial for the sugars binding to the column and the detection method, as the electrode detects the products of the sugars oxyanions oxidation [55,56].

Specific methods and columns able to handle sulfate from H2SO4 hydrolysis of plant materials have been developed to give better resolution of the typical monosaccharides derived from PCW [50,56,57] and have been applied in various studies, including the analysis of the cell wall carbohydrates of multiple plants, such as broccoli, carrot, tomato, sisal waste, cotton, sesame, and grass, to name a few [58–61]. Thus, HPAEC-PAD is becoming a well-established analytical technique for the PCW glycosyl residue composition characterization.

However, few drawbacks are observed during PCW sugar analysis. For the characterization of sugars from PCW, satisfactory separation of rhamnose from arabinose and xylose from mannose has been one of the main challenges of this technique [62]. The almost coelution of xylose and mannose, especially for samples that are rich in xylan and/or xyloglucan, was observed by Biswal and coauthors [61], although this can be overcome by method optimization [50,62]. Also, a baseline draft could occur when using acetate anions as pushers, which is mainly observed when analyzing oligosaccharides [63]. Furthermore, the lack of ready compatibility with mass spectrometry to further investigate the peaks can be considered another disadvantage of this technique when dealing with plant samples of unknown composition or unknown peaks derived from plant biomass acid hydrolysates [50,61]. Nonetheless, advantages related to the needlessness derivatization, the selectivity, and sensitivity of the detection method, almost 200 times higher than HPLC-RID analysis [64], place HPAEC-PAD as one of the best methods of choice for the direct analysis of PCW glycosyl residues composition [65].

3.3: Gas chromatography—The alditol acetate (AA) and trimethylsilyl (TMS) derivatives

The quantitative analysis of sugars in PCW polysaccharides by GC has been employed since the 1960s [24], with the flame ionization detector (FID) being the most common detector. Nowadays, this tool is, in most cases, coupled with a mass spectrometry detection (GC-MS) [66] to enhance the specificity for an accurate analysis of the glycosyl residues.

As the method relies on evaluating volatile compounds and sugars have finite volatility, it is necessary to derivatize the monosaccharides in a volatile form previously. A specific and detailed review of the main derivatization techniques of carbohydrates for GC and GC-MS analyses can be found in the report of Ruiz-Matute and collaborators [67]. It is important to note that the chosen methodology must be adequate to the type of biomass sample and its expected carbohydrate content. Currently, the common derivatives used are methyl ethers, acetates, trifluoroacetate, and trimethylsilyl ethers. Here, we will present the most common sugar composition analysis methods for PCW samples: the alditol acetate (AA) and trimethylsilyl (TMS) methods [68]. Before the derivatization for GC analysis, samples must be hydrolyzed, and any trace of acids used should be removed.

Various techniques to acetylate the PCW monosaccharides are variances of the original AA method [24,69,70]. In the most straightforward protocol detailed by Pettolino et al. [13], the monosaccharides are reduced to alditols using NaBH4 or NaBD4 and sequentially acetylated using acetic anhydride. Prior to acetylation, it is important to entirely remove boron since borate can form complexes with the hydroxyl groups resulting in underestimated levels of monosaccharides. This is usually done by treatment with methanol followed by evaporation [71]. The hydroxyl groups from alditols readily react with acetic anhydride in the presence of a catalyst, being the most common sodium acetate, pyridine, and methylimidazole [72]. The produced volatile compounds can be analyzed by GC, whose results are single peaks for each sugar identified by their retention time relative to the internal standard, usually myo-inositol or scyllo-inositol.

However, it has been reported that glucose content derived from the hydrolysis of various lignocellulosic biomass was slightly underestimated by AA-GC analysis compared to HPLC-RID, while it gave a better response to hemicellulose-derived sugars [41]. Also, the AA classic method is unsuitable for the acidic sugars, impairing the characterization of structural polysaccharides constituted by these sugars, such as pectin and glucuronoxylan [61]. The entire composition can be achieved after carbodiimide activation and reduction of the carboxyl groups using NaBD4, which yields the 6,6-dideuterio neutral sugars of galacturonic and glucuronic acids, to be analyzed by monosaccharide analysis by AA/GC-MS method [13,73]. Additionally, the reduction of carboxyl groups eliminates hydrolysis's difficulties, enabling a more reliable quantification. Alternatively, a spectrophotometric method [74] can be used to provide the total uronic acid content but with no identification of the uronic acid kind.

In contrast, the TMS method has mainly been applied to PCW neutral sugars and acidic sugars analysis [75]. The methodology, in the majority, follows the procedure description of Sweeley et al. [76] with some adaptations and new silylating reagents. With a simpler sample preparation compared with the AA method, hydrolyzed glycosyl residues can be submitted to sequential methanolysis and trimethylsilylation, resulting in TMS-methyl glycosides with favorable volatility and stability [61], and GC is used to separate TMS derivatives. Nevertheless, the interpretation of the GC profiles is difficult due to the presence of multiple peaks derived from each monosaccharide derivative, of both the α- and β-anomeric configurations and the pyranose and furanose ring forms [77]. Furthermore, the TMS method is not indicated for insoluble polysaccharides’ analysis, such as cellulose and linear mannan, since using a harsh hydrolysis step can result in the loss of less stable portions of the sugars [78].

The comparison of carbodiimide method/AA and TMS approaches efficiency in analyzing the glycosyl residues composition from dicot and grass species has shown that the acid conditions regularly employed are not enough to hydrolyze the cellulose; therefore, quantification is usually restricted to noncellulosic sugar content [61]. Both methods provided comparable results for neutral and acidic sugars present in the PCWs, but the TMS method gave a slightly higher yield for most sugars.

In summary, the GC-MS technique provides further refinement in identifying PCW carbohydrates with a good sensitivity, resolution, and robustness with simple instrumentation [28]. However, the laborious derivatization procedures are subject to significant experimental errors, are time-consuming, and are not very practical for routine analysis in an industrial environment, such as biorefineries for biomass processing. Despite these constraints, AA and TMS methods have been recommended by the American Society for Testing and Materials [68] to analyze sugars in plant biomass. It has been demonstrated that the carbodiimide method/AA, TMS, and HPAEC approaches provided highly comparable results and are suitable for investigating the PCW polysaccharides [61]. Therefore, technicians should critically evaluate the available toolkit for PCW glycosyl composition analysis to select the most suitable technique (or combinations of methods) by considering the factors.

4: Structural analysis of plant cell wall polysaccharides

The analysis of the monomeric sugar composition of PCW samples can provide a lot of helpful information to support studies and applications. However, some scientific endeavors and industrial uses require more detailed molecular knowledge to understand structure-property relationships [6]. Therefore, elucidating anomeric configuration, linkage pattern, and side chains distribution will often be necessary.

The specific nature of carbohydrates allows innumerable structural possibilities, from multiple branching and substitution, furanose or pyranose ring sizes, and different configurations (D or L, and the anomeric α- or β-glycosidic linkages). Hence, it is challenging to contemplate structural polysaccharides’ analysis to cover a broad range of structural aspects [7].

However, there are currently some analytical tools for analyzing these complex PCW polysaccharides, although not every method is suitable for all polysaccharides in the PCW, which leads to the use of different complementary techniques for a complete characterization [6,79].

This section will overview the use of nuclear magnetic resonance, methylation analysis, and immunological approaches, as those methodologies have been commonly used for detailed structural characterization of PCW polysaccharides (Fig. 5).

Fig. 5

Fig. 5 Analytical methods for the identification of glycosyl residues constituents of PCW polysaccharides; (A) NMR spectroscopy; (B) methylation analysis by gas chromatography; (C) glycome profile/immunolocalization.

4.1: Nuclear magnetic resonance spectroscopy (NMR)

NMR is one of the main techniques applied for the structural characterization of complex PCW polysaccharides, giving information at an atomic and molecular level to elucidate the structure and conformation. It enables confirmation of glycosyl residues composition, the identification of α- or β-anomeric configurations, the position of substituent groups, linkage patterns, and conformation [80,81]. Structural analysis of PCW polysaccharides by NMR is typically performed to various degrees by choosing different 1D and 2D NMR experimental strategies that usually rely on NMR nuclei ¹H and ¹³C. This section will briefly comment on the solution-state NMR, the most used in carbohydrate analysis, but it will also encompass solid-state NMR.

Advances in NMR technology have made the analysis of purified polysaccharides’ fractions and whole PCW samples possible. The study of the whole PCW by NMR can improve the understanding of polysaccharides’ interactions with each other and with other biomolecules (such as lignin), but the increased complexity in spectra interpretation limits the complete characterization of PCW polysaccharides [82,83]. Solid-state NMR can be applied to evaluate intact cell walls with minimal perturbance of the physical state and molecular interactions of polysaccharides [84,85] but with limited resolution. As an alternative to improve spectral resolution, solution-state NMR can be applied to analyze full dissolved PCW without prior polysaccharide purification. For this purpose, the PCW material is derivatized through acetylation or methylation and then dissolved in a common perdeuterated organic solvent or an appropriate solvent system, such as DMSO-d6/NMI-d6, 6 wt% LiCl-DMSO, or an ionic liquid, which are used to directly dissolve the ball-milled plant cell wall [83,86–88].

In general, NMR spectra of carbohydrates have several crowded and overlapped signals, particularly in the nonanomeric region. In addition, the ionic liquids or extensive ball-milling used in the analyses of whole PCW can cause modification on the cell wall components, which could lead to inaccurate interpretation [83,88]. Thus, despite the great importance of whole PCW studies by NMR, this section will focus on characterization studies of isolated PCW polysaccharides (after fractionation of the AIR), as this strategy reduces the number of signals and overlapping to make the interpretation less difficult, resulting in rich information for atomic-level structure determination [89].

In this context, the characterization of PCW soluble polysaccharides by NMR in solution-state is commonly used. The sample preparation requires exchanging the water for a solvent whose proton does not have NMR absorptions detected under the same conditions as the sample. For this, the solvent most used for carbohydrate analysis is deuterium oxide (D2O), but others, such as DMSO-d6, have also been used [90,91]. In some cases, for less soluble polysaccharides in pure D2O as linear mannan, 50% urea D2O can improve solubility [92]. The sample should be prepared with minimal or no content of residual water, which requires three or more steps of solubilization/lyophilization, also ensuring that hydroxyl groups of carbohydrate are fully deuterium exchanged. It is desirable to analyze the sample in the highest concentration possible, as it directly affects the time required for data acquisition and the signal-to-noise ratio [81]. However, the high viscosity of the sample can be a constraint for getting a high concentration solution. In this case, partial hydrolysis has been used to obtain less viscous solutions [93]. In addition, partial hydrolysis, mainly by enzymatic processing, is sometimes used to produce representative oligosaccharides whose spectra are easier to elucidate the structure than the native PCW polysaccharides [91].

The determination of the primary structure of isolated and soluble PCW polysaccharides usually starts with identifying each glycosyl residue in the polymer. The structural analyses by 1D ¹H NMR and ¹³C NMR can detect α- or β-configuration of the anomeric proton and carbon in a sugar residue, respectively [94]. It is noteworthy that, except for mannose and rhamnose, most PCW monosaccharides in the pyranose form have α-configuration of the anomeric proton in 5.1–5.8 ppm and β-configuration among 4.3–4.8 ppm, while α- and β-configurations of the anomeric carbon are in 98–103 ppm and 103–106 ppm, respectively [95,96]. However, in both ¹H NMR and ¹³C NMR, common functional groups in PCW polysaccharides, such as the O-acetyl group and O-alkyl group, can change the chemical shifts of adjacent protons and carbon [96]. This can be an issue in the case of rhamnogalacturonan II, which is only a minor component of PCW [19]. On the other hand, one bond ¹³C-¹H coupling constants are an option to determine the anomeric configuration unequivocally [97].

Apart from the anomeric protons, acetyl (δ 2.0–2.1), and methyl (δ 1.2) groups, other signals are not well resolved in the ¹H spectra of carbohydrates. Thus, 1D ¹H NMR is not appropriate to fully characterize the structure of PCW polysaccharides. Nevertheless, it has been used to determine the degree of methyl-esterification and acetylation of pectins [98,99], and it can be used to determine the molar ratios of monomers by integrating intensities of anomeric protons signals [81,100]. The ¹³C resonances of carbohydrates are spread out over a broader range than ¹H, and for simpler structures, 1D ¹³C NMR spectra can provide structural characterization. In many studies, identifying typical signals in an uncharacterized sample can be enough to determine structural features by comparing the identified signals with available literature. For example, the structure of a linear [1,4]-linked-d-mannan in the sample from a Brazilian plant was confirmed, based on six characteristic signals in the ¹³C NMR spectra for this polysaccharide, at 101.8 (C-1), 78.1 (C-4), 76.5 (C-5), 73.0 (C-3), 71.6 (C-2), and 62.0 (C-6) [101].

However, due to the limitations of 1D NMR, 2D NMR technology plays an essential role in the structural analysis of more complex polysaccharides by establishing correlations between different nuclei of the molecule. For example, the ¹H NMR spectra of xylooligosaccharides obtained by enzymatic hydrolysis of xylans from various monocots allowed the diagnosis of glucuronic acid and 4-O-methylated glucuronic acid residues bearing a substituent at O-2, while 2D NMR experiments established that the substituted glucuronic acid was attached to O-2 of a 4-linked xylopyranose [91].

Thus, the complexity of the target PCW polysaccharide will dictate the NMR experimental strategy, where a series of 2D experiments (or even 3D) can be combined to help elucidate structural aspects. Here, to exemplify a strategy for the structural analysis of a complex PCW soluble polysaccharide, we will take as an example the study of a highly branched arabinoxylan from dicot seeds [93]. Firstly, the highly viscous polysaccharide was subjected to partial acid hydrolysis to be analyzed by NMR. 1D ¹H and ¹³C NMR spectra were acquired, and the ¹H NMR spectrum allowed identifying regions of α-linked Araf and β-linked Xylp, while the ¹³C NMR spectrum enabled the identification of a methyl carbon of rhamnose and nine anomeric carbon signals relative to different residues. Then, by associating these data with the 2D experiment HSQC (heteronuclear single quantum coherence), it was possible to identify the signals of the anomeric protons directly bound to the anomeric carbons, as HSQC allows establishing the correlation between hydrogens directly bonded to carbons [81]. After that, each of the nine residues had its protons and carbons chemicals shifts completely assigned by combining information from HSQC, TOCSY (total correlation spectroscopy), and COSY (correlation spectroscopy), thus allowing assured identification of the sugar residues. In this strategy, TOCSY and COSY were used to assign all other protons besides the anomeric protons that had already been identified, and carbons’ chemical shifts were then assigned from HSQC. The rationale behind this strategy is based on the fact that TOCSY provides correlations among all the hydrogens of the same monosaccharide; therefore, by using the anomeric hydrogen as a starting point, it is possible to assign the other hydrogens. COSY is used for the same purpose, but its analysis together with TOCSY helps the assignment of the hydrogens, as COSY allows establishing a correlation between a hydrogen and the adjacent hydrogen (up to three bonds) in the same sugar residue [81]. Then, by having information regarding all protons from the sugars’ residues, the assignment of the carbons from HSQC spectra was facilitated. Lastly, information regarding the sequence of glycosyl residues in the arabinoxylan structure was determined by the HMBC spectrum (heteronuclear multiple bond correlation), which makes possible the identification of bonds between adjacent sugar residues through the coupling between hydrogens and carbons two or three bonds apart. Thus, by following this experimental strategy, combined with methylation data analysis (see Section 4.2), it was possible to propose the structure of a highly branched arabinoxylan with a β-1,4-linked Xylp backbone, with short side chains attached to its O-2 (β-1,2,4-linked Xylp) or O-3 (β-1,3,4-linked Xylp) positions, containing α-T-linked Araf, β-T-linked Xylp, and α-T-linked GlcAp as main terminal residues [93]. However, it is important to emphasize that the ideal strategy will depend on the complexity of the soluble PCW polysaccharide and that NMR information can be combined with other techniques when not all data can be resolved solely by this methodology.

Nevertheless, the evaluation of cellulose by solution-state NMR methodologies is more complex due to its insolubility in D2O. Other solvent systems have been evaluated to overcome solubility limitations, such as DMSO-d6/pyridine-d5, allowing the assignment of chemical shifts for nonderivatized amorphous cellulose [92,102]. In addition, techniques to derivatize the polysaccharides to facilitate the dissolution of both soluble and insoluble polysaccharide fractions of PCW are also available [87].

On the other hand, solid-state ¹³C NMR is an alternative to investigate structural features of cellulose in an intact state, providing not only information on crystallinity index but also about the ultrastructure of cellulose [103]. The evaluation of the cellulose ultrastructure by ¹³C solid-state NMR allows characterizing other forms of cellulose, such as paracrystalline cellulose and amorphous cellulose fibril surfaces, as peaks corresponding to the crystalline structure (δ 86–92 ppm) differ from the one of the amorphous region (δ 80–86 ppm).

With all the advances in the NMR spectroscopy technique, the method has been an easy and accurate choice with high reproducibility and structure-sensitivity to determine the anomeric configuration, location and degree of substitution, and the monosaccharide linkage sequence of polysaccharides from PCW [79]. Nowadays, carbohydrate identification can be facilitated by bioinformatic tools and a collection of NMR data of polysaccharides from PCW available in the literature and online databases [104,105]. Despite the advantages, the complete elucidation of most structurally complex polysaccharides will require combined data for specific characterization, such as the complementary methylation analysis by GC-MS for the linkage patterns confirmation.

4.2: Gas chromatography—Methylation analysis

Methylation analysis coupled with detection by GC-MS is a widely used method for determining the linkage structure of PCW polysaccharides based on the identification of partially methylated sugar derivatives. Since the first preparation of O-methylated sugars in the early 1900s [106], several methods of polysaccharide methylation have been developed with different solvents and basic agents, which are reviewed elsewhere [107,108]. Here, two of the most commonly used methylation methods applied to characterize PCW samples will be discussed (the methods of Hakomori and Ciucanu and Kerek) [109,110].

Independently of the methodology used, in this procedure, the reaction of a methylating agent with sugars residues in the presence of a strong base converts all free hydroxyl groups of a polysaccharide (i.e., hydroxyl groups which are not involved in the glycosidic linkage) into methyl ethers, producing complete methylation (per-O-methylation). The base is required to produce the alkoxide that makes a nucleophilic attack on the methylating reagent. Methyl iodide is the usual methylating agent, but dimethyl sulfate has also been used in the Haworth method [108,111].

The methylation analysis encompasses the methylation procedure, total acid hydrolysis of permethylated polysaccharides to release partially methylated sugars that are subsequently reduced to alditols, which are further acetylated, resulting in partially methylated alditol acetates (PMAAs). For the reduction, NaBD4 is used to tag the anomeric carbon with deuterium and differentiate symmetrical derivatives that are unresolved chromatographically [112]. The PMAAs are analyzed by GC-MS using a combination of their retention time and mass spectra, and the linkage analysis in the polysaccharide is deduced from the identification of these derivatives [13,113].

The method of Hakomori [109] and Ciucanu and Kerek [110] will differ in the initial methylation step by the use of a different base. The choice of one method over the other will be dictated by the samples’ characteristics, as methylation conditions are not universally applicable to all carbohydrate types [114]. In the Hakomori method, the base is the methylsulfinyl carbanion (CH3-SO-CH2−), also known as dimsyl anion, which is prepared by dissolving sodium hydride or potassium hydride in DMSO (NaH/DMSO) under a nitrogen stream at room temperature [109,112]. The dimsyl anion is sensitive to oxygen, carbon dioxide, and water, thus requiring an inert and anhydrous atmosphere during reaction and storage, and it suffers exothermic decomposition at relatively low temperatures, generating noncondensable gases that can cause explosions, posing a significant safety concern [115]. This method also presents the disadvantage of producing many side products, such as ethyl methyl sulfoxide, disulfur derivatives, and further condensation and methylation derivatives, which can hamper the separation and identification of PMMAs