Glucan Chains Research Articles

Structural factors affecting 13C NMR chemical shifts of cellulose: a computational study

The doublet C4 peaks at ~ 85 and ~ 89 ppm in solid-state 13C NMR spectra of native cellulose have been attributed to signals of C4 atoms on the surface (solvent-exposed) and in the interior of microfibrils, designated as sC4 and iC4, respectively. The relative intensity ratios of sC4 and iC4 observed in NMR spectra of cellulose have been used to estimate the degree of crystallinity of cellulose and the number of glucan chains in cellulose microfibrils. However, the molecular structures of cellulose responsible for the specific surface and interior C4 peaks have not been positively confirmed. Using density functional theory (DFT) methods and structures produced from classical molecular dynamics simulations, we investigated how the following four factors affect 13C NMR chemical shifts in cellulose: conformations of exocyclic groups at C6 (tg, gt and gg), H2O molecules H-bonded on the surface of the microfibril, glycosidic bond angles (Φ, Ψ) and the distances between H4 and HO3 atoms. We focus on changes in the δ13C4 value because it is the most significant observable for the same C atom within the cellulose structure. DFT results indicate that different conformations of the exocyclic groups at C6 have the greatest influence on δ13C4 peak separation, while the other three factors have secondary effects that increase the spread of the calculated C4 interior and surface peaks.

Crystal structure of the flexible tandem repeat domain of bacterial cellulose synthesis subunit C

Bacterial cellulose (BC) is synthesized and exported through the cell membrane via a large protein complex (terminal complex) that consists of three or four subunits. BcsC is a little-studied subunit considered to export BC to the extracellular matrix. It is predicted to have two domains: a tetratrico peptide repeat (TPR) domain and a β-barrelled outer membrane domain. Here we report the crystal structure of the N-terminal part of BcsC-TPR domain (Asp24–Arg272) derived from Enterobacter CJF-002. Unlike most TPR-containing proteins which have continuous TPR motifs, this structure has an extra α-helix between two clusters of TPR motifs. Five independent molecules in the crystal had three different conformations that varied at the hinge of the inserted α-helix. Such structural feature indicates that the inserted α-helix confers flexibility to the chain and changes the direction of the TPR super-helix, which was also suggested by structural analysis of BcsC-TPR (Asp24–Leu664) in solution by size exclusion chromatography-small-angle X-ray scattering. The flexibility at the α-helical hinge may play important role for exporting glucan chains.

Characterization of crystalline linear (1 → 3)-α-d-glucan synthesized in vitro

We investigated the crystal structure and molecular arrangement of the linear (1→3)-α-d-glucan synthesized by glucosyltransferase GtfJ cloned from Streptococcus salivarius using sucrose as a substrate. The synthetic products had two morphologies: wavy fibril-like crystals as major and thin lamellae as minor products. Their structures were analyzed using electron microdiffraction, synchrotron X-ray powder diffraction, and solid-state 13C NMR spectroscopy. The fibrils and lamellae had the same allomorphic form but different molecular arrangements. The wet crystals were in a hydrated form, which converted into an anhydrous form with a significant decrease in crystallinity on drying. The hydrated and anhydrous forms had an extended-chain conformation with 2/1 helix, and the hydrated form was estimated to contain one water molecule per glucose residue. The long glucan chains were folded in the fibril crystals, while the short, extended chains were arranged perpendicular to the base plane of the lamellae.

Small-granule starches from sweet corn and cow cockle: Physical properties and amylopectin branching pattern

Small-granule starches (SGS) attract growing interests with their unique structure and functionalities, such as fat replacement and texture modifications. To explore new resources of SGS, sweet corn starch (SCS) and cow cockle starch (CCS) were studied for their physical properties and branching pattern, with normal corn starch, waxy corn starch, normal rice starch, and waxy rice starch as comparisons. Hydrothermal and wide-angle X-ray powder diffraction analyses showed the much lower content of ordered structure (melting enthalpy of 5.6 J/g) and lower crystallinity (28%) for SCS than for normal corn starch (14.4 J/g and 34%, respectively). The rapid viscosity analyzer analysis showed very low viscosity for SCS through the pasting process and high resistance of CCS against the shear force. The results of β-limit dextrin structure analyses showed highly branched clusters of SCS (about 15 glucan chains per cluster) and a lack of long inter-cluster chains that connect 3 or more clusters. In contrast, CCS displayed a branching pattern similar to that of corn starches, however, with fewer short intra-cluster chains. It was considered that the low crystallinity and viscosity of SCS were attributed to its high branching, which reduced the formation of crystalline structure during the genesis of starch and decreased the interactions among swollen granules. On the other hand, the submicron size made CCS granules less susceptible to the shear force applied during pasting, leading to negligible breakdown. Using SCS and CCS as models, this study connected the physical properties and branching pattern of SGS, thus supporting their potential applications.

Simultaneous silencing of isoamylases ISA1, ISA2 and ISA3 by multi-target RNAi in potato tubers leads to decreased starch content and an early sprouting phenotype.

Isoamylases hydrolyse (1–6)-alpha-D-glucosidic linkages in starch and are involved in both starch granule formation and starch degradation. In plants, three isoamylase isoforms with distinct functions in starch synthesis (ISA1 and ISA2) and degradation (ISA3) have been described. Here, we created transgenic potato plants with simultaneously decreased expression of all three isoamylases using a chimeric RNAi construct targeting all three isoforms. Constitutive expression of the hairpin RNA using the 35S CaMV promoter resulted in efficient silencing of all three isoforms in leaves, growing tubers, and sprouting tubers. Neither plant growth nor tuber yield was effected in isoamylase-deficient potato lines. Interestingly, starch metabolism was found to be impaired in a tissue-specific manner. While leaf starch content was unaffected, tuber starch was significantly reduced. The reduction in tuber starch content in the transgenic plants was accompanied by a decrease in starch granules size, an increased sucrose content and decreased hexose levels. Despite the effects on granule size, only little changes in chain length composition of soluble and insoluble glucose polymers were detected. The transgenic tubers displayed an early sprouting phenotype that was accompanied by an increased level of sucrose in parenchyma cells below the outgrowing bud. Since high sucrose levels promote sprouting, we propose that the increased number of small starch granules may cause an accelerated turnover of glucan chains and hence a more rapid synthesis of sucrose. This observation links alterations in starch structure/degradation with developmental processes like meristem activation and sprout outgrowth in potato tubers.

2H–13C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates

Site-specific determination of molecular motion and water accessibility by indirect detection of 2H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (RESPIRATIONCP) technique was developed, which allowed efficient transfer of 2H magnetization to 13C at moderate 2H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the 2H-13C magnetization transfer characteristics of one-bond perdeuterated CD n spin systems and two-bond H/D exchanged C-(O)-D and C-(N)-D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband 2H-13C polarization transfer can be achieved using 2H radiofrequency fields of ~50kHz, relatively short contact times of 1.3-1.7ms, and with sufficiently high sensitivity to enable 2D 2H-13C correlation experiments with undistorted 2H spectra in the indirect dimension. To demonstrate the utility of this 2H-13C technique for studying molecular motion, we show 2H-13C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 2H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5-C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the 2H-13C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O-D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50kHz. The latter O-D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C-D dipolar order parameters of 0.46-0.55 for pectins, suggesting that additional motions exist at the C-O bonds in the wall polysaccharides. 2H-13C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1.

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes.

Previously we have reported that the Gram-negative bacterium Azotobacter chroococcum NCIMB 8003 uses the 4,6-α-glucanotransferase GtfD to convert maltodextrins and starch into a reuteran-like polymer consisting of (α1→4) glucan chains connected by alternating (α1→4)/(α1→6) linkages and (α1→4,6) branching points. This enzyme constituted the single evidence for this reaction and product specificity in the GH70 family, mostly containing glucansucrases encoded by lactic acid bacteria (http://www.CAZy.org). In this work, 4 additional GtfD-like proteins were identified in taxonomically diverse plant-associated bacteria forming a new GH70 subfamily with intermediate characteristics between the evolutionary related GH13 and GH70 families. The GtfD enzyme encoded by Paenibacillus beijingensis DSM 24997 was characterized providing the first example of a reuteran-like polymer synthesizing 4,6-α-glucanotransferase in a Gram-positive bacterium. Whereas the A. chroococcum GtfD activity on amylose resulted in the synthesis of a high molecular polymer, in addition to maltose and other small oligosaccharides, two reuteran-like polymer distributions are produced by P. beijingensis GtfD: a high-molecular mass polymer and a low-molecular mass polymer with an average Mw of 27 MDa and 19 kDa, respectively. Compared to the A. chroooccum GtfD product, both P. beijingensis GtfD polymers contain longer linear (α1→4) sequences in their structure reflecting a preference for transfer of even longer glucan chains by this enzyme. Overall, this study provides new insights into the evolutionary history of GH70 enzymes, and enlarges the diversity of natural enzymes that can be applied for modification of the starch present in food into less and/or more slowly digestible carbohydrate structures.

Bound Substrate in the Structure of Cyanobacterial Branching Enzyme Supports a New Mechanistic Model

Branching enzyme (BE) catalyzes the formation of α-1,6-glucosidic linkages in amylopectin and glycogen. The reaction products are variable, depending on the organism sources, and the mechanistic basis for these different outcomes is unclear. Although most cyanobacteria have only one BE isoform belonging to glycoside hydrolase family 13, Cyanothece sp. ATCC 51142 has three isoforms (BE1, BE2, and BE3) with distinct enzymatic properties, suggesting that investigations of these enzymes might provide unique insights into this system. Here, we report the crystal structure of ligand-free wild-type BE1 (residues 5-759 of 1-773) at 1.85 Å resolution. The enzyme consists of four domains, including domain N, carbohydrate-binding module family 48 (CBM48), domain A containing the catalytic site, and domain C. The central domain A displays a (β/α)8-barrel fold, whereas the other domains adopt β-sandwich folds. Domain N was found in a new location at the back of the protein, forming hydrogen bonds and hydrophobic interactions with CBM48 and domain A. Site-directed mutational analysis identified a mutant (W610N) that bound maltoheptaose with sufficient affinity to enable structure determination at 2.30 Å resolution. In this structure, maltoheptaose was bound in the active site cleft, allowing us to assign subsites -7 to -1. Moreover, seven oligosaccharide-binding sites were identified on the protein surface, and we postulated that two of these in domain A served as the entrance and exit of the donor/acceptor glucan chains, respectively. Based on these structures, we propose a substrate binding model explaining the mechanism of glycosylation/deglycosylation reactions catalyzed by BE.

Evidence of a Transition from Diffusive to Super-Diffusive Motion of Cellulose Synthase Complexes in Living Plants

The polysaccharide cellulose is the main component of plant cell walls, so it is the most abundant polymer on the earth. While it is widely used in industry due to its remarkable properties, such as renewability and biodegradability, its biosynthesis is still not well understood. The large transmembrane protein complex responsible for synthesizing cellulose contains several cellulose synthase A (CESA) subunits that polymerize UDP glucose into the constituent glucan chains of cellulose. Here, we used variable angle epi-fluorescence microscopy in combination with single-particle tracking to characterize the motion of GFP labeled CESA complexes in the root and mesocotyl of Brachypodium distachyon seedlings that are a 3 to 4 days old. We show that CESA complexes move through the plasma membrane at approximately 165 nm/minute. Their motion is known to be guided by cortical microtubules, but no molecular motors are involved. Rather, the motion is thought to be driven by the polymerization and crystallization of the cellulose. A mean-squared displacement analysis shows that CESA complexes move diffusively on short time scales and undergo a transition to super-diffusive motion on a time scale of about 10 s. We also report on the effect of actin and microtubule inhibitors on CESA motion. This work was funded through NSF grant PHY- 1205989.

The impact of antibiotics on bacterial cellulose in vivo

This study investigated how antibiotics, to which Gluconacetobacter hansenii is naturally resistant, impact cellulose crystallinity, allomorph, aggregation into bundles and layers, cellulose yield, and cell morphology. G. hansenii was exposed to 100 μg/mL ampicillin, chloramphenicol, and kanamycin for 7 days, and cellulose structure was analyzed using scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. Biomass and cellulose weights were also assessed. Ampicillin increased bundle thickness, and the bundles also showed nodular deposits indicative of non-cellulosic exopolysaccharide deposition. Ampicillin also yielded the lowest amount of cellulose per gram of biomass (p < 0.01) and induced significant filamentation behavior. Chloramphenicol inhibited biomass production (p < 0.01), increased the I-α allomorph content (p < 0.01), and also induced filamentation, though not as profusely as ampicillin. We hypothesize that defects in the peptidoglycan layer and in protein production lowered cellulose yield and promoted cells to undergo filamentation as a survival tactic. Additionally, we hypothesize that antibiotic stress caused additional exopolysaccharides to be produced and that they likely enhanced glucan chain aggregation into higher-order structures. Our findings have significant implications for downstream applications such as genetically engineering G. hansenii to produce bacterial cellulose with modified properties.

Synthesis and self-assembly of octenyl succinic anhydride modified short glucan chains based amphiphilic biopolymer: Micelles, ultrasmall micelles, vesicles, and lutein encapsulation/release

The amphiphilic polymers are widely used to prepare nanocarriers by self-assembly with crucial implications for biomedical, biotechnological, and food applications. An amphiphilic polymer of octenyl succinic anhydride modified short glucan chains (OSA-SGC) with various degrees of substitution (DS) were first synthesized in this work. OSA-SGC polymer spontaneously formed vesicles, micelles, and ultrasmall micelles in a phosphate buffer solution. The properties of self-assembled nanoparticles were studied using fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The critical aggregation concentrations (CAC) of OSA-SGC polymer were in the range of 0.0316–0.1260 mg/mL and decreased with increasing DS. Lutein, a hydrophobic functional ingredients, was incorporated into the OSA-SGC micelles and vesicles. Lutein-loaded nanoparticles exhibited high encapsulation efficiency (∼85%) and loading content (∼8%). Thus, the study suggested that self-aggregated nanoparticles of OSA-SGC polymer could have potential applications in the health food, cosmetics, and pharmaceutical fields because they can aid in the delivery of hydrophobic functional ingredients or drugs.

Functional Analysis of Cellulose Synthase (CESA) Protein Class Specificity.

The cellulose synthase complex (CSC) exhibits a 6-fold symmetry and is known as a "rosette." Each CSC is believed to contain between 18 and 24 CESA proteins that each synthesize an individual glucan chain. These chains form the microfibrils that confer the remarkable structural properties of cellulose. At least three different classes of CESA proteins are essential to form the CSC However, while organization of the CSC determines microfibril structure, how individual CESA proteins are organized within the CSC remains unclear. Parts of the plant CESA proteins map sufficiently well onto the bacterial CESA (BcsA) structure, indicating that they are likely to share a common catalytic mechanism. However, plant CESA proteins are much larger than the bacterial BcsA protein, prompting the suggestion that these plant-specific regions are important for interactions between CESA proteins and for conferring CESA class specificity. In this study, we have undertaken a comprehensive analysis of well-defined regions of secondary cell wall CESA proteins, with the aim of defining what distinguishes different CESA proteins and hence what determines the specificity of each CESA class. Our results demonstrate that CESA class specificity extends throughout the protein and not just in the highly variable regions. Furthermore, we find that different CESA isoforms vary greatly in their levels of site specificity and this is likely to be determined by the constraints imposed by their position within the CSC rather than their primary structure.

Amylopectin small chain glucans form structure fingerprint that determines botanical origin of starch

Starch granule size, shape and structure of amylopectin are species specific and influence starch properties and end-use of starch. Amylopectin glucan chain structure was used to predict the starch botanical sources. Mathematical probability for accumulation of small glucan chains DP 6–10 reveal exponential fit curve with maximum R2 in smallest granule size starches (Chlamydomonas, quinoa, buckwheat). Cereal and cassava showed R2 of 0.81–0.96 while in pulses and tubers it was less than 0.7. The amylopectin small glucan chains form a unique ‘finger print region’ that identified starch botanical source. Differential amylopectin chain length distribution (APCLD) graphs between DP 6–80 of all species from Chlamydomonas starch distinguished five structural groups that clustered the 31 analyzed starches into four major patterns. APCLD analyses of amylopectin combined with characteristic pattern of small linear DP (6–9) glucan chains predicted the starch botanical source.

Preparation and characterization of essential oil-loaded starch nanoparticles formed by short glucan chains.

Essential oils (EOs), including menthone, oregano, cinnamon, lavender, and citral, are natural products that have antimicrobial and antioxidant activities. However, extremely low water solubility, and easy degradation by heat, restrict their application. The aim of this work was to evaluate the enhancement in antioxidative and antimicrobial activities of EOs encapsulated in starch nanoparticles (SNPs) prepared by short glucan chains. For the first time, we have successfully fabricated menthone-loaded SNPs (SNPs-M) at different complexation temperatures (30, 60, and 90°C) by an in situ nanoprecipitation method. The SNPs-M displayed spherical shapes, and the particle sizes ranged from 93 to 113nm. The encapsulation efficiency (EE) of SNPs-M increased significantly with an increase in complexation temperature, and the maximum EE was 86.6%. The SNPs-M formed at 90°C had high crystallization and thermal stability. The durations of the antioxidant and antimicrobial activities of EOs was extended by their encapsulation in the SNPs.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

Elaboration and characterization of corn starch films incorporating silver nanoparticles obtained using short glucan chains

Nanocomposites are being used as a new type of packaging that has good biodegradability and antibacterial ability and that has wide applicability in the preservation and storage field. Transmission electron microscopy results demonstrated that the AgNPs were spherical in shape with the diameter of 2–6 nm for 48 h reaction time. The mechanical properties of composite films increased after the incorporation of AgNPs. Differential scanning calorimetric and thermogravimetric analysis results showed that the thermal stability of silver–starch composite film was increased through incorporation with AgNPs. The composite films exhibited strong antibacterial activity against Escherichia coli and Staphylococcus aureus, and the composite films were more effective against Gram-negative bacteria (E. coli).

Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex.

A six-lobed membrane spanning cellulose synthesis complex (CSC) containing multiple cellulose synthase (CESA) glycosyltransferases mediates cellulose microfibril formation. The number of CESAs in the CSC has been debated for decades in light of changing estimates of the diameter of the smallest microfibril formed from the β-1,4 glucan chains synthesized by one CSC. We obtained more direct evidence through generating improved transmission electron microscopy (TEM) images and image averages of the rosette-type CSC, revealing the frequent triangularity and average cross-sectional area in the plasma membrane of its individual lobes. Trimeric oligomers of two alternative CESA computational models corresponded well with individual lobe geometry. A six-fold assembly of the trimeric computational oligomer had the lowest potential energy per monomer and was consistent with rosette CSC morphology. Negative stain TEM and image averaging showed the triangularity of a recombinant CESA cytosolic domain, consistent with previous modeling of its trimeric nature from small angle scattering (SAXS) data. Six trimeric SAXS models nearly filled the space below an average FF-TEM image of the rosette CSC. In summary, the multifaceted data support a rosette CSC with 18 CESAs that mediates the synthesis of a fundamental microfibril composed of 18 glucan chains.

In vitro digestion of nanoscale starch particles and evolution of thermal, morphological, and structural characteristics

In vitro digestions of starch nanocrystals (SNCs), prepared by acid hydrolysis of native starch and starch nanoparticles (SNPs) fabricated by self-assembly of short glucan chains, were investigated for kinetics of enzymatic hydrolysis. Their thermal, morphological, and structural properties during amylolysis were also compared. The kinetics of enzymatic hydrolysis indicated that the hydrolysis rate of SNPs was lowest in that of SNCs, cooked starch, and native starch. Onset, peak, conclusion temperatures, and enthalpy of gelatinization of SNPs and SNCs decreased during digestion. The A-type crystallization of SNCs contributed to their higher rate of hydrolysis than that of SNPs, which was B-type. The relative crystallinity of SNCs during hydrolysis decreased but that of SNPs increased. The results suggest that SNPs exhibit higher resistance to digestion than SNCs. This finding provides information for diverse potential applications of SNCs and SNPs.

Proteomic profiling of cellulase-aid-extracted membrane proteins for functional identification of cellulose synthase complexes and their potential associated- components in cotton fibers.

Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.

Cellulosic Microfibril and Its EmbeddingMatrix within Plant Cell Wall

Cellulose is the homopolymer of (1→4)-β-D-glucopyranose. Glucan chains are aggregated to form microfibrils with cross dimensions ranged from 2 to 20 nm. The variation in each of microfibrilluar dimension, hydrogen-bonding network and molecular orientation depends on their parent nature. Cellulosic microfibrils isolated from bacteria and certain algae (10–25 nm in diameter) were found to be thicker than those extracted from the primary cell walls in higher plants ( 3–10 nm in diameter).Three biopolymers, namely hemicellulose, lignin and/or pectin contribute to the embedding matrix enveloping the cellulosic microfibrils.The secondary cell wall was found to be differentiated into three consecutive layers (S1, S2, S3). Within each layer, cellulose microfibrils are found parallel and of high order with a different microfibrilluar angle. The bundling process of cellulose microfibrils in both primary and secondary cell walls may involve the aggregation of the closely arranged cellulosic microfibrils. Some models for the microfibril construction have been proposed. Along the longitudinal microfibril’s axe, crystalline regions are alternated with amorphous ones. The most important methods of estimating the crystallinity of cellulose microfibrils such as Xray diffraction (XRD), 13C solid-state NMR spectroscopy (13C-NMR), small-angle neutron scattering (SANS) and sum frequency generation (SFG) spectroscopy were concluded.