Crystalline Forms Of Cellulose Research Articles

Functional characterization of a lytic polysaccharide monooxygenase from Schizophyllum commune that degrades non-crystalline substrates

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that use O2 or H2O2 to oxidatively cleave glycosidic bonds. LPMOs are prevalent in nature, and the functional variation among these enzymes is a topic of great interest. We present the functional characterization of one of the 22 putative AA9-type LPMOs from the fungus Schizophyllum commune, ScLPMO9A. The enzyme, expressed in Escherichia coli, showed C4-oxidative cleavage of amorphous cellulose and soluble cello-oligosaccharides. Activity on xyloglucan, mixed-linkage β-glucan, and glucomannan was also observed, and product profiles differed compared to the well-studied C4-oxidizing NcLPMO9C from Neurospora crassa. While NcLPMO9C is also active on more crystalline forms of cellulose, ScLPMO9A is not. Differences between the two enzymes were also revealed by nuclear magnetic resonance (NMR) titration studies showing that, in contrast to NcLPMO9C, ScLPMO9A has higher affinity for linear substrates compared to branched substrates. Studies of H2O2-fueled degradation of amorphous cellulose showed that ScLPMO9A catalyzes a fast and specific peroxygenase reaction that is at least two orders of magnitude faster than the apparent monooxygenase reaction. Together, these results show that ScLPMO9A is an efficient LPMO with a broad substrate range, which, rather than acting on cellulose, has evolved to act on amorphous and soluble glucans.

Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara

BackgroundCellulases play a key role in the enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. Thus, the discovery of novel cellulases from exotic biological niches is of great interest as they may present properties that are valuable in the biorefining of lignocellulosic biomass.ResultsWe have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multi-domain enzyme from the unusual gill endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The catalytic GH5 domain, was cloned and recombinantly produced with or without a C-terminal family 10 carbohydrate-binding module (CBM). Both variants showed hydrolytic endo-activity on soluble substrates such as β-glucan, carboxymethylcellulose and konjac glucomannan, respectively. However, low activity was observed towards the crystalline form of cellulose. Interestingly, when co-incubated with a cellulose-active LPMO, a clear synergy was observed that boosted the overall hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 Å resolution and revealed a substrate binding cleft extension containing a putative + 3 subsite, which is uncommon in this enzyme family. The enzyme was active in a wide range of pH, temperatures and showed high tolerance for NaCl.ConclusionsThis study provides significant knowledge in the discovery of new enzymes from shipworm gill endosymbionts and sheds new light on biochemical and structural characterization of cellulolytic cellulase. Study demonstrated a boost in the hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose-active LPMO. These findings will be relevant for the development of future enzyme cocktails that may be useful for the biotechnological conversion of lignocellulose.

Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR

Enzymatic deconstruction of lignocellulosic biomass is crucial to establishment of the renewable biofuel and bioproduct economy. Better understanding of these enzymes, including their catalytic and binding domains, and other features offer potential avenues for improvement. Glycoside hydrolase family 9 (GH9) enzymes are attractive targets because they have members that exhibit exo- and endo-cellulolytic activity, processivity of reaction, and thermostability. This study examines a GH9 from Acetovibrio thermocellus ATCC 27405, AtCelR containing a catalytic domain and a carbohydrate binding module (CBM3c). Crystal structures of the enzyme without substrate, bound to cellohexaose (substrate) or cellobiose (product), show the positioning of ligands to calcium and adjacent residues in the catalytic domain that may contribute to substrate binding and facilitate product release. We also investigated the properties of the enzyme engineered to contain an additional carbohydrate binding module (CBM3a). Relative to the catalytic domain alone, CBM3a gave improved binding for Avicel (a crystalline form of cellulose), and catalytic efficiency (kcat/KM) was improved 40×with both CBM3c and CBM3a present. However, because of the molecular weight added by CBM3a, the specific activity of the engineered enzyme was not increased relative to the native construct consisting of only the catalytic and CBM3c domains. This work provides new insight into a potential role of the conserved calcium in the catalytic domain and identifies contributions and limitations of domain engineering for AtCelR and perhaps other GH9 enzymes.

Investigation of chemical linkages between lignin and carbohydrates in cultured poplar cambium tissues via double isotope labeling

Lignin precursor labeled with 13C (coniferin-13Cα), carbohydrate precursor labeled with D (6,6-D2-glucose) were put into cambium tissue stripped from a growing poplar. The tissue was further cultured in vitro for 18d. Then, the isotopic abundance was determined. The results showed that the labeled precursors could be normally involved in the formation of new xylem. The labeled new xylem tissue was fractionated by ionic liquid DMSO/TBAH system to obtain two components: glucan-lignin complex (GL) and xylan-lignin complex (XL). The X-ray diffraction (XRD) results indicated that the crystalline form of cellulose in the GL component was transformed from type I to type II after the ionic liquid separation. Then the GL and XL were purified and modified by enzymatic and chemical methods, and their structures were elucidated by nuclear magnetic resonance (NMR) spectroscopy. The results showed that lignin subunits in the cultured tissues were mainly connected by β-5 and β-O-4 linkages, of which the β-O-4 substructure unit predominated. Lignin and carbohydrates were mainly connected by acetal bonds, ether bonds, and ester bonds. Combined with the carbohydrate composition and XRD analysis results, the GL components also confirmed the existence of acetal bonds, ester bonds and ether bonds between lignin and cellulose.

Study on the effects of different pectinase/cellulase ratios and pretreatment times on the preparation of nanocellulose by ultrasound-assisted bio-enzyme heat treatment.

With the development of science and technology, efficient, fast and green methods are increasingly being pursued. The production of nanocellulose by green methods, such as bio-enzymes-assisted ultrasound treatment, has been the focus of many studies. However, the yield of cellulose nanocrystals prepared by this method is very low. In this paper, by pretreatment of microcrystalline cellulose (MCC), nanocellulose was prepared by heating and stirring + pectinase/cellulase + ultrasonic treatment (HSt - P/C - Ultr). The effects of the ratios of pectinase and cellulase and the hydrolysis time on the yield of nanocellulose were studied. FTIR, XRD, SEM, TEM and TG were used to determine the structure, crystallinity, morphology and thermal stability of nanocellulose. The results showed that optimal hydrolysis conditions were determined as a pectinase : cellulase ratio of 1 : 1, 90 min and 50 °C. The yield of nanocellulose was about 32.0%. The yield of pectinase cellulase = 1 : 1 was higher than that of microcrystalline cellulose (MCC) treated by a single bio-enzyme. This indicated that the synergistic effects of pectinase and cellulase have a certain effect on the formation of nanocellulose. During the preparation, the crystalline form of cellulose did not change. It was still cellulose I with a crystallinity of 73.5%, which is 9.50% higher than that of microcrystalline cellulose (MCC), a width of 20-50 nm, a high aspect ratio and a winding network structure. Therefore, nanocellulose prepared by this method is an ideal toughening material for manufacturing composite materials.

All‐cellulose composites prepared by partially dissolving cellulose using NaOH/thiourea aqueous solution

AbstractAll‐cellulose composites (ACCs) were prepared by partially dissolving cellulose in the filter paper using NaOH/thiourea aqueous solution. The effects of dissolution time, thiourea ratio, and temperature on the properties of ACCs were investigated. ACCs were characterized by scanning electron microscope, attenuated total reflectance Fourier transform infrared spectroscopy, Raman spectroscopy, X‐ray diffraction, and tensile tests. The results revealed that the fibers in ACCs were tightly intertwined. The crystalline form of cellulose in ACCs was transformed from type I to type I/II mixture, and the crystallinity decreased from 77.32 to 51.40%. The tensile strength of ACCs was remarkably improved to 23.16 MPa. The results confirmed ACCs had a high potential for practical applications in the packaging field.

Fabrication and characterization of antibacterial epsilon-poly-L-lysine anchored dicarboxyl cellulose beads

Pathogens in the food and environment pose a great threat to human health. To solve this problem, we described a novel route to synthesize antibacterial epsilon-poly-L-lysine (EPL) anchored dicarboxyl cellulose beads. Cellulose beads were prepared via a sol-gel transition method and oxidized by sodium periodate and sodium chlorite to form carboxyl groups. EPL was anchored on the beads using carbodiimide mediated amidation. The structure and morphology of beads were characterized by FTIR, XPS, XRD, SEM, and TGA. After dissolution and regeneration, the crystalline form of cellulose is transformed from cellulose I to cellulose II. The thermal degradation temperature of the beads is 200∼300 °C.The samples displayed excellent antimicrobial activity against Staphylococcus aureus, Alicyclobacillus acidoterrestris and Escherichia coli within 12 h. The beads could be biodegraded in soil after 20 days. The biodegradable beads exhibited great potential in food and environmental applications.

Stability and Ligand Promiscuity of Type A Carbohydrate-binding Modules Are Illustrated by the Structure of Spirochaeta thermophila StCBM64C

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

The crystal structure of the endoglucanase Cel10, a family 8 glycosyl hydrolase from Klebsiella pneumoniae.

Cellulases are produced by microorganisms that grow on cellulose biomass. Here, a cellulase, Cel10, was identified in a strain of Klebsiella pneumoniae isolated from Chinese bamboo rat gut. Analysis of substrate specificity showed that Cel10 is able to hydrolyze amorphous carboxymethyl cellulose (CMC) and crystalline forms of cellulose (Avicel and xylan) but is unable to hydrolyze p-nitrophenol β-D-glucopyranoside (p-NPG), proving that Cel10 is an endoglucanase. A phylogenetic tree analysis indicates that Cel10 belongs to the glycoside hydrolase 8 (GH8) subfamily. In order to further understanding of its substrate specificity, the structure of Cel10 was solved by molecular replacement and refined to 1.76 Å resolution. The overall fold is distinct from those of most other enzymes belonging to the GH8 subfamily. Although it forms the typical (α/α)6-barrel motif fold, like Acetobacterxylinum CMCax, one helix is missing. Structural comparisons with Clostridium thermocellum CelA (CtCelA), the best characterized GH8 endoglucanase, revealed that sugar-recognition subsite -3 is completely missing in Cel10. The absence of this subsite correlates to a more open substrate-binding cleft on the cellooligosaccharide reducing-end side.

Characterization of dissolving pulp by fibre swelling in dilute cupriethylenediamine (CUEN) solution in a MorFi analyser

Abstract The swelling of dissolving pulps has been investigated by a new method based on the MorfiR analyser, which is measuring the width variation of thousands of fibres in a cupriethylenediamine (CUEN) solution in a few minutes. Pulps from various origins were analysed coming from softwood, birch wood, eucalyptus wood, kraft pulps, sulphite pulps and ECF and TCF bleached pulps, which were modified by several treatments including chemical (cold caustic extraction, hypochlorite) or enzymatic (cellulase) methods. The swelling was much affected by the crystalline form of cellulose and the hemicellulose content but did not depend neither on the cellulose DP nor on the fibre structure (hardwood vs. softwood). The dissolving pulp reactivity in the viscose process was also assessed by swelling in dilute solutions of cupriethylenediamine (CUEN) instead of the Fock’s method.

TEMPO/NaBr/NaClO-Mediated Surface Oxidation of Nanocrystalline Cellulose and Its Microparticulate Retention System with Cationic Polyacrylamide

TEMPO/NaBr/NaClO-mediated surface oxidation of NCC, acid-extracted from aspen kraft pulp, was studied, and the properties of nanocellulose whiskers before and after oxidation were characterized by conductimetry, Fourier transform infrared spectroscopy, X-ray diffraction, and atomic force microscopy. The resulting products with varied oxidation degrees were then applied in the deinked pulp to evaluate the improvements of fines retention and pulp drainage. It was found that TEMPO-oxidized NCC maintained its crystalline form of cellulose I, while it showed better dispersibility and smaller dimension due to the high level of carboxyl content and degree of oxidation. By adding NCC and TONCC to the deinked pulp, the retention was improved while the drainage rate was decreased to some extent. When TONCC samples were applied together with cationic polyacrylamide to constitute a microparticulate retention system, both fines retention and pulp drainage were apparently improved. Further study showed that the retention and drainage rate were significantly influenced by the degree of oxidation. TONCC sample with the highest DO (0.134) gave the highest retention and drainage rate, 89.6% and 9.41 mL/s, respectively.

Preliminary Study on the Preparation of Bamboo Cellulose Carbamate

In this paper, bamboo fibers were utilized in synthesizing the cellulose carbamate (CC) together with urea for the first time. This paper explored the impact of a serious of factors on Nitrogen content of CC, including the concentration of sodium hydroxide solution used for activating, activation time, aging time, urea socking time, the concentration of urea solution, reaction time and reaction temperatures. Moreover, the analysis and characterizations of CC with different nitrogen contents were conducted by using FTIR, DSC, XRD and SEM. The result indicates that CC was generated as the cellulose reacted with urea, and the crystalline form of cellulose has changed after the esterification reaction. The surface morphology of fibers became wrinkle and rough after the reaction, thereby improving its solubility.

Graft copolymerization of cellulose and 2-hydroxyethyl methacrylate in an ionic liquid

Dissolution and homogeneous graft copolymerization of cellulose were performed in an ionic liquid 1-allyl-3-methylimidazolium chloride (AmimCl) with 2-hydroxyethyl methacrylate. The synthesized AmimCl and cellulose graft copolymers were characterized by FTIR, 1H-NMR and XRD spectroscopy. The results show that AmimCl dissolved cellulose directly by destroying intermolecular and intramolecular hydrogen bonds in cellulose and the crystalline form of cellulose was transformed from cellulose I to cellulose II after regeneration from AmimCl. The best synthetic condition of the cellulose-graft-P (2-hydroxyethyl methacrylate) was that cellulose 0.5 g, 2-hydroxyethyl methacrylate 3.00 g and initiator ammonium persulfate 0.05 g reacted for 180 min at 60°C; the rate of grafting reached 77.3%.

Measurement and calculation of 13C chemical shift tensors in α-glucose and α-glucose monohydrate

The 13C chemical shift tensors of two crystalline forms of glucose (α-glucose and α-glucose·H2O) were determined from one-dimensional (1D) and two-dimensional (2D) solid-state nuclear magnetic resonance (NMR) spectroscopy experiments. The experimental values determined from 1D and 2D methods are in very good agreement. Quantum chemical calculations were also carried out using the gauge-including projector augmented wave (GIPAW) method for plane-wave density functional theory (DFT) as implemented in the CAmbridge Serial Total Energy Package (CASTEP). The calculated 13C chemical shifts were found to be in excellent agreement with experimental values for crystal structures that had their hydrogen atoms optimized and after an appropriate calibration was applied to convert calculated chemical shieldings into chemical shifts. The work presented here lays an important foundation for future solid-state NMR and quantum chemical calculation investigations of the various crystalline forms of cellulose.

Solid State NMR Studies on Cellulose Crystallinity in Fines and Bulk Fibres Separated from Refined Kraft Pulp

Summary Solid state NMR spectroscopy has been used in this work to investigate crystallinity of cellulose in spruce wood before and after kraft pulping and TCF bleaching. Effects of refining of the spruce kraft pulp in water and in weak alkali have been studied by determination of the crystallinity of isolated fines and corresponding bulk fibres in order to find out whether the crystallinity in the fibre surface material and inside the fibre wall differ from each other. Also the possible effect of enzymatic endo-1,4-β-galactanase treatment on the crystallinity of some refined pulps has been investigated. It was found that the Iα crystalline form of cellulose predominates over the Iβ form in native spruce and vice versa in all the pulps studied. In pulping part of the cellulose Iα is converted to the more stable Iβ form mainly by heat. Any measurable changes in the degree of crystallinity or in the relative proportions of different crystalline forms of cellulose could not be seen in pulps after refining, TCF-bleaching or galactanase treatment. However, the degree of crystallinity was found to be considerably lower in the fines compared to the corresponding bulk fibres. Therefore it is suggested that the cellulose crystallinity is lower or the size of crystallites smaller on the fibre surface than inside the fibre wall. Slightly higher contents of lignin and hemicelluloses, mainly xylan and mannan, were also found in the fines compared to the bulk fibres.

The cell wall-anchored Streptomyces reticuli avicel-binding protein (AbpS) and its gene.

Streptomyces reticuli produces a 35-kDa cellulose-binding protein (AbpS) which interacts strongly with crystalline forms of cellulose (Avicel, bacterial microcrystalline cellulose, and tunicin cellulose); other polysaccharides are recognized on weakly (chitin and Valonia cellulose) or not at all (xylan, starch, and agar). The protein could be purified to homogeneity due to its affinity to Avicel. After we sequenced internal peptides, the corresponding gene was identified by reverse genetics. In vivo labelling experiments with fluorescein isothiocyanate (FITC), FITC-labelled secondary antibodies, or proteinase K treatment revealed that the anchored AbpS protrudes from the surfaces of the hyphae. When we investigated the hydrophobicity of the deduced AbpS, one putative transmembrane segment was predicted at the C terminus. By analysis of the secondary structure, a large centrally located alpha-helix which has weak homology to the tropomyosin protein family was found. Physiological studies showed that AbpS is synthesized during the late logarithmic phase, independently of the carbon source.

Synergistic interaction of the cellulosome integrating protein (CipA) from Clostridium thermocellum with a cellulosomal endoglucanase

Activity of a cellulosomal endoglucanase (endoglucanase E; EGE) from Clostridium thermocellum against two crystalline forms of cellulose was enhanced by combination with the cellulosome integrating protein (CipA), but CipA did not enhance EGE activity against amorphous cellulose, even though it was able to bind to it. Similarly, CipA added in trans to genetically truncated EGE that was unable to combine with it nevertheless enhanced EGE activity against crystalline cellulose. These results indicate that the CipA cellulose binding domain does not mediate an increase in activity solely by bringing the catalytic subunits of the cellulosome complex into intimate contact with the substrate.

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237.

Thermostable alkaline cellulase (endo-1,4-beta-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val- Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6-9.0 and displayed maximum activity at 45 degrees C. The alkaline enzyme was stable up to 50 degrees C and more than 30% of the original activity was detectable after heating at 100 degrees C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (beta-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H3PO4-swollen cellulose, NaOH-swollen cellulose, curdlan (beta-1,3-linkage), laminarin (beta-1,3;1,6-linkage), and xylan were barely hydrolyzed at all.

Fine structure and tensile properties of ramie fibres in the crystalline form of cellulose I, II, III I and IV I

This study focuses on the change in the fine structure and tensile properties of ramie fibres by the treatment for crystalline conversion. Through the conversion from cellulose I into II, III I and IV I, the integral crystallinity index and the crystallite size decreased and the internal surface area increased while Young's modulus of the fibre decreased and the ultimate strain increased. Two phase parallel, series and parallel-series models which comprise two kinds of springs with different constants representing the crystalline and amorphous components, respectively, were applied to the tensile force-strain relationship of the fibres. The parallel model could not be applied to the samples because the crystal lattice strains were smaller than the fibre strains; the former strains were less than one fourth of the latter ones, at any given tensile load. On the basis of the series model, Young's modulus of crystalline component of cellulose III I was the highest and the next was that of cellulose I, followed by II and IV I. Young's modulus of amorphous component of cellulose I was the highest and the next was that of II, followed by III I and IV I. Applying a parallel series model, the values for Young's moduli of crystalline and amorphous components were found to depend both on the feature of crystalline component and the density of amorphous region.

Purification and characterization of a protease-resistant cellulase from Aspergillus niger

An endo-β-1,4-glucanase (EC 3.2.1.4) was purified from a culture filtrate of Aspergillus niger IFO31125 by column chromatography through TSK-gel DEAE-3SW and TSK-gel DEAE-5PW, and by gel filtration through TSK-gel G2000SW by high performance liquid chromatography. The enzyme was estimated to have a molecular weight of about 40 kDa by both gel filtration and SDS-polyacrylamide gel electrophoresis, and appeared to consist of a monomeric protein. It contained 8.9% carbohydrate. The optimal pH for activity was 6.0–7.0, and the stable pH range was 5.0–10.0. The optimum temperature at pH 6.0 was around 70°C. The enzyme was very thermally stable and no loss of original activity was found on incubation at 60°C for 2 h. The enzyme efficiently hydrolyzed carboxymethylcellulose and lichenan, but crystalline forms of cellulose, curdlan, laminarin, cellobiose, p-nitrophenyl-β- d-glucopyranoside and p-nitrophenyl-β- d-cellobioside were barely hydrolyzed. The activity of the enzyme was inhibited by Hg 2+ and Cu 2+ but was not affected by other inhibitors of thiol enzymes such as p-chloromercuribenzoate and N-ethylmaleimide. N-Bromosuccinimide showed a strong inhibitory effect, suggesting that a tryptophan residue is essential for the activity of the enzyme. The N-terminal amino acid sequence of the enzyme showed considerable homology to those of endo-β-1,4-glucanases from some other microorganisms, including Sclerotinia sclerotiorum and Schizophyllum commune. The enzyme had very strong protease-resistance, and showed no loss of activity when incubated with proteases such as Savinase at 40°C, even for 2 weeks.