Chitin fractionation and characterization in N,N-dimethylacetamide/lithium chloride solvent system

A detailed study of the interaction of chitin molecular species with the solvent system N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl) allowed the development of a new method for chitin fractionation by coacervate extraction. The controlled increase of the extracting power of the solvent was carried out using slight modification of the solvent composition. Partial extractions of molecular species were done between coacervation and complete dissolution limits using different mixtures of DMAc/LiCl of increasing extracting power. Fractions were characterized in DMAc/LiCl 5% (w/w) by viscometry and size exclusion chromatography with refractive index and multi-angle laser light scattering detectors. Fractions obtained by coacervate extraction range from 80,000 to 710,000 g mol⁻¹ with polydispersity index between 1.28 and 1.44. The Mark–Houwink–Sakurada equation constants a and K for chitin in DMAc/LiCl 5% (w/w) were found to be 0.95 and 7.6×10⁻⁵ dl g⁻¹, respectively.     

Effect of solvent exchange on the solid structure and dissolution behavior of cellulose

Effects of solvent exchange and milling on the solid structure of cellulose were investigated, using small- and wide-angle X-ray scattering and solid-state NMR. The solvent exchange facilitated the dissolution of cellulose in LiCl/DMAc with no change of the crystalline structure of cellulose. In contrast, the milling never facilitated the dissolution of cellulose, though the crystalline structure was almost destroyed. These facts show that the crystalline structure of cellulose hardly affects the dissolution in LiCl/DMAc. The fractal dimensions determined by the small-angle X-ray scattering measurements were increased by the solvent exchange, suggesting that the aggregation state of the cellulose microfibril is affected. It was also suggested by the NMR (1)H spin relaxation time measurements that the solvent exchange enhances the molecular mobility of cellulose and shortens the characteristic length along the microfibril, which allows easier access of the solvent molecule to cellulose.     

微波条件下LiCl/DMAc溶解稻秆纤维素制备还原糖

利用氯化锂/N,N-二甲基乙酰胺(LiCl/DMAc)溶剂体系在微波控制条件下对稻秆纤维素进行溶解预处理以提高纤维素酶解糖化效率。考察了微波时间和微波强度对产糖量及还原糖转化率的影响。通过扫描电镜(SEM)和热重分析仪(TG)对预处理前后稻秆纤维素的微观形貌及热稳定性进行表征,并利用高效液相色谱仪(HPLC)对酶解糖液进行糖成分鉴定和含量分析。结果表明,微波加热能够有效促进LiCl/DMAc对稻秆纤维素的溶解。与原生稻秆相比,经微波-LiCl/DMAc法溶解后再生纤维素出现明显解聚,热分解温度由290℃降至220℃。在微波功率为385 W、加热溶解时间为7 min时,所得稻秆纤维素还原糖转化率由30.90%上升至98.67%;HPLC谱图表明,糖液中主要成分为葡萄糖和木糖,分别占所得还原糖总量的43.74%和48.55%。     

Influence of the supramolecular structure and physicochemical properties of cellulose on its dissolution in a lithium chloride/N,N-dimethylacetamide solvent system

The present work deals with the effects of structural variables of celluloses on their dissolution in the solvent system LiCl/N,N-dimethylacetamide, LiCl/DMAc. Celluloses from fast growing sources (sisal and linters), as well as microcrystalline cellulose (avicel PH-101) were studied. The following structural variables were investigated: index of crystallinity, I(c); crystallite size; polymer porosity; and degree of polymerization determined by viscosity, DPv. Mercerization of fibrous celluloses was found to decrease DPv, I(c), the specific surface area, and the ratio pore volume/radius. The relevance of the structural properties of cellulose to its dissolution is discussed. Rate constants and activation parameters of cellulose decrystallization, prior to its solubilization, have been determined under nonisothermal conditions. The kinetic parameters calculated showed that dissolution is accompanied with small, negative enthalpy and a large, negative entropy of activation.     

Preparation of C-6 substituted chitin derivatives under homogeneous conditions

Tosylation of chitin under homogeneous conditions was achieved by the reaction of tosyl chloride with chitin in a DMAc/LiCl solvent system. The resultant tosyl-chitin was fully N-acetylated with acetic anhydride in methanol. The fully acetylated tosyl-chitin was subsequently reacted with the sodium salts of ethyl p-hydroxybenzoate, diethyl malonate, and diethyl phosphite in DMAc to give the corresponding chitin derivatives of 6-O-ethyl benzoate-chitin, 6-deoxy-diethyl malonate-chitin, and 6-(deoxydiethyl) phosphite-chitin, respectively. Subsequent hydrolysis of the chitin-ester derivatives with tert-butoxide in dimethyl sulfoxide (DMSO) generated 6-O-carboxyphenyl-chitin and 6-(deoxydicarboxy)methyl-chitin. The structures of the chitin derivatives were assessed by FT-IR, (13)C NMR, and (31)P NMR, while the degree of substitution of the S(N)2 reaction was estimated by elemental analysis. All the chitin derivatives were found to be soluble or swellable in water, DMAc, or DMSO.     

A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 2. Validation and applications

Fluorescence labeling with the marker carbazole-9-carboxylic acid [2-(2-aminooxyethoxy)ethoxy]amide was shown to be a promising approach toward the accurate determination of carbonyls in cellulosic materials. Combined with gel permeation chromatography in DMAc/LiCl with fluorescence/multiple-angle laser light scattering/refractive index detection, the method yields carbonyl profiles relative to the molecular weight of the cellulosic material. The derivatization procedure can be carried out either homogeneously in DMAc/LiCl or advantageously as heterogeneous derivatization in aqueous buffer. The heterogeneous carbonyl group determination, offering shorter reaction times and increased simplicity as compared to the homogeneous approach, was comprehensively validated. The carbonyl content in numerous dissolving pulps of different provenience has been determined, including pulps with carbonyl contents additionally increased by oxidative treatment. The method was also applied to follow bleaching sequences and oxidative treatments of pulps.     

Effect of solvent exchange on the supramolecular structure, the molecular mobility and the dissolution behavior of cellulose in LiCl/DMAc

We investigated the effect of solvent exchange on the supramolecular structure and the molecular mobility of the cellulose molecule to clarify the mechanism of the dissolution of cellulose in lithium chloride/N,N-dimethylacetamide (LiCl/DMAc). Among the celluloses that were solvent exchanged in different ways, the DMAc-treated celluloses dissolved most rapidly. Dissolution of the acetone-treated celluloses was much slower than the DMAc-treated ones, but considerably faster than the untreated one. Such differences in the dissolution behavior were well explained by the differences in the surface fractal dimension calculated from the small-angle X-ray scattering profiles and in the (1)H spin-lattice and spin-spin relaxation times estimated from the solid-state NMR spectroscopic measurements. Furthermore, it was suggested from the IR spectra and the (13)C spin-lattice relaxation times of cellulose that DMAc is adsorbed on the surface of cellulose even after vacuum-drying and affects the molecular mobility and hydrogen-bonding state of cellulose.     

Preparation and characterization of physical gels and beads from chitin solutions

A detailed account of physical bulk gel and bead formation from various chitin solutions and nonsolvents is given. Instant gel formation occurs upon contact of chitin solutions in dimethylacetamide (DMAc)/lithium chloride (LiCl) or N-methyl-pyrrolidinone (NMP)/LiCl solvents and nonsolvents such as water, ethanol, or acetone. Ethanol was found to be the optimal nonsolvent for homogeneous spherical bead formation from chitin solutions. Similarly, DMAc-based chitin solutions proved to yield higher quality beads compared to NMP-based solutions. The differences in bead morphology, crystallinity, and thermal degradation are explained in light of the attainment of a balance between attractive hydrogen bonding in the chitin gel network and segment–nonsolvent interactions. The dependence of swelling of chitin gels on pH indicated a maximum of swelling ratio value of 4.3 at pH 11 in aqueous solutions while the equilibrium swelling ratio value of chitin beads formed with ethanol reached a maximum of 2.4. Bulk gels formed under favorable conditions were demonstrated to be recyclable after solvent separation and drying.     

Preparation and enzymatic behavior of surfactant-enveloped enzymes for glycosynthesis in nonaqueous aprotic media

Surfactant-enveloped enzymes (SEEs) were prepared from pure cellulases, cellobiohydrolase I and endoglucanase I (Cel7A and Cel7B, respectively), via simply freeze-drying water-in-oil emulsions, wherein the aqueous phase containing each cellulase was stabilized with the nonionic surfactant, dioleyl-N-d-glucona-l-glutamate. The enzymatic tolerance of SEEs to various nonaqueous solvents was investigated, aiming at a novel synthetic approach in biocatalytic glycoengineering. SEE-Cel7A preserved ca. 67% of the original activity after 3 h incubation in lithium chloride (LiCl)/dimethylacetamide (DMAc) that is a good solvent for carbohydrates but completely deactivates intact enzymes. This excellent enzymatic durability depended on the preparation conditions of SEEs, e.g. pH and salt species of the aqueous phase during SEE preparation. SEE-Cel7A or SEE-Cel7B was applied as a biocatalyst to synthesize cellulose, a sugar polymer which is insoluble in common solvents but dissolves in LiCl/DMAc. Both SEEs could catalyze the direct dehydration of cellobiose without any activation of the anomeric carbon, a property that is indispensable for conventional chemo-enzymatic synthesis. The SEE-Cel7A provided short-chain cellulose with the degree of polymerization (DP) ca. 20, and longer-chain cellulose with DP ca. 60 was preferentially obtained by the SEE-Cel7B, possibly through preferential reverse hydrolysis instead of inherent hydrolysis. Nonaqueous SEE-mediated biocatalysis using inexpensive glycohydrolases and sugars that do not need to be chemically modified beforehand would have potentially wide applications in glycoengineering.     

Structural comparison and enhanced enzymatic hydrolysis of the cellulosic preparation from Populus tomentosa Carr., by different cellulose-soluble solvent systems

This study aims to establish an efficient pretreatment process using cellulose-dissolution solvents to enhance the enzymatic saccharification. LiOH/urea, LiCl/DMAc, concentrated phosphoric acid, ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and N-methyl-morpholine-N-oxide (NMMO) were selected as the cellulose dissolution agents. Except the cellulosic sample regenerated from LiCl/DMAc system, all the other treated samples exhibited lower cellulose crystallinity and degree of polymerization (DP), and consequently, exhibited a significant enhancement on enzymatic hydrolysis kinetic. Ionic liquid pretreatment offered unique advantages in the hydrolysis rate in the first 10 h, probably due to the extensively structural transformation of cellulose from the crystalline to the amorphous region. Meanwhile, the regenerated cellulose from concentrated phosphoric acid almost completely consisted of cellulose II, and achieved the highest saccharification yield.     

Cellulose acetates from linters and sisal: correlation between synthesis conditions in DMAc/LiCl and product properties

We report the acetylation of celluloses from sisal (untreated and alkali treated) and cotton linters (alkali treated), under homogeneous solution conditions, using DMAc/LiCl as solvent system. Our target was to evaluate the effects of cellulose dissolution and reactions conditions on the product properties. The products were characterized in terms of degree of substitution (DS) by 1H NMR, and molar weight distribution (MWD) by size exclusion chromatography. Changes in the DS of the products were correlated with reaction conditions and solution properties. It was found that the dissolution of celluloses and degree of substitution of cellulose derivatives depends on a fine adjustment of the dissolution/derivatization conditions, as well as on the origin (sisal or linters) of celluloses.     

Photobactericidal plastic films based on cellulose esterified by chloroacetate and a cationic porphyrin

The synthesis and characterisation of pyridinium porphyrinic chloroacetyl cellulose ester chlorides, where photosensitizing agents are covalently bounded to the polymeric chain, is presented in this paper. First, cellulose was homogenously converted into chloroacetate cellulose ester in DMAc/LiCl solvent by using chloroacetyl chloride. The complete substitution of cellulose was achieved using 7equiv of chloroacetyl chloride for a 2h reaction at room temperature. The absence of base did not prove detrimental to reaction. The grafting of monopyridyltritolylporphyrin onto chloroacetate cellulose ester was then realised by alkylation of the photosensitizer in DMF. These new plastic films were found to be thermostable up to 55 degrees C; higher temperatures led to progressive deacetylation. First results of their photobactericidal activity against Staphylococcus aureus and Escherichia coli strains are very encouraging. Such materials could find applications in medical environments as an alternative to overcome the rampant bacterial multiresistance to classical antibiotics.     

Rheological properties and molecular structure of tunicate cellulose in LiCl/1,3-dimethyl-2-imidazolidinone

Solution properties and molecular structure of tunicate cellulose (TC), an animal cellulose from Halocynthia roretzi, were investigated in terms of rheological and dilute solution properties. The solvent used is 8 wt % LiCl/1,3-dimethyl-2-imidazolidinone (DMI). A solution of dissolving pulp (DP), derived from plant, was also used for comparison. The weight-average molecular weight, Mw, and the limiting viscosity number, [eta], of the TC were evaluated to be 413 x 10(6) and 2645 mL/g, respectively. The TC solution showed the same concentration dependence of GN (GN=5.49 x 10(6)phiw(2.1)4 Pa; phiw: weight fraction of cellulose in solution; GN: plateau modulus) as the DP solution and, moreover, also as the solution of cotton linter (CC) in 8 wt % LiCl/N,N-dimethylacetamide (DMAc). This exponent of 2.1(4) indicates that network structure by entanglements was formed in these solutions. According to the theory of Fetters et al., moreover, such identity means that all of these celluloses have the identical chain structure though their biological origins are far different. On the other hand, the phiw-dependence of eta0-etas (eta0=zero shear rate viscosity of solution; etas=solvent viscosity) was different between the TC and the DP solution in the semidilute regime: the TC solution exhibited eta0-etas proportional, variant phiw(7.5) and the DP solution eta0-etas proportional, variant phiw4. According to the theory of Doi-Edwards, this exponent of 4 (the DP solution) indicates that the DP behaves as flexible polymers in the solution. In contrast, the dependence for the TC solution seems unexplainable on the basis of molecular theories. This difference probably signifies the difference in the relaxation process or mechanism in entanglement systems.     

SEC-MALS-QELS study on the molecular conformation of cellulose in LiCl/amide solutions

The SEC-MALS-QELS (size-exclusion chromatography equipped with multiangle light scattering and quasi-elastic light scattering detectors) method using lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) and LiCl/1,3-dimethyl-2-imidazolidinone (LiCl/DMI) as mobile phases was applied to cellulose and cellulose tricarbanilate (CTC) samples with various average degree of polymerization (DP) values. Molecular conformations of cellulose and CTC in the solvents were then discussed and compared on the basis of the relationships between the radii of gyration (R(g,z) or S(2)(z)(1/2)), the hydrodynamic radii (R(h,z)), and weight-average DP (DP(w)) or the contour lengths (L(w)). The Benoit-Doty theory for wormlike polymer chains was applied to the R(g) vs L(w) data obtained, and the theoretical curves with Kuhn segment lengths l(K) of around 18 nm were found to fit the data of both cellulose and CTC molecules in the solvents. It was concluded from the obtained results that both cellulose and CTC molecules have conformations essentially identical to each other in the solvents; they behave as typical semiflexible chains in good solvents.     

Novel cellulose derivatives. Part VI. Preparation and thermal analysis of two novel cellulose esters with fluorine-containing substituents

Two novel cellulose esters were prepared with fluorine (F)-containing substituents using homogeneous phase reaction chemistry in DMAc/LiCl. The partially substituted derivatives and their corresponding perpropionates proved to be thermoplastic polymers. The 2,2-difluoroethoxy and 2,2,3,3,4,4,5,5-octafluoropentoxy substituents were easily identified by ¹H- and ¹⁹F-NMR spectroscopy without disclosing their precise location on the anhydroglucose unit. Thermal analysis revealed modest or no crystallinity; glass transition temperatures between 53 and 113°C; and improved thermal stability as compared to their F-free counterparts.     

Reentrant condensation of lysozyme: Implications for studying dynamics of lysozyme in aqueous solutions of lithium chloride

Recent studies have outlined the use of eutectic solutions of lithium chloride in water to study microscopic dynamics of lysozyme in an aqueous solvent that is remarkably similar to pure water in many respects, yet allows experiments over a wide temperature range without solvent crystallization. The eutectic point in a (H₂O)_R(LiCl) system corresponds to R ≈ 7.3, and it is of interest to investigate whether less‐concentrated aqueous solutions of LiCl could be used in low‐temperature studies of a solvated protein. We have investigated a range of concentrations of lysozyme and LiCl in aqueous solutions to identify systems that do not show phase separation and avoid solvent crystallization on cooling down. Compared to the lysozyme concentration in solution, the concentration of LiCl in the aqueous solvent plays the major role in determining systems suitable for low‐temperature studies. We have observed interesting and rich phase behavior reminiscent of reentrant condensation of proteins. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 624–629, 2014.     

Chemical modification of silk sericin in lithium chloride/dimethyl sulfoxide solvent with 4-cyanophenyl isocyanate

This paper reports chemical modification of silk sericin in LiCl/dimethyl sulfoxide (DMSO) solvent with 4-cyanophenyl isocyanate. Sericin is a highly hydrophilic protein secreted by Bombyx mori, serving as a protein glue in a cocoon. LiCl/DMSO was found to be a good solvent of sericin and useful for homogeneous modification of its abundant hydroxyl groups under nonaqueous condition. Fourier transform infrared (FTIR) analysis of the modified sericins revealed that 4-cyanophenyl groups were incorporated into sericin molecules mainly through urethane linkages. Several characteristics of the modified sericins such as solubility characteristic, hygroscopic property, and thermal stability were investigated. Secondary structure analysis using FTIR spectra suggested that formation of strong intermolecular hydrogen bonds was inhibited by the modification that is probably attributable to the incorporation of bulky 4-cyanophenyl groups. These results demonstrate that chemical modification of sericin using LiCl/DMSO solvent markedly alters its characteristics.     

Concurrent production of chitin from shrimp shells and fungi

Crustacean shells constitute the traditional and current commercial source of chitin. Conversely, the control of fungal fermentation processes to produce quality chitin makes fungal mycelia an attractive alternative source. Therefore, the exploitation of both of these sources to produce chitin in a concurrent process should be advantageous and is reported here. Three proteolytic Aspergillus niger (strains 0576, 0307 and 0474) were selected from a screening for protease activity from among 34 zygomycete and deuteromycete strains. When fungi and shrimp shell powder were combined in a single reactor, the release of protease by the fungi facilitated the deproteinization of shrimp-shell powder and the release of hydrolyzed proteins. The hydrolyzed proteins in turn were utilized as a nitrogen source for fungal growth, leading to a lowering of the pH of the fermentation medium, thereby further enhancing the demineralization of the shrimp-shell powder. The shrimp-shell powders and fungal mycelia were separated after fermentation and extracted for chitin with 5% LiCl/DMAc solvent. Chitin isolates from the shells were found to have a protein content of less than 5%, while chitin isolates from the three fungal mycelia strains had protein content in the range of 10-15%. The relative molecular weights as estimated by GPC for all chitin samples were in the 10(5) dalton range. All samples displayed characteristic profiles for chitin in their FTIR and solid-state NMR spectra. All chitin samples evaluated with MTT and Neutral Red assays with three commercial cell lines did not display cytotoxic effects.     

Selective and facile cyclization of N-chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems.

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72, 857-867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N-chloroacetylated, C-cysteine amide peptides from the C4 domain of HIV-1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed-phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS-PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.     

Effect of some factors on fabrication of poly(L-lactic acid) microporous foams by thermally induced phase separation using N,N-dimethylacetamide as solvent

Biocompatible, highly interconnected microporous poly(L-lactic acid) (PLLA) foams or scaffolds with nano-fibrous structure, containing pores with diameters of 0.1-3.5 μm and fibers with diameters of 300-700 nm scale, were prepared through the thermally induced liquid-liquid phase separation (TIPS) method using N,N'-dimethyl acetamide (DMAc) as solvent. Various foam morphologies were obtained by changing parameters involved in the TIPS process, such as polymer concentration, solvent composition, and quenching temperatures. The morphology of different foams was examined by scanning electron microscopy, whereas the pore size and the pore size distribution were calculated. The results showed that most porous foams presented nano-fibrous structure with interconnected open pores. In the case of using DMAc as solvent, with increasing polymer concentration, either the average pore diameter or the pore size distribution exhibited a maximum value at 0.05 g/mL polymer concentration and quenching temperature of -30°C. It was found that all the pore size distribution fit the F-distribution equation. With increasing the quenching temperature from -30°C to -10°C, the maximum average pore diameter of the foams decreased and the pore size distribution became narrower, whereas the polymer concentration exhibiting the maximum pore size and widest pore size distribution increased from 0.05 g/mL to 0.07 g/mL. In the case of using the mixed solvent of DMAc/DOX (1,4-dioxane) from 9/1 to 7/3 (v/v) there appeared a maximum value of average pore diameter and a widest pore size distribution all at 0.05 g/mL PLLA concentration and quenching temperature of -30°C. The maximum pore size tends to increase with increasing DOX content.