Fibers 2014, 2(1), 34-44; doi:10.3390/fib2010034

Article
Hierarchically Self-Assembled Nanofiber Films from Amylose-Grafted Carboxymethyl Cellulose
Daisuke Hatanaka 1, Yasutaka Takemoto 1, Kazuya Yamamoto 1 and Jun-ichi Kadokawa 1,2,*
1
Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan; E-Mails: k3559474@kadai.jp (D.H.); k3181684@kadai.jp (Y.T.); yamamoto@eng.kagoshima-u.ac.jp (K.Y.); kadokawa@eng.kagoshima-u.ac.jp (J.K.)
2
Research Center for Environmentally Friendly Materials Engineering, Muroran Institute of Technology, 27-1 Mizumoto-cho, Muroran, Hokkaido 050-8585, Japan
*
Author to whom correspondence should be addressed; E-Mail: kadokawa@eng.kagoshima-u.ac.jp; Tel.: +81-99-285-7743; Fax: +81-99-285-3253.
Received: 18 November 2013; in revised form: 21 January 2014 / Accepted: 22 January 2014 /
Published: 28 January 2014

Abstract

: In this paper, we report the formation of hierarchically self-assembled nanofiber films from amylose-grafted sodium carboxymethyl celluloses (NaCMCs) that were synthesized by a chemoenzymatic approach. First, maltooligosaccharide primer-grafted NaCMCs were prepared by a chemical reaction using two kinds of NaCMCs with different degrees of polymerization (DPs) from Avicel and cotton sources. Then, phosphorylase-catalyzed enzymatic polymerization of α-d-glucose 1-phosphate from the nonreducing ends of the primer chains on the products was conducted to produce the prescribed amylose-grafted NaCMCs. The films were obtained by drying aqueous alkaline solutions of the amylose-grafted NaCMCs. The scanning electron microscopy (SEM) image of the film fabricated from the material with the higher DP from the cotton source showed a clear, self-assembled, highly condensed tangle of nanofibers. The SEM image of the material with the lower DP from the Avicel source, on the other hand, showed an unclear nanofiber morphology. These results indicate that the DPs of the main chains in the materials strongly affected the hierarchically self-assembled nanofiber formation. The SEM images of the films after washing out the alkali, furthermore, showed that the fibers partially merged with each other at the interfacial area owing to the double helix formation between the amylose-grafted chains. The mechanical properties of the films under tensile mode also depended on the self-assembled morphologies of the amylose-grafted NaCMCs from the different sources.
Keywords:
self-assembled nanofiber; amylose; carboxymethyl cellulose; chemoenzymatic

1. Introduction

Cellulose is the most abundant biological macromolecule, with a polysaccharide structure consisting of a chain of β-(1→4)-linked glucose residues [1,2], and is a very important renewable resource used in furniture, clothing, and medical products. Considerable efforts are also being devoted to developing new material applications of cellulose because of its biodegradable and eco-friendly properties. Self-assembled fibrillar nanostructures from cellulose, so-called nanofibers, are promising materials for practical applications in bio-related research fields such as tissue engineering [3,4,5]. Conventional approaches to the production of cellulose nanofibers are mainly top-down procedures that break down the starting bulk materials from natural cellulose resources [6,7,8].

In a previous study, we found that the self-assembly of amylose-grafted carboxymethyl cellulose sodium salt (NaCMC) forms nanofiber films upon drying its alkaline aqueous solution [9]. Carboxymethyl cellulose (CMC), an anionic water-soluble polysaccharide, is one of the most widely used cellulose derivatives, and its sodium salt (NaCMC) has a number of COONa groups that promote water solubility [10]. Our method for the formation of nanofibers from amylose-grafted NaCMCs is completely different from the aforementioned conventional top-down procedures because our method is a hierarchically self-assembling generative (bottom-up) route, in which fibrillar nanostructures are produced by regeneration from the solutions of the substrates.

Amylose-grafted NaCMC (3) was synthesized by a chemoenzymatic technique according to Scheme 1, which was combined of phosphorylase-catalyzed enzymatic polymerization with chemical reaction [11,12,13,14,15,16,17,18,19]. Because the enzymatic polymerization of α-d-glucose 1-phosphate (G-1-P) is initiated at the nonreducing end of the maltooligosaccharide primer and produces amylose by the following propagation [20,21,22,23,24,25,26,27], the primer was first introduced on the NaCMC chain by the condensation of an amine-functionalized maltooligosaccharide (1) with carboxylates in NaCMC to give a maltooligosaccharide-grafted NaCMC. Then, the phosphorylase-catalyzed polymerization of G-1-P was conducted using the product to give the prescribed material, 3. The introduction of amylose-graft chains contributed to the construction of a rigid NaCMC main chain, resulting in a nanofiber film upon drying the alkaline solution of the product. Furthermore, the long amylose-graft chains formed double helixes in the intermolecular NaCMC chains by washing out alkali from the film to produce a robust film with the merged nanofiber morphology.

In this paper, we describe the effect of the degree of polymerization (DP) of the NaCMC main chains on the formation behaviors of hierarchically self-assembled nanofiber films from 3. For this purpose, the materials were synthesized by the aforementioned chemoenzymatic method using NaCMCs having similar degrees of carboxymethylation (DC). The NaCMCs were prepared from two kinds of cellulose with different DPs (microcrystalline cellulose (Avicel No. 2331), DP = ca. 230; cotton, DP = ca. 2500) [28,29].

Fibers 02 00034 g006 200
Scheme 1. Chemoenzymatic synthesis of amylose-grafted NaCMC (3).

Click here to enlarge figure

Scheme 1. Chemoenzymatic synthesis of amylose-grafted NaCMC (3).
Fibers 02 00034 g006 1024

2. Experimental Section

2.1. Materials

Microcrystalline cellulose from Merck (Avicel, No. 2331) and absorbent cotton from Kakui Co. Ltd. (Kagoshima, Japan) were used. Carboxymethylation of the cellulose was carried out by the reaction of cellulose with sodium chloroacetate according to the literature procedure [30]. The DC values were estimated by the titration method described in the literature [31]. Thermostable phosphorylase (Aquifex aeolicus VF5) was supplied by Ezaki Glico Co. Ltd., Osaka, Japan [23,32,33]. An amine-functionalized maltooligosaccharide (1) was prepared according to the literature procedure [16]. Other reagents and solvents were used as received.

2.2. Synthesis of Maltooligosaccharide-Grafted NaCMC (2)

To a solution of NaCMC (from Avicel, DC = 0.46, 0.020 g, 0.0101 mmol) in water (3.0 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (0.0387 g, 0.202 mmol) and N-hydroxysuccinimide (NHS) (0.0232 g, 0.202 mmol), and the mixture was stirred at room temperature for 1 h. Then, 1 (0.245 g, 0.202 mmol) was added to the solution and the mixture was further stirred at room temperature for 24 h. After the reaction solution was dialyzed in a dialysis bag (molecular cut off: 12,000–14,000) against water overnight, the obtained material was purified further by precipitation into methanol (300 mL). The precipitate was isolated by filtration, washed with dimethyl sulfoxide (DMSO) and methanol, and dried under reduced pressure to give maltooligosaccharide-grafted NaCMC (2) (0.133 g); 1H NMR (D2O) δ 3.00–4.44 (sugar protons of H2-H6, NCH2CH2N), 4.44–4.68 (H1 of NaCMC), 5.17, 5.34 (H1 of maltooligosaccharide). The degree of substitution (DS) for the grafting was determined by the integrated ratio of the H1 signal of maltooligosaccharide to that of NaCMC to be 0.074. Maltooligosaccharide-grafted NaCMC from cotton was synthesized according to a similar procedure (DC = 0.43, DS = 0.070).

2.3. Synthesis of Amylose-Grafted NaCMC (3)

The aforementioned 2 (from Avicel, 0.0080 g, 0.0212 mmol) was dissolved in an aqueous sodium acetate buffer solution (0.2 mol/L, pH 6.2, 3.0 mL) and G-1-P disodium salt (0.486 g, 1.60 mmol) was added to the solution. After the pH value was adjusted to 6.2 by the addition of 0.2 mol/L aqueous acetic acid, thermostable phosphorylase (16 units) was added to this solution, which was then maintained at 45 °C for 20 h with stirring. After the resulting gelic mixture was immersed in water (100 mL) for 3 h, the gel was lyophilized to give amylose-grafted NaCMC (3, 0.107 g); 1H NMR (1 mol/L NaOD/D2O) δ 3.00–4.44 (sugar protons of H2–H6, NCH2CH2N), 4.44–4.68 (H1 of NaCMC), 5.13, 5.27 (H1 of amylose). Amylose-grafted NaCMC from cotton was synthesized according to a similar procedure.

2.4. Formation of Nanofiber Film from 3

Amylose-grafted NaCMC 3 (0.040 g) was first dissolved in a 0.50 mol/L NaOH aqueous solution (1.5 mL) by stirring the mixture at room temperature. The solution was thinly cast onto a glass plate and dried under ambient conditions to give a film. The resulting film was immersed twice in water (10 mL for 10 min and 5 mL for 5 min) to remove the NaOH and dried under ambient conditions.

2.5. Measurements

1H NMR spectra were recorded on a JEOL ECX-400 spectrometer. Scanning electron microscopy (SEM) images were obtained using an Hitachi SU-70 electron microscope. X-ray diffraction (XRD) measurements were conducted using PANalytical X’Pert Pro MPD with Ni-filtered Cu Kα radiation (λ = 0.15418 nm). The stress–strain curves under tensile mode were measured using a tensile tester (Little Senster LSC-1/30, Tokyo Testing Machine).

3. Results and Discussion

3.1. Chemoenzymatic Synthesis of Amylose-Grafted NaCMC 3

For the chemoenzymatic synthesis of the amylose-grafted NaCMCs (3), in this study, two kinds of NaCMCs with similar DC values and different DP values were prepared by the carboxymethylation of Avicel and cotton [30]. The NaCMC from Avicel had DC and DP of 0.46 and 230, respectively, and the NaCMC from cotton had DC and DP of 0.43 and 2500, respectively [28,29]. The introduction ofmaltooligosaccharides onto the NaCMC chains was performed by the condensation of 1 with carboxylates in the NaCMCs using the EDC/NHS condensing agent in water to give 2 (Scheme 1). The ratios of the introduced maltooligosaccharide chains to the repeating units (functionality) in the products from the two kinds of NaCMCs were adjusted to have similar values (DS = 0.074 from Avicel and DS = 0.070 from cotton) by appropriate reaction conditions, which were determined by the integrated ratios of the H1 signal of the maltooligosaccharides to that of the NaCMCs in the 1H NMR spectra in D2O.

The amylose-grafted NaCMCs were synthesized by the phosphorylase-catalyzed polymerization of G-1-P from the nonreducing ends of the maltooligosaccharide (primer) graft chains on 2 in the G-1-P/primer feed ratio of 500 (Scheme 1). As the polymerization progressed, the gelation of the reaction mixtures took place. The gelic products were immersed in water and the resulting gels were lyophilized to give 3. The isolated products were insoluble in water but soluble in aqueous alkaline solution. Thus, the structures of the products were characterized by the 1H NMR spectra measured in 1 mol/L NaOD/D2O (Figure 1, from the cotton source), which showed an obvious increase in the integrated ratios of the H1 signal of amylose to the H1 signal of NaCMC as compared with that in the 1H NMR spectra of 2. The average DPs of the amylose-graft chains in 3 from Avicel and cotton were calculated on the basis of the elemental analysis data, and the functionalities of the maltooligosaccharide chain (the DS values) in 2 were found to be 187 and 218 for Avicel and cotton sources, respectively.

Fibers 02 00034 g001 200
Figure 1. 1H NMR spectrum of amylose-grafted NaCMC (3) from cotton (NaOD/D2O).

Click here to enlarge figure

Figure 1. 1H NMR spectrum of amylose-grafted NaCMC (3) from cotton (NaOD/D2O).
Fibers 02 00034 g001 1024

3.2. Formation and Characterization of Self-Assembled Nanofiber Films from 3

We previously reported that amylose-grafted NaCMCs (3) with DPs of 140–214 and an amylose-graft chain functionality of 35.4 synthesized from commercially available NaCMC (DP = ca. 1200 and DC = 0.7) formed self-assembled nanofiber films after drying their aqueous alkaline solutions. In the present study, we evaluated the effect of the DPs of the NaCMC main chains in 3 on the formation behavior of the self-assembled nanofiber films using two materials synthesized as aforementioned. Solutions of the materials in 0.50 mol/L NaOH aq. were cast onto a glass plate and dried under ambient conditions to give the product films. It was confirmed from the SEM image that the film of 3 from the cotton source was constructed from nanofibers arranged in a highly condensed tangle (Figure 2b). On the other hand, such a clear nanofiber morphology was not seen in the SEM image of the film fabricated from the Avicel source, although it showed some broad fibrillar entanglement (Figure 2a). The SEM images of the films after washing out the alkali showed that the fibrils partially merged with each other at interfacial areas with the remaining fibrillar morphologies (Figure 2c,d). The SEM results suggest that the longer NaCMC chain in 3 is favorable for producing the regularly controlled self-assembly needed to construct the clear nanofiber morphology.

Fibers 02 00034 g002 200
Figure 2. SEM images of films prepared from alkaline solutions of 3 from Avicel and cotton ((a,b), respectively) and respective films after washing out alkali ((c,d), respectively).

Click here to enlarge figure

Figure 2. SEM images of films prepared from alkaline solutions of 3 from Avicel and cotton ((a,b), respectively) and respective films after washing out alkali ((c,d), respectively).
Fibers 02 00034 g002 1024

XRD measurements of the films were conducted to evaluate the hierarchically self-assembled structure of 3. The XRD profile of the film of 3 from cotton before washing out the alkali slightly show diffraction peaks due to amylose helix at around 17 and 23° [34] besides NaOH crystalline peaks (Figure 3a), indicating that the amylose graft chains only partially formed double helix conformation in the film. Because the aqueous alkaline solution is a good solvent for amylose, the formation of an amylose double helix is mostly prevented during the drying process of the solution. After washing out the alkali from the film, the XRD profile exhibited diffraction peaks obviously due to the amylose helix (Figure 3b,c), indicated with shadows), suggesting the progress of double helix formation during the washing process.

Fibers 02 00034 g003 200
Figure 3. XRD profiles of films of 3 from cotton (a) before and (b) after washing out the alkali and (c) amylose.

Click here to enlarge figure

Figure 3. XRD profiles of films of 3 from cotton (a) before and (b) after washing out the alkali and (c) amylose.
Fibers 02 00034 g003 1024

On the basis of the above results, the self-assembling process of 3 is proposed to lead to the formation of the nanofiber film (Figure 4). As already reported in our previous paper [9], the introduction of saccharide-graft chains on NaCMC prevented the construction of a random-coil conformation, resulting in the rigid nature of the NaCMC chain. While drying the aqueous alkaline solution of 3, some of these rigid materials regularly assembled to induce nanofibrillation with the slight double helix formation from amylose graft chains on NaCMCs, but they did not construct large aggregates because the double helix formation of the most of the amylose chains was prevented due to the alkaline conditions. By washing out alkali from the film, the double helix was able to form on the nanofibers, leading to merging on the surface of the fibers. The average DP value of the NaCMC main chain of 3 from the cotton source (ca. 2500) was much larger than that of the amylose-graft chain (218), resulting in nanofibrillation with a high aspect ratio. On the other hand, because the two DP values of the main and graft chains in 3 from the Avicel source were comparable (230 and 187), the self-assembled nanofibers were not clearly formed.

Fibers 02 00034 g004 200
Figure 4. Proposed self-assembling process of 3 under alkaline conditions leading to nanofiber film.

Click here to enlarge figure

Figure 4. Proposed self-assembling process of 3 under alkaline conditions leading to nanofiber film.
Fibers 02 00034 g004 1024

Finally, the mechanical properties of the films of 3 from the Avicel and cotton sources after washing out alkali were evaluated by tensile testing (Figure 5). The stress–strain curve of the film from the cotton source showed larger fracture stress and strain values than the film from the Avicel source. This result indicates that the DP of the main chain in 3 strongly affects the mechanical properties of the present nanofiber film.

Fibers 02 00034 g005 200
Figure 5. Stress–strain curves of films of 3 from (a) Avicel and (b) cotton after washing out alkali.

Click here to enlarge figure

Figure 5. Stress–strain curves of films of 3 from (a) Avicel and (b) cotton after washing out alkali.
Fibers 02 00034 g005 1024

4. Conclusions

This paper reports the formation of hierarchically self-assembled nanofiber films from amylose-grafted NaCMC films (3) with different DPs. The materials were synthesized by the chemoenzymatic method using NaCMCs from different sources, Avicel and cotton. The film with the clear nanofiber morphology was formed from the material with the higher DP (cotton) upon drying its aqueous alkaline solution, whereas the clear nanofiber morphology was not obtained in the film from the material with the lower DP (Avicel). By washing out the alkali from the films, the fibers merged at their interfacial areas. The obvious formation of the hierarchically self-assembled nanofibers in the film strengthened the mechanical properties under tensile mode.

Acknowledgments

A donation of phosphorylase by Ezaki Glico Co. Ltd., Osaka, Japan is gratefully acknowledged.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Klemm, D.; Heublein, B.; Fink, H.-P.; Bohn, A. Cellulose: Fascinating biopolymers and sustainable raw material. Angew. Chem. Int. Ed. 2005, 44, 3358–3393, doi:10.1002/anie.200460587.
  2. Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Nanocelluloses: A new family of nature-based materials. Angew. Chem. Int. Ed. 2011, 50, 5438–5466, doi:10.1002/anie.201001273.
  3. Beglou, M.J.; Haghi, A.K. Electrospun biodegdadable and biocompatible natural nanofibers: A detailed review. Cellul. Chem. Technol. 2008, 42, 441–462.
  4. Isogai, A.; Saito, T.; Fukuzumi, H. TEMPO-oxidized cellulose nanofibers. Nanoscale 2011, 3, 71–85, doi:10.1039/c0nr00583e.
  5. Abdul Khalil, H.P.S.; Bhat, A.H.; Ireana Yusra, A.F. Green composites from sustainable cellulose nanofibrils: A review. Carbohydr. Polym. 2012, 87, 963–979, doi:10.1016/j.carbpol.2011.08.078.
  6. Saito, T.; Nishiyama, Y.; Putaux, J.L.; Vignon, M.; Isogai, A. Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of native cellulose. Biomacromolecules 2006, 7, 1687–1691, doi:10.1021/bm060154s.
  7. Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Cellulose nanofibers prepared by TEMPO-mediated oxidation of native cellulose. Biomacromolecules 2007, 8, 2485–2491, doi:10.1021/bm0703970.
  8. Abe, K.; Iwamoto, S.; Yano, H. Obtaining cellulose nanofibers with a uniform width of 15 nm from wood. Biomacromolecules 2007, 8, 3276–3278, doi:10.1021/bm700624p.
  9. Kadokawa, J.; Arimura, T.; Takemoto, Y.; Yamamoto, K. Self-assembly of amylose-grafted carboxymethyl cellulose. Carbohydr. Polym. 2012, 90, 1371–1377, doi:10.1016/j.carbpol.2012.07.006.
  10. Stephen, A.M.; Philips, G.O.; Williams, P.A. Food Polysaccharides and Their Applications; Taylor & Francis: London, UK, 1995.
  11. Matsuda, S.; Kaneko, Y.; Kadokawa, J. Chemoenzymatic synthesis of amylose-grafted chitosan. Macromol. Rapid Commun. 2007, 28, 863–867, doi:10.1002/marc.200600821.
  12. Kaneko, Y.; Matsuda, S.; Kadokawa, J. Chemoenzymatic syntheses of amylose-grafted chitin and chitosan. Biomacromolecules 2007, 8, 3959–3964, doi:10.1021/bm701000t.
  13. Omagari, Y.; Matsuda, S.; Kaneko, Y.; Kadokawa, J. Chemoenzymatic synthesis of amylose-grafted cellulose. Macromol. Biosci. 2009, 9, 450–455, doi:10.1002/mabi.200800237.
  14. Kaneko, Y.; Kadokawa, J. Chemoenzymatic Synthesis of Amylose-Grafted Polymers. In Handbook of Carbohydrate Polymers: Development, Properties and Applications; Ito, R., Matsuo, Y., Eds.; Nova Science Publishers, Inc.: Hauppauge, NY, USA, 2009. Chapter 23; pp. 671–691.
  15. Omagari, Y.; Kaneko, Y.; Kadokawa, J. Chemoenzymatic synthesis of amylose-grafted alginate and its formation of enzymatic disintegratable beads. Carbohydr. Polym. 2010, 82, 394–400, doi:10.1016/j.carbpol.2010.04.078.
  16. Arimura, T.; Omagari, Y.; Yamamoto, K.; Kadokawa, J. Chemoenzymatic synthesis and hydrogelation of amylose-grafted xanthan gums. Int. J. Biol. Macromol. 2011, 49, 498–503, doi:10.1016/j.ijbiomac.2011.06.003.
  17. Omagari, Y.; Kadokawa, J. Synthesis of heteropolysaccharides having amylose chains using phosphorylase-catalyzed enzymatic polymerization. Kobunshi Ronbunshu 2011, 68, 242–249, doi:10.1295/koron.68.242.
  18. Kadokawa, J. Synthesis of Amylose-Grafted Polysaccharide Materials by Phosphorylase-Catalyzed Enzymatic Polymerization. In Biobased Monomers, Polymers, and Materials; Smith, P.B., Gross, R.A., Eds.; ACS Symposium Series: Washington, DC, USA, 2012. Chapter 15; pp. 237–255.
  19. Kadokawa, J.; Kaneko, Y. Engineering of Polysaccharide Materials—By Phosphorylase-Catalyzed Enzymatic Chain-Elongation; Pan Stanford Publishing Pte. Ltd.: Singapore, Singapore, 2013.
  20. Ziegast, G.; Pfannemüller, B. Phosphorolytic syntheses with di-, oligo- and multi-functional primers. Carbohydr. Res. 1987, 160, 185–204, doi:10.1016/0008-6215(87)80311-7.
  21. Kitaoka, M.; Hayashi, K. Carbohydrate-processing phosphorolytic enzymes. Trends Glycosci. Glycotechnol. 2002, 14, 35–50, doi:10.4052/tigg.14.35.
  22. Fujii, K.; Takata, H.; Yanase, M.; Terada, Y.; Ohdan, K.; Takaha, T.; Okada, S.; Kuriki, T. Bioengineering and application of novel glucose polymers. Biocatal. Biotransform. 2003, 21, 167–172.
  23. Yanase, M.; Takaha, T.; Kuriki, T. α-Glucan phosphorylase and its use in carbohydrate engineering. J. Food Agric. 2006, 86, 1631–1635, doi:10.1002/jsfa.2513.
  24. Ohdan, K.; Fujii, K.; Yanase, M.; Takaha, T.; Kuriki, T. Enzymatic synthesis of amylose. Biocatal. Biotransform. 2006, 24, 77–81.
  25. Seibel, J.; Jördening, H.-J.; Buchholz, K. Glycosylation with activated sugars using glycosyltransferases and transglycosidases. Biocatal. Biotranform. 2006, 24, 311–342.
  26. Kobayashi, S.; Makino, A. Enzymatic polymer synthesis: An opportunity for green polymer chemistry. Chem. Rev. 2009, 109, 5288–5353, doi:10.1021/cr900165z.
  27. Kadokawa, J.; Kobayashi, S. Polymer synthesis by enzymatic catalysis. Curr. Opin. Chem. Biol. 2010, 14, 145–153, doi:10.1016/j.cbpa.2009.11.020.
  28. Sasaki, M.; Sekiguchi, G.; Adschiri, T.; Arai, K. Rapid and selective conversion of cellulose to valuable chemical intermediates using supercritical water. In Proceedings of the 6th International Symposium on Supercritical Fluids, 2003, Versailles, France, 28–30 April 2003; Volume 2, pp. 1417–1422.
  29. Khullar, R.; Varshney, V.K.; Naithani, S.; Heinze, T.; Soni, P.L. Carboxymethylation of cellulosic material (average degree of polymerization 2600) isolated from cotton (Gossypium) linters with respect to degree of substitution and rheological behavior. J. Appl. Polym. Sci. 2005, 96, 1477–1482, doi:10.1002/app.21645.
  30. Olaru, N.; Olaru, L. Influence of organic diluents on cellulose carboxymethylation. Macromol. Chem. Phys. 2001, 202, 207–211, doi:10.1002/1521-3935(20010101)202:1<207::AID-MACP207>3.0.CO;2-Q.
  31. Hosokawa, K.; Hanada, N.; Sato, S. Cationized carboxymethyl cellulose sodium salts with good chemical resistance and dispersibility. Japanese Patent 2002-201202. Filed 28 December 2000, Issued 19 July 2002.
  32. Bhuiyan, S.H.; Rus’d, A.A.; Kitaoka, M.; Hayashi, K. Characterization of a hyperthermostable glycogen phosphorylase from Aquifex aeolicus expressed in Escherichia coli. J. Mol. Catal. B: Enzym. 2003, 22, 173–180, doi:10.1016/S1381-1177(03)00029-8.
  33. Yanase, M.; Takata, H.; Fujii, K.; Takaha, T.; Kuriki, T. Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L α-glucan phosphorylase. Appl. Environ. Microbiol. 2003, 71, 5433–5439.
  34. Zobel, H.F. Starch crystal transformations and their industrial importance. Starch 1988, 40, 1–7, doi:10.1002/star.19880400102.
Fibers EISSN 2079-6439 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert