Effect of Cellulase Enzyme on Cellulose Nano

PHYSICAL CHEMISTRY

W 2009 Carl Hanser Verlag, Munich, Germany

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A. Calvimontes, M. Stamm and V. Dutschk

Effect of Cellulase Enzyme on Cellulose Nano-topography In this study, modification effects of cellulose foil by cellulase enzyme are studied in respect to topography changes by means of atomic force microscopy (AFM). The results provide useful information to understand the enzyme action in the amorphous and crystalline regions of cellulose. It was revealed, that the treatment of cellulose with the cellulase enzyme depends on its concentration, which selectively attacks amorphous cellulose regions. At lower concentration, a random surface roughening of the cellulose foil was observed. With increasing enzyme concentration, the cellulose surface became smoother. On the basis of this knowledge, a conceptual model to describe the conditioning effect by cellulase on the cleanability of cotton fabrics was developed. With the help of this model, we are able to estimate changes in the nanoporosity of cotton fibres. Key words: Cellulose, enzyme cellulase, Celluclean, Cellophan, topography, morphology, porosity

Der Effekt von Cellulase auf die Nanotopographie von Cellulose. Im Rahmen dieser Arbeit wurden die Effekte der Modifikation von Cellulose mit dem Enzym Cellulase auf die Topographieänderungen mittels Rasterkraftmikroskopie (AFM) untersucht. Die Ergebnisse bieten ausreichende Auskunft zum Verständnis der Enzymwirkung auf die Änderungen in amorphen und kristallinen Regionen der Cellulose. Es wurde gezeigt, dass die Modifizierung der Cellulose mit Cellulase abhängig von der Enzymkonzentration selektiv amorphe Bereiche der Cellulose angreift. Bei niedrigen Konzentrationen wurde eine zufällige Oberflächenanrauung der Cellulose-Folie beobachtet. Bei höheren Konzentrationen wird die Celluloseoberfläche glatter. Auf der Basis der gewonnenen Ergebnisse wurde ein konzeptuelles Modell zur Beschreibung des Aufbereitungseffekts der Cellulose bei Cellulase auf die Auswaschbarkeit von Baumwolle entwi-

ckelt. Unter Zuhilfenahme des Modells ist eine Bewertung von Änderungen der Nanoporosität von Baumwollfasern möglich. Stichwörter: Cellulose, Cellulase-Enzym, Celluclean, Cellophan, Topographie, Morphologie, Porosität

1 Introduction

Enzymes are successfully used in all household laundry detergents to boost the removal of organic-based stains caused by blood, grass, starch, fats, and food thickeners [1]. It is well-known, that enzymes also offer care for fabrics and their colour. Some special enzymes, such as cellulases, act directly on cotton-based laundry materials to facilitate the cleaning of fabric fibres. Cellulases have been used for household washing for nearly three decades to preserve fabric colour, ensure anti-greying, and offer satisfying whiteness [2]. Celluclean, a brand new enzyme developed by Novozymes, is a superior cleaning cellulase giving a remarkable stain removal and anti-greying effects in addition to maintaining whiteness under wash-relevant conditions [1]. This product is a unique enzyme solution that removes beta-glucan stains, which can form a film on the fabric, attracting dirt. According to product information, this enzyme also removes particulate soil from cotton fabrics, preventing its re-deposition. Wash tests performed by detergents with and without Celluclean have clearly proven that Celluclean offers antigreying effect and makes whites whiter and coloured clothes brighter [1, 2]. In textile structures, fibres are not completely fixed compared to solid materials. Washing and drying processes

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Figure 1 Influence of the enzyme concentration on cellulose film roughness (a); influence of the enzyme on porosity and actual surface – Wenzel roughness factor (b)

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usually lead to significant changes in the surface topography such as relaxation/shrinkage and pilling. During the investigations of the effect of Celluclean on cotton textiles by washing and drying, we observed that for an appropriate analyz-

ing and understanding the enzymatic effect on the surface topography, it is indispensable to separate it from the mechanical effect due to washing and drying processes. For this reason, modification of a cellulose film by cellulase enzyme is studied in respect to topography effects by means atomic force microscopy (AFM). The results provide useful information to understand the enzyme action in the amorphous and crystalline regions of cellulose. Changes in topography were studied depending on enzyme concentration. 2 Experimental

Figure 2 Comparison of profiles at cellulose film surfaces after treatment with lower and higher enzyme concentrations

Cellophane films (Cellophane, DuPont) were used as a cellulose surface. Cellophane is a thin, transparent sheet, also known as “regenerated cellulose film”; it is identical to cellulose in chemical structure and is usually made from viscose [3]. Unmodified and modified surfaces were examined topographically using an atomic force microscope (AFM), Dimension 3100 (Veeco Instruments, USA) in contact mode. Changes in the crystallinity of the original cellulose occur already by the Cellophan manufacturing process. Chen et al. [4] reported an infrared crystallinity index of 0.23 and 0.62 for Cellophane (DuPont) and cotton, respectively. Nevertheless, the aim of our experiment was to identify morphologi-

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A. Calvimontes et al.: Effect of cellulase enzyme on cellulose nano-topography


Figure 3 Schematisation of topographic changes after treatment with low enzyme concentration. Rz increases while Ra decreases

Figure 4

2D and 3D representations of a unmodified cellulose film surface


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Figure 5 Topographic changes in the amorphous regions of cellulose films due to the enzyme action: (a) Cellulose surface without treatment – an 9 · 9 lm AFM image (left); an 2.5 · 2.5 lm AFM image (right); (b) Cellulose surface after treatment with 0.2 ppm Celluclean; an 9 · 9 lm AFM image (left); an 2.5 · 2.5 lm AFM image (right) (c) Profiles measured using an AFM at unmodified and differently modified cellulose surface

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Figure 6

Conceptual model to a mechanistic understanding the effect of Celluclean on cellulose

cal differences on a cellulose surface, to relate it to the crystalline and amorphous regions and to study topographical changes due to the enzymatic action. With this purpouse, we performed experiments by measuring the topography of cellulose foils before and after impregnation/drying with Celluclean solutions. 3 Results

The nanotopography of Cellophan surfaces were modified by the action of Celluclean solutions. Arithmetic mean roughness Ra of a surface is defined as the average of all distances between z-coordinate (height points) and a calculated mean-height plane. Mean rough height Rz is commonly named “mean roughness depth”. For its calculation, the surface has to be devided into 25 sub-areas, where the arithmetic average of the maximal amplitudes (maximal distance between highest elevation and deepest point) of the subareas is calculated. Ra is statistically oriented to a mean value, then exceptional higher or deeper points do not have any important impacts on it. Rz characterizes the highest and deepest points of a surface, therefore, it is strong dependent of larger surface inhomogeneities. Fig. 1a shows, that the enzyme polishes the cellulose surface (sequential decrease of Ra). However, at lower enzyme concentration, peaks are formed on the surface (increase of Rz). At higher enzyme concentrations, these peaks are removed. Porosity, which is defined as the void volume contained between the true surface and the calculated mean-height plane per unit area, is strongly dependent of Rz in this case, due to surface inhomogeneities produced by the enzyme action. However, Wenzel roughness factor calculated from the experimental topographic data, defined as the ratio between the real (true, actual) surface area and the geometric projected (plain) area, shows its maximal point at medium enzyme conentration (Fig. 1b). The formation of peaks after treatment with lower enzyme concentration, their posterior remove and polishing of the surface are evident by comparing the respective surface profiles measured along 9 lm (Fig. 2). The simultaneous Ra decrease and Rz increase, at lower enzyme concentration, is the result of important morphologic changes, as schematises Fig. 3. Morphologically, we identified 2 different regions in the surface topography of cellulose films: a relative smooth matrix containing small hills (Fig. 4). According to our results, the formation of peaks at lower enzyme concentrations and the smoothing process in general, are produced principally on the hills surface (cf. Fig. 5). For this reason, we presume that these formations correpond to the amorphous cellulose.


Consequently the almost smooth phase corresponds to the crystalline region. 4 Conclusion

Celluclean selectively attacks the amorphous cellulose regions which consist of small hills in a matrix of flat crystalline regions. According to the measured nano-topographic changes, at lower concentrations of Celluclean the formation of peaks and an increase of a real surface (in terms of Wenzel roughness factor) indicate that the enzymatic effect is not complete and, therefore, not all the amorphous fractioned parts were removed. At higher enzyme concentrations, amorphous cellulose regions are polished. On the basis of this knowledge, a conceptual model to describe the effect of conditioning by cellulase on the micro-structure of cotton yarns surface and, as a consequence, on cleanability of cotton fabrics can be developed, as shown in Fig. 6. Acknowledgement

We would like to acknowledge and thank our partner Procter & Gamble by the financial support of our research. References 1. Ryom, N. M., Baltsen, L. E. T., Jokumsen, K. V. and Salzmann, P.: A New Cellulase for Whiteness Maintenance Under Wash Relevant Conditions, oral contribution, SEPAWA Congress, Würzburg, 10 – 12 October 2007. 2. Hauthal, H. G.: Basics, Ingredients, Detergents, Product Safety and Sustainability, Third European Detergents Conference Report., Tenside Surf. Det. 45 (2008) 1, 30 – 42. 3. Young, R.: Cellulose structure modification and hydrolysis, New York, Wiley, 1986. ISBN 0471827614. 4. Chen, Y.-S., Yang, K. S. and Cuculo, J. A.: Formation and Characterization of Cellulose Films from a Liquid Crystalline Solution of Cellulose in Ammonia/ Ammonium Thiocyanate, Journal of Applied Polymer Science, 41 (1990) 587 – 594. Received: 19. 05. 2009 Revised: 18. 08. 2009

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Correspondence to Alfredo Calvimontes Leibniz-Institut für Polymerforschung Dresden e. V. Hohe Strasse 6 01069 Dresden Germany E-Mail: [email protected]

The authors of this paper M. Sc. Alfredo Calvimontes was born in 1965 and graduate from the La Salle University (Mexico), Faculty of Chemical Engineering in 1989. During twelve years he worked as a manufacturing engineer in the chemical and textile industries in Mexico

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Prof. Dr. Manfred Stamm studied Physics from 1968 to 1974 at the University of Frankfurt am Main, received his PhD at the Institute of Physical Chemistry of the University of Mainz and finalised his Habilitation on Investigation of polymer surfaces and interfaces with nanometer resolution. Between 1979 and 1985 he worked as staff scientist at Institute of Solid State Research in Jülich. Between 1984 and 1985 he was visiting scientist at Brookhaven national laboratory in the United States. Between 1985 and 1999 he was staff scientist and project leader at the Max-Planck Institute of Polymer Research in Mainz. From 1999 Manfred Stamm is professor for Physical Chemistry of Polymeric Materials at the Technical University Dresden and head of IPF-Institute of Physical Chemistry and Physics of Polymers at Leibniz Institute of Polymer Research in Dresden. Prof. Stamm is member of editorial board of Coll.Interf.Sci., referee of several journals and funding agencies, member of board of “Dresden Center of Science and Art” DZWK and of “Dresden Materials Research Science Association” MFD. In 2004 received the International Belgian Polymer Group Award.

Dr.-Ing. Victoria Dutschk was born in 1966 in Bendery (Moldova) and graduate from the Odessa State University, Faculty of Mechanics and Mathematics in 1988. She obtained her PhD in 2000 at the Technische Universität Dresden, Faculty of Mechanical Engineering, Department Material Science under the guidande of Prof. Wolfgang Pompe. Since 1994 she has been working as a research associate at the Leibniz Institute of Polymer Research Dresden in the field interfacial engineering focussing specifically on surface active agents.

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and Bolivia. In 2004 he received his Master Degree in Textile and Clothing Engineering from the Dresden University of Technology. At the present time he works at the Leibniz Institute of Polymer Research Dresden.

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