A new method for measuring scouring efficiency of natural ... - CiteSeerX

Journal of Biotechnology 107 (2004) 265–273

A new method for measuring scouring efficiency of natural fibers based on the cellulose-binding domain-␤-glucuronidase fused protein Ofir Degani a,b , Shimon Gepstein b , Carlos G. Dosoretz c,∗ a

Department of Environmental Biotechnology, MIGAL-Galilee Technology Center, South Industrial Zone, Kiryat Shmona 10200, Israel b Department of Plant Physiology, Faculty of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel c Faculty of Civil and Environmental Engineering, Division of Environmental, Water and Agricultural Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel Received 7 April 2003; received in revised form 13 October 2003; accepted 20 October 2003

Abstract Cellulose-binding domains (CBDs) are characterized by their ability to strongly bind to different forms of cellulose. This study examined the use of a recombinant CBD fused to the reporter enzyme ␤-glucuronidase (CBD-GUS) to determine the extent of removal of the water-repellent waxy component of cotton fiber cuticles following hydrolytic treatment, i.e., scouring. The CBD-GUS test displayed higher sensitivity and repeatability than conventional water absorb techniques applied in the textile industry. Increases in the levels of CBD-GUS bound to the exposed cellulose correlated to increases in the fabric’s hydrophilicity as a function of the severity of the scouring treatment applied, clearly indicating that the amount of bound enzyme increases proportionally with the amount of available binding sites. The binding of CBD-GUS also gave measurable and repeatable results when used on untreated or raw fabrics in comparison with conventional water drop techniques. The quantitative response of the reaction as bound enzyme activity was optimized for fully wettable fabrics. A minimal free enzyme concentration-to-swatch weight ratio of 75:1 was found to be necessary to ensure enzyme saturation (i.e., a linear response), corresponding to a free enzyme-to-bound enzyme ratio of at least 3:5. © 2003 Elsevier B.V. All rights reserved. Keywords: Water absorbency; Wettability tests; Cellulose-binding domain; ␤-Glucuronidase; Scouring; Cotton fiber

1. Introduction Cotton fiber has a multilayered structure that has been studied and characterized for nearly a century. The structure of the primary cell wall of the ∗ Corresponding author. Tel.: +972-4-8294962; fax: +972-4-8228898. E-mail address: [email protected] (C.G. Dosoretz).

cotton fiber, and particularly the outermost surface layer-the cuticle, has a determinant influence on textile-manufacturing processes, properties, and use (Hardin and Akin, 1998; Cavaco-Paulo, 1998). Scouring is practiced in the textile industry to remove the non-cellulosic hydrophobic cuticle constituents. This improves wettability of the fibers, which facilitates uniform dyeing and finishing. Conventionally, scouring is performed by hot hydrolysis with NaOH, which

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O. Degani et al. / Journal of Biotechnology 107 (2004) 265–273

involves large quantities of water and energy and requires special handling of the strong alkaline effluents (Cavaco-Paulo, 1998). The search for environmentally friendly alternatives to scouring gave rise to the use of enzymes for this purpose (Husain et al., 1999). Extracellular lytic enzymes involved in degrading the cell’s outer layer during invasion of plants by phytopathogenic fungi and bacteria have been considered as candidates for this ‘bioscouring’ (Cavaco-Paulo, 1998; Degani et al., 2002; Sawada and Ueda, 2001). This work is the first to examine the use of cellulose-binding domain-␤-glucuronidase (CBDGUS) fused protein as an analytical tool to quantitatively determine the water absorbency of natural fibers, i.e., the amount of wax removal, by a given chemical or enzymatic treatment. CBD provides a specific means of linking enzymes or other protein to cellulose (Shoseyov and Warren, 1997; Levy and Shoseyov, 2002), because it is a discrete protein module that possesses the intrinsic ability to bind strongly to different forms of cellulose. CBDs are found in nature in enzymes (cellulases and xylanases) as well as in other proteins without hydrolytic activity. Because they are discrete structural and functional units that fold independently, they can easily be produced in isolation or fused to a chosen target protein by genetic manipulation (Tomme et al., 1998). Many CBD fusion proteins have been produced, among them CBD fused to the reporter enzyme GUS. For quantitative studies, the most widely used assay is the fluorometric, in which 4-methylumbelliferyl-␤-d-glucuronide (MUG), when cleaved by GUS, liberates the fluorescent product methylumbelliferone (Draper et al., 1988). Today, the most common way of evaluating wettability of cotton in fabric form is based on American Association of Textile Chemists and Colorists (AATCC) test method no. 39-1980 (AATCC, 1980). The time (in seconds) between the contact of a water drop, carefully deposited onto the fabric’s surface, and its disappearance into the fabric matrix, i.e., the time required for the specular reflection of the water, is recorded as the fabric wetting time. For cotton in fiber form, the standard test method for fabrics is inapplicable. To test water absorbency of fibers, a bundle of cotton fibers is dropped onto the surface of water in a beaker. The time it takes for the bundle to sink to the bottom of the beaker is generally considered to be proportional to the water absorbency

(Li and Hardin, 1997). Another less common reported technique consists of measuring the absorbency of a 1% direct red 81 solution, expressed as color index, relative to the respective weight loss (Traore and Buschle-Diller, 1999). The lower edge of a sample (2.54 by 20.32 cm) is placed vertically at the surface of 100 ml of dye solution at room temperature. The time allowed for the dye front to move is set to 5 min. The dyed areas of the fabric strip, as well as the length of the absorbed waterfront, are recorded. The common disadvantage to all of these techniques is that they are basically qualitative and inaccurate when the scouring is incomplete or when the wax removal is low in uniformity. More accurate techniques, such as water contact angle (Hartzell and Hsieh, 1998) or surface-tension methods based on measuring water absorbency of a single fiber or woven fiber, require expensive instrumentation and expertise. The use of CBD-GUS for measuring cotton scouring, reported herein, provides a quantitative method for determining the wettability of natural fabrics and fibers, which combines both simplicity and sensitivity. The removal of the water-repellent components of the plant cell cuticle during scouring results in enhanced cellulose accessibility. Consequently, the increase of cellulose accessibility as a function of scouring intensity will be reflected by a proportional increase of CBD-GUS binding. 2. Experimental procedures 2.1. Chemicals Purified cutinase (CUT) (ISC-02-BE1) from Pseudomonas mandocino was obtained from InterSpex Products Inc (San Mateo, California). Pectin lyase (PL) (PectoZym HF) was obtained from Rakuto Kasei Ltd. (Yokneam, Israel). Detergents and raw cotton fabrics were kindly supplied by AvcoChem Ltd. (TelAviv, Israel). 4-Methylumbelliferyl-␤-d-glucuronide, methylumbelliferone and 5-bromo-4-chloro-3-idolyl␤-d-glucuronic acid were obtained from Duchefa Biochemie (Haarlem, The Netherlands). 2.2. Scouring treatments Scouring treatments were performed as described by Degani et al. (2002). Prior to treatment, raw


fabrics were cut into square swatches of 0.05–1 g, dried at 105 ◦ C for 2 h and precisely weighed. Enzymatic treatments were performed by incubating one swatch of fabric in reaction mixture at ratio of 1 g to 10 ml, in a 15 ml polycarbonate screw-cap test tube placed diagonally in a rotary shaker, at 18.3 rad/s and 37 ◦ C for 20 h. The reaction mixture contained 0.1% (w/v) non-ionic detergent composed of a blend of polyoxyethylene fatty alcohols (AvcoPal-AWS, AvcoChem ltd.), 100 mM phosphate buffer pH 8 and the indicated enzyme. Controls of the enzymatic treatments were incubated under the same conditions but without enzyme. Enzyme activity determination was as indicated in each case. Chemical scouring was carried out by digesting dried and weighed fabrics at a ratio of 1 mg fabric to 50 ml boiling NaOH solution, in the concentration range of 0–2 M, for 30 min. Fabrics were boiled in double-distilled water (DDW) for 30 min as a control. Following incubation or digestion, the fabric samples were separated from the reaction fluid (squeezed through 50 ml syringe), then exhaustively rinsed with DDW and squeezed. Rinsing and squeezing was repeated three times. The rinsed samples were then dried at 105 ◦ C and weighed for calculation of weight loss, and further used for water absorbency determinations and CBD-GUS tests. Each treatment point consisted of at least three replicates. 2.3. Expression and recovery of the recombinant CBD-GUS fusion protein Escherichia coli BL21 (DE3) carrying the CBDGUS fused protein expression system (pET-CBD) was kindly supplied by CBD Technologies, Ltd. (Rehovot, Israel). Inoculum was prepared by growing the cells overnight (30 ◦ C in a rotary shaker at 18.3 rad/s) in Luria–Bertani (LB) medium (10 g NaCl, 10 g trypton, 5 g yeast extract dissolved in 1 l of DDW) containing 50 mg l−1 kanamycin A. After dilution to a volumetric ratio of 1:1000 in fresh LB medium containing 50 mg l−1 kanamycin A, cells were grown in shaking 250 ml flasks at 18.3 rad/s, 30 ◦ C overnight. The cells were harvested by centrifugation at 1600 × g, resuspended in 10 ml phosphate-buffered saline (PBS) buffer (pH 7.2) and sonicated (3 × 30 s). The dissolved protein was separated from the cell debris by

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centrifugation at 7000 × g and concentrated by ultrafiltration, using a 10 kDa-cutoff PM-10 membrane (Amicon, Danvers, MA). 2.4. Assay of soluble CBD-GUS activity GUS activity of CBD-GUS in the solution was determined with 4-methylumbelliferyl-␤-d-glucuronide as the substrate according to the supplier’s instructions, with some modifications. The reaction was performed at 37 ◦ C. The assay buffer solution contained 50 mM sodium phosphate buffer pH 7.0, 10 mM dithiothreitol (DTT), 1 mM Na2 EDTA, 0.1% (w/v) sodium lauryl sarcosine, 0.1% (w/v) Triton® X-100 and 1 mM MUG. A 200 ␮l aliquot of CBD-GUS solution was mixed with 800 ␮l of GUS assay buffer and the increase in fluorescence was followed for 60 s at excitation and emission wavelengths of 365 and 455 nm, respectively, in a Hitachi F-2000 spectrofluorimeter. One unit of activity was defined as the amount of enzyme required for the release of 1 nmol methylumbelliferone per minute at 37 ◦ C. A calibration curve of MU, in the range of 0–5 ␮M, dissolved in the assay buffer without MUG, was performed to convert fluorescence readings into MU concentrations. 2.5. Determination of scouring efficiency 2.5.1. Water absorbency The treated fabric swatches, after washing and drying, were tested for wettability according to AATCC Test method no 27-1977, applying a 20 ␮l water drop (AATCC, 1980). Each fabric swatch was tested in at least nine different areas and the mean time was calculated. 2.5.2. Determination of wettability using the CBD-GUS assay Unless otherwise specified, weighed treated fabric swatches, after washing and drying, were immersed in 2 ml CBD-GUS solution in PBS buffer (pH 7.2) at the indicated activity (expressed in U ml−1 ) and shaken (18.3 rad/s) in an orbital shaker at room temperature for 60 min. The swatches were then washed twice for 10 min each in 2-ml PBS buffer containing 0.05% (w/v) TweenTM 80, in a rotary shaker (18.3 rad/s), and then one more time in 2 ml PBS buffer alone

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under the same conditions. The excess washing buffer was removed by inverting the tubes on absorbent paper. The activity of CBD-GUS bound to the cellulose in the fabric, representing the extent of scouring, was quantified by incubating the swatches for 15 min in the assay buffer solution for GUS activity (see above) at 37 ◦ C in a rotary shaker (18.3 rad/s). Then, 100 ␮l of the reaction fluid were mixed with 900 ␮l of 2 M Na2 CO3 to stop the reaction and MU was spectrofluorometrically determined as described.

2.6. Histochemical assay of fabric-bound CBD-GUS A histochemical test of scoured swatches was performed with 5-bromo-4-chloro-3-indolyl-b-dglucuronic acid (X-GlcA). Cotton fabric swatches (50 mg) were treated with CBD-GUS as already described. After washing away the excess CBD-GUS, the swatches were incubated overnight in 1 ml test solution containing 5 mg ml−1 X-GlcA in PBS buffer at 37 ◦ C in a rotary shaker (18.3 rad/s). The excess test solution was disposed of by inverting the treatment tubes and the fabrics were dried at 105 ◦ C for at least

Fig. 1. Qualitative histochemical staining of cotton fabrics treated with CBD-GUS using X-GlcA as the substrate for GUS activity. Swatches (50 mg) were enzymatically scoured with 10.4 U ml−1 cutinase (top panel) or chemically scoured with 1 M NaOH (bottom panel). Controls represent raw, untreated fabric swatches. The fabrics were incubated in 1 ml test solution containing 5 mg ml−1 X-GlcA in PBS buffer in a rotary shaker (18.3 rad/s). The dark (green) stain indicates substantial CBD-GUS-binding to the fabrics after chemical or enzymatic treatment.


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2 h. The stained swatches were then digitized using a Hewlett Packard Scanjet 5100 scanner. 2.7. Reverse phase (RP)-HPLC analysis HPLC analysis of the hydrolysis fluid was performed as previously described (Degani et al., 2002) in a Hewlett Packard HPLC (HP1100 series) equipped with UV, using a Luna C18 column (25 cm × 4.60 mm i.d., 5 ␮m; Phenomenex, Torrance, CA). The UV detector was set at 210 nm. Elution was performed using a gradient system consisting of solvent A (acetonitrile), solvent B (1 mM trifluoroacetic acid solution) and solvent C (tetrahydrofuran) with the following program (in percent v/v): initially 50A:25B:25C; linear gradient over 30 min to 60A:20B:20C; linear gradient over 5 min to 60A:5B:35C, then held isocratically for a further 15 min. The flow rate was maintained at 0.85 ml min−1 . All solvents were of far-UV-quality HPLC-grade purity.

3. Results The ability of CBD-GUS to bind to the exposed cellulose upon chemical (1 M NaOH) or enzymatic (10.4 U ml−1 cutinase) scouring of cotton fabrics was first examined by qualitative histochemical staining using X-GlcA as a substrate for GUS activity (Fig. 1). As can be seen from the stained swatches, highly discernible staining (dark green color) was observed on the treated fibers (Fig. 1, right panels) compared to the untreated ones (Fig. 1, left panels), indicating a substantial difference in CBD-GUS binding which correlates with the water absorbence achieved by the scouring treatments. To evaluate the binding of CBD-GUS, the assay for activity of the free enzyme was adapted to that of the enzyme bound to scoured fabrics (1 M NaOH) using the fluorogenic substrate MUG. The scoured fabrics were treated with a CBD-GUS solution for 60 min, washed, and incubated for increasing periods of time with assay reaction solution containing MUG; the released fluorophore MU was detected by spectrofluorimetry (Fig. 2). A typical time profile of the reaction shows it to be linear for almost 30 min of incubation. Based on these results, an incubation time of 15 min was used in subsequent experiments. The

Fig. 2. Time profile of MU release by scoured cotton fabric-bound GUS during incubation with MUG. Fabric swatches (100 mg) were scoured with 1 M NaOH and incubated for 60 min with 2 ml CBD-GUS solution in a rotary shaker (18.3 rad/s). After discarding excess CBD-GUS and extensive washing with PBS buffer, the fabrics were incubated at the indicated times in 2 ml assay buffer (MUG solution) at 37 ◦ C with shaking and fluorescence was measured (excitation = 365 nm, emission = 455 nm). Inset shows the time profile for CBD-GUS binding to dry or pre-wetted swatches following scouring. (): dry swatch; (䊐): pre-wetted swatch. GUS activity is defined as the amount of CBD-GUS needed to release 1 nmol MU per minute. Values represent averages of three independent replicates.

time required for CBD-GUS to bind to the scoured cotton fabric (i.e., to reach equilibrium) was approximately 5 min, regardless of whether a dried or water pre-wetted swatch had been immersed in the solution (Fig. 2, inset). The time required for complete water absorbency (i.e., the time required for a drop of water to penetrate the fabric) under the same conditions was 1.4 min. To ensure equilibration in all subsequent experiments, a reaction time of 60 min was applied. The quantitative response of the reaction was then evaluated by measuring the bound enzyme concentration as a function of the free enzyme concentration for a fixed swatch weight (50 mg) and as a function of the swatch weight for a fixed free enzyme concentration, using easily wettable fabrics following chemical scouring (Fig. 3). Applying a free enzyme concentration in the range of 0–120 U ml−1 , which corresponded to a ratio of initial unbound enzyme-to-swatch weight of 0–4.8, a linear response was observed (Fig. 3, upper panel). Although these results proved that a quantitative response is attainable, the enzyme was below saturating concentrations. Fig. 3, bottom panel, shows

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Fig. 4. CBD-GUS and water drop wettability tests of cotton fabrics chemically scoured at different NaOH concentrations. (䊉): GUS activity; (䊊): wetting time. Scouring reactions were carried out for 30 min at 100 ◦ C in NaOH solution at the indicated concentration. Control treatments (0 M NaOH) consisted of boiling DDW for 30 min. Following scouring, the fabrics (20 mg) were washed with DDW and incubated in a 1.5 ml CBD-GUS solution (1750 U ml−1 ) in a rotary shaker (18.3 rad/s) for 60 min. MUG incubation and analysis were as described in legend to Fig. 2. Values represent averages of three independent replicates. Bars indicate standard deviation.

Fig. 3. Influence of initial CBD-GUS activity and fabric weight on bound-enzyme activity in easily wettable fabrics following chemical scouring. Top panel: bound enzyme concentration as a function of the free enzyme concentration for a fixed swatch weight (50 mg). The data correlated to a linear fit with r 2 = 0.995. Bottom panel: bound enzyme concentration as a function of swatch weight for a fixed free enzyme concentration (1900 U ml−1 ). The data correlated to a Michaelis–Menten hyperbolic fit (r2 = 0.983). The fabrics were chemically scoured (1 M NaOH), washed with DDW and incubated in 2 ml CBD-GUS solution in a rotary shaker (18.3 rad/s) for 60 min. MUG incubation and analysis was as described in legend to Fig. 2. Values represent averages of three independent replicates. Bars indicate standard deviation.

the change of bound enzyme concentration within a swatch weight range of 0–100 mg applying an initial unbound enzyme concentration of approximately 1900 U ml−1 . A saturation curve was obtained, indicating that the amount of bound enzyme increases proportionally with the amount of available binding sites, with a linear dependency (i.e., initial slope) observable in the range of 0–20 mg bound enzyme per 1 mg

swatch, which corresponds to a saturating free enzyme concentration-to-swatch weight ratio of 75. Furthermore, these results can be interpreted as follows: a ratio of free enzyme (U mg−1 swatch) to bound enzyme (U mg−1 swatch) equal to 3.5 or higher indicates a saturating environment, ensuring a quantitative measurement of the amount of available binding sites on the cellulose surface. Following the characterization of the binding reaction, the fused enzyme was applied to a quantitative evaluation of the efficiency of scouring achieved by subjecting cotton fabrics to increasing strengths of chemical hydrolysis, in comparison with the conventional contact water drop technique (Fig. 4). To ensure saturation, a free enzyme-to-swatch weight ratio of 130 U mg−1 was applied. An increase in the levels of the CBD-GUS bound to the exposed cellulose was observed, which correlated with the increase in fabric hydrophilicity, reaching an asymptotic value of approximately 32 U of bound enzyme per 1 mg swatch for 2 M NaOH. This value corresponded to a free enzyme (U mg−1 swatch)-to-bound enzyme (U mg−1 swatch) ratio of 4. A decreasing trend was observed for the water drop technique, reaching an unreadable value (i.e., zero) for the 1 M NaOH treatment. As


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Fig. 5. Effect of enzymatic scouring of cotton fabrics with pectin lyase (PL), cutinase (CUT) and their mixture (PL+CUT), on water absorbency and release of hydrolysis products. Top panel: CBD-GUS and water drop wettability tests. Bottom panel: RP-HPLC separation of hydrolysis products. ( ), GUS activity; (䊐), wetting time. Enzymatic scouring was performed by incubating the fabric swatches for 4 h at 37 ◦ C and pH 8 (100 mM phosphate buffer) with 300 U ml−1 PL, or 1.7 U ml−1 CUT, or their mixture, in the absence of detergent. MUG incubation and analysis was as described in legend to Fig. 2. Values represent averages of two independent replicates.

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can be deduced from the opposite trends obtained by the two techniques, the major advantage of the CBD-GUS method is that increasing values are obtained as the wettability increases, enhancing the accuracy of the measurement. In contrast, the water drops technique, which displays zero values for highly wettable conditions, could not discern between the effects of 1 versus 2 M NaOH treatment, while a 10-fold increase in wettability was observable using the CBD-GUS method. Furthermore, since the measurable activity of GUS depends on the binding of CBD to exposed cellulose, this method, in contrast to others, gives a complete picture of the degree of dewaxing achieved and not just an indication of water penetration. The advantage of the CBD-GUS binding test, i.e., high readings for short wetting times, were also shown by measuring the efficiency of mild scouring by enzymatic hydrolysis of the cotton fiber cuticle with pectin lyase (300 U ml−1 ) alone, or in the presence of a small concentration of cutinase (1.7 U ml−1 ). As can be seen in Fig. 5, upper panel, whereas pectin lyase hydrolysis resulted in effective scouring, its combination with a minute amount of cutinase, which alone caused an insignificant change, resulted in a 20% increase of scouring efficiency, suggesting a synergistic interaction between the two enzymes. The release of specific hydrolysis products by each of the enzymes as well as their combination is shown in Fig. 5, bottom panel, in RP18-HPLC chromatograms of the water-soluble products upon hydrolysis.

4. Discussion The major goal of any scouring process is to improve the water absorbency of natural fibers by removing the water-repellent components of the fiber cuticle which facilitates uniform dyeing and finishing (Cavaco-Paulo, 1998). Since the cuticle is cross-linked to the primary cell wall by esterified pectic substances, efficient scouring correlates with a considerable removal of both waxy and pectic substances (Hartzell, and Hsieh, 1998). Absorbency is quantitatively estimated by measuring the time required for a water drop to be absorbed or the distance traveled by a waterfront after a predetermined time. Although simple to perform, these techniques lack accuracy and cannot

discern between highly efficient scouring techniques, especially when the fiber becomes rapidly wettable and insignificant times are being recorded. At the opposite extreme are staining techniques targeted to bind to the newly accessible hydrophilic components, such as ruthenium red for pectin or congo red for cellulose, but these give only qualitative estimates and often do not display clear differences between the treatments (Degani et al., 2002; Traore and Buschle-Diller, 1999). This research examined the use of CBD fused to the reporter protein GUS to determine the amount of cellulose exposure with a high degree of sensitivity and repeatability, allowing for the distinction between very slight differences in scouring efficiencies in contrast to conventional water absorbency techniques. An increase in CBD-GUS bound to the exposed cellulose correlated with an increase in the fabric’s hydrophilicity, from what a fabric’s weight loss was observed as function of the severity of the scouring treatment applied, clearly indicating that the bound enzyme increases proportionally with the amount of available binding sites. This proportionality indicates that the CBD-GUS assay reported in this work is a direct evaluation of the scouring efficiency rather than a simple measurement of wettability. The binding of CBD-GUS also gave accurate and repeatable estimates for untreated or raw fabrics, in comparison to conventional water drop techniques. The ability of CBD to bind to an untreated fabric is supported by previous scanning electron microscopy findings showing that the cotton fiber cuticle contains micropores or cracks which allow diffusion of enzymes (Hardin and Akin, 1998). Since the CBD-GUS method is based on penetration of the enzyme into the cellulosic matrix, it gives a general value of scouring efficiency and uniformity; however, it appears that CBD-binding to cellulose in the cotton cell wall correlates well with the time frame of the waterfront’s course. Crystalline cellulose, the major constituent of cotton fibers, consists of closely packed cellulose chains that are stabilized by hydrogen-bonding to form a tight, regular array which shields many of the glycosidic bonds from enzymatic attack (Focher et al., 1981). Goldstein et al. (1993) reported the ability of a unique sequence of the cellulase complex of Clostridium cellulovorans to bind at high efficiency to cotton crystalline cellulose (6.4 ␮mol of CBD per gram) in comparison


to fibrous cellulose (0.2 ␮mol g−1 ). Although cellulase penetration might appear to be diffusion-limited, Kaya et al. (1994) found that mild mixing enhances cellulase-binding to cellulose fibers during scouring, but increasing shear results in reduced binding and activity. Based on this, mild shaking was applied during all incubations of the fabric samples with CBD-GUS. The benefits of using CBD-GUS to determine water absorbency of scoured fibers may be summarized as follows: 1. The same method is applicable to both fiber and fabric forms of cotton. 2. It gives an overall estimate of scouring efficiency, by determining both water absorbency and cellulose accessibility. 3. The results of this measurement technique are measurable, even if the cotton is only partly scoured or if it has not been treated at all. 4. The test is simple and versatile. Where there is no possibility of measuring the fluorescent substrate, the same assay can be preformed with spectrophotometric or histochemical substrates. In conclusion, the use of CBD-fused protein may serve as an efficient tool for both analytical and practical applications in the cellulose-based industry, such as paper manufacturing, in general, and textiles in particular. Preliminary experiments conducted with CBD fused to green fluorescence protein (CBD-GFP) offer a simple tool for estimating the process’ uniformity (e.g., scouring, staining) as well as structural changes in the fibers as a result of the enzymatic or chemical treatment (results not shown). In addition, binding CBD to scouring enzymes such as pectinases and cutinase may allow their precise targeting to the cotton fiber cell wall or even to the cuticle micropores. This would reduce the required enzyme dosages and the time needed to scour the cotton cuticle.

Acknowledgements We thank Oded Shoseyov from the Hebrew University of Jerusalem and Stanly Hirsch from CBD Technologies Ltd., for all of their kind help with the CBD-GUS.

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