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Effect of a nonionic surfactant on


Cellulose 9: 83–89, 2002. ? 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Effect of a nonionic surfactant on Trichoderma cellulase treatments of regenerated cellulose and cotton yarns
Chiyomi Mizutani 1,*, Kandan Sethumadhavan 2, Phyllis Howley 2 and Noelie Bertoniere 2
Heian Jogakuin (St. Agnes’) College, 5-81-1 Nanpeidai, 569-1092 Takatsuki, Japan; 2Southern Regional Research Center, USDA, ARS, 1100 Robert E. Lee, New Orleans, LA 70124, USA; *Author for correspondence
Received 24 January 2001; accepted in revised form 4 December 2001
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Key words: Cellulase, Cellulosic materials, Hydrolysis, Surfactant, Tensile strength Abstract It has been shown that some surfactants affect the hydrolysis of cellulose by cellulase. In this study, the effect of the surfactant Tween 20 on the hydrolysis of different cellulosic ?bers was investigated and related to the cellulose ?ber structure. It was found that this non-ionic surfactant enhanced the enzymatic sacchari?cation of highly crystalline cellulosics such as Avicel, Tencel and cotton but not of cuprammonium rayon. The enhanced sacchari?cation effected by the surfactant is attributed to inhibition of non-productive sorption of the endoglucanase of the cellulose surface which gives greater access to the cellulose chain ends by the exoglucanase. Although all three ?bers lost tensile strength as a result of the enzymatic treatment, no further decrease was effected by the presence of the surfactant. Introduction Enzyme treatments have been of interest in cotton ?nishing to improve fabric softness, effect a washed appearance in denims, impart a smooth appearance by removing surface fuzz ?bers, or remove tangled bundles of surface ?bers that do not pick up dyes and thus result in white specks. They also have potential for simplifying other steps in the manufacturing processes (Tyndall 1992). In recent years, much attention has been paid to the enzyme treatment of cotton and rayon fabrics to improve their softness without damaging their intrinsic characteristics (Kamide et al. 1992; Buschle-Diller et al. 1994). The relationship between weight loss and tensile strength with mixed enzyme systems was studied (Buschle-Diller et al. 1999; Miettinen-Oinonen et al. 2001). Tensile strength was shown to decrease with an increase in hydrolysis. Thus, one of the most important problems in the enzymatic treatment of cellulosic fabrics is to balance the improvement in hand and the loss of tensile strength. It is generally accepted that the enzymatic hydrolysis of cellulosic ?bers is initiated by the random attack of an endocellulase (1,4-?-D-glucan 4-glucanohydrolase) [EC3.2.1.4] on the internal glucosidic bonds of an intact glucan chain. A newly created nonreducing chain terminus is susceptible to attack by an exocellulase (1,4-?-D-glucan cellobiohydrolases) [EC3.2.1.91]. Finally, the cellobiose produced is hydrolyzed to glucose by the action of ?-glucosidase (Flickinger 1980; Hoshino et al. 1997). It has been shown (Henrissat et al. 1985) that synergism between cellobiohydrolase I and endoglucanase I or II depends on the structural and ultrastructural features of the substrate. However, the exact mode of the action of these cellulase systems has not been fully elucidated and further investigation will be required. The interactions between cellulases and cellulose have been reviewed (Henrissat 1994). Recently interesting studies have been reported on the effects of several surfactants on the enzymatic hydrolysis of cellulose. Several surfactants were found to be bene?cial (Castanon and Wilke 1981; Ooshima et al. 1986; Park et al. 1992; Helle et al.

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Table 1. Enzymatic characterization of Cellusoft L endo Average of cellulase activity (?g/mg/min) Ratio (%) Molecular weight (Kda) c 161.7 a 91 52.9 exo 11.8 b 9 74.2

a Endo cellulase activity was determined by the method of Miller (1959). b Exo cellulase activity was determined by the method of Somogi (1952) and Nelson (1944). c Molecular weights were measured by SDS-polyacrylamide gel electrophoresis.

Figure 1. Chemical structure of Tween 20.

1993; Wu and Ju 1998), including nonionic Tween 20, 80 and 81, cationic Q-86W, and anionic Neopelex F-25 at low concentrations. However, inhibitory effects were observed with the cationic Q-86W at higher concentration and with an anionic surfactant, Neopelex F-25. Non-ionic surfactants were found to be more suitable for enhancing cellulose hydrolysis, as demonstrated by sugar formation (Ooshima et al. 1986). In these investigations, the effects of several surfactants were studied with cellulosic materials such as ?lter paper and microcrystalline cellulose powder as substrates. A change in the relative sorption of endo- and exocellulase due to the presence of the surfactant is proposed to account for the observed effect. It has also been suggested (Castanon and Wilke 1981) that the surfactant renders the cellulose readily wettable by the cellulase solution, bringing the substrate into intimate contact with the enzymes and allowing the enzymes to reach otherwise inaccessible places. Few studies have been reported on the effect of surfactants on the hydrolysis of cellulosic ?bers such as cotton or regenerated cellulosic ?bers by cellulases and the effect of the combined treatment on their properties after treatment. In this investigation, the effect of the action of a whole cellulase on three cellulosic ?bers that differed in structure was assessed. The impact of these treatments on textile performance was also determined.

Materials and methods Materials The source of the cellulase enzyme used in these experiments was Cellusoft L (produced by Novo Nordisk, Denmark) from Trichoderma reesei; it was used without further puri?cation. The cellulase activities and molecular weights of the endo- and exocellulase components were determined and the results are shown in Table 1. The surfactant, polyoxyethylene sorbitan monolaurate, Tween 20 (Figure 1), was obtained from Aldrich (Milwaukee, WI). The regenerated cellulosic spun yarns such as viscose rayon, cuprammonium rayon, Tencel and cotton (Pima J-6 variety from USDA laboratory) were used as substrates. The microcrystalline cellulose powder, Avicel PH101 from Fluka (Milwaukee, WI), was also used. Cotton was boiled in 0.5% (w/w) aq. NaOH solution for 30 min, then rinsed under distilled water, and air-dried. Regenerated cellulosic yarns were puri?ed for 24 h in a Soxhlet extractor using ethanol:benzene (1:1), and air-dried. Treatment method Cellulase enzyme was diluted with 50 mM of sodium acetate buffer pH 4.8 to a concentration of 1 mg/mL and used as a treatment solution. The substrate (1 g) was placed in the treatment solution at a ratio of 1:20.

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Figure 2. Effect of surfactant (Tween 20) on hydrolysis of Avicel. The incubation mixture contained 1 g of Avicel, 1% (w/v) of cellulases in 20 mL of 50 mM acetate buffer, pH 4.8, and kept at 50 °C at various time periods.

The reaction was carried out in an Erlenmeyer ?ask at 50 °C (+/?1 °C). A range of surfactant concentrations (0–1.25%) was used in the incubation mixture in order to determine the glucose release at various time intervals. After treatment, substrates were washed with distilled water several times before airdrying. Evaluations Reducing sugar as glucose: Reducing sugars, calculated as glucose, were determined by a method that utilizes dinitrosalicylic acid (DNS) reagent (Ghose 1987). The % increase in reducing power, calculated as glucose, upon inclusion of surfactant was calculated by the following equation: [(B?A)/A] × 100, where A = glucose release without surfactant and B = glucose release with surfactant included. The degree of crystallinity: The degree of crystallinity was estimated by X-ray diffraction using a Regaku Corporation model RU-3h X-ray diffractometer with a ?at camera and Ni-?ltered CuK? radiation. Degree of crystallinity of the sample was calculated from a ratio of the peak areas of the amorphous and crystalline regions on X-ray diffraction curves (Kamide et al. 1992). Tensile strength: Yarn tensile strength was determined by ASTM D2256. The gauge length was 250

mm and tests were performed at a 300 mm/min rate of extension. Critical micelle concentration: The critical micelle concentration of surfactant was measured with a surface tension balance of the Du Nuyi type (Shimadzu, Japan) that was adapted for the ring method. For our experiments, the surfactant was mixed with 50 mM sodium acetate buffer.

Results In this study, the commercial cellulase from Trichoderma reesei was reacted with cellulosic yarns spun from cotton, cuprammonium rayon, Tencel and crystalline cellulose (Avicel) in the presence and absence of Tween 20. Figure 2 shows the effect of 0.1% nonionic surfactant Tween 20 on the hydrolysis of 1 g of Avicel with 1% cellulase. The initial incubation time of 4 h showed no effect; however, after 12 h it was found that 0.1% Tween 20 greatly enhanced the hydrolysis of the Avicel compared to the control containing no Tween 20. Therefore, a long incubation time is necessary to get a positive effect from surfactant use. Figure 3 shows glucose release at various concentrations of Tween 20 at different incubation times. After 4 h of incubation time there was no difference in the glucose release due to Tween 20 at any of its

86 factant might associate with the enzyme protein, thus affecting enzyme activity. The effects of Tween 20 on the hydrolysis of cellulosic yarns prepared from cotton, cuprammonium rayon, Tencel and cotton are compared in Table 2. The order of formation of reducing sugars, calculated as glucose, is cuprammonium rayon > Tencel > cotton, which we attribute to the different crystalline/ amorphous ratios for these celluloses. Cuprammonium rayon, Tencel and cotton are 30%, 50% and 55% crystalline, respectively. The order of hydrolysis is thus inversely related to the crystallinity of the cellulose. This suggests that the enzyme is attacking the amorphous regions of the cellulose yarns. Data for Avicel are included in the table and a high degree of sacchari?cation was detected. However, because of the high surface area in this microcrystalline cellulose powder, comparison with the cellulosic yarns is not meaningful. In contrast, the % increase in glucose release upon addition of Tween 20 was unaltered in the case of cuprammonium rayon, whereas it was 19% higher with cotton and 15% higher with Tencel. The relationship between the crystallinity of the cellulosic materials and the % increase in glucose release effected by the presence of the surfactant is plotted in Figure 5. The crystallinity of the cellulosic materials is directly proportional to the % increase in glucose release effected by Tween 20. This suggests an important role for the surfactant in the hydrolysis of crystalline cellulose. Table 3 shows the effects of Tween 20 on tensile strength of the cuprammonium rayon, Tencel and cotton yarns. The tensile strength of these cellulosic yarns is reduced extensively after treatment with the present concentrations of exo- and endocellulase from Trichoderma reesei, due primarily to the long incubation time. The order of reduced tensile strength was cotton (76%) > cuprammonium rayon (35%) > Tencel (31%). This suggests that the susceptibility to breaking strength reduction is markedly affected by differences in cellulose ?ne structure, but is not correlated with crystallinity.

Figure 3. Release of glucose at various concentrations of Tween 20 and incubation times. 1 g of Avicel was used as substrate with 1% (w/v) of cellulases containing Tween 20 in 20 mL of 50 mM acetate buffer at 50 °C and pH 4.8. Reaction times are 1 h (?), 4 h (?), 8 h (‰), 24 h (?), 48 h (?) and 72 h (?).

concentrations. This mirrored the effect shown in Figure 2. Among the Tween 20 concentrations evaluated between 8 to 72 h incubation, maximum glucose release was observed at 0.1%. The relationship between optimum glucose release and critical micelle concentration for Tween 20 was investigated. Figure 4 shows surface tension curves for Tween 20 in 50 mM acetate buffer, pH 4.8. As the concentration of surfactant was increased, the surface tension continued to decrease until the critical micelle concentration was reached. As shown in Figure 4, the critical micelle concentration for Tween 20 was 0.03%. Data on glucose release were only collected at all reaction times for 0.0, 0.1, 0.25, 0.5, 0.75 and 1.0% surfactant. In these cases the maximum glucose release from Avicel was at 0.1% Tween 20. However, at 48 h reaction time data were collected at 0.05% surfactant for Avicel and the other three cellulosic yarns (Table 2). At this time we see that the same maximum was reached at 0.05% surfactant. This is in close agreement with the critical micelle concentration. Above the critical micelle concentration, the sur-

Discussion The surface area of cellulose that is accessible to cellulase enzymes is the most important factor in determining initial rates of hydrolysis. We see this here, where sugar formation upon exposure to cellulases is directly related to ?ber crystallinity. However, highly

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Figure 4. Surface tension curve of Tween 20 in 50 mM acetate buffer, pH 4.8. Table 2. Effect of surfactant on hydrolysis of various cellulosic materials Concentration of surfactant a (%) Reducing sugar as glucose (mg) Cupra rayon b 0 0.05 0.1
a

Tencel 64.4 73.0 74.2

Cotton 61.7 73.5 73.4

Avicel 107.0 153.1 153.0

216.5 226.3 220.5

The incubation was carried out on 1 g of substrate, 1% (w/v) of cellulase containing Tween 20 in 20 mL of 50 mM acetate buffer at 50 °C and pH 4.8 for 48 h. b Cupra rayon is cuprammonium rayon.

crystalline cellulose such as cotton has reduced accessibility because the cellulase molecules cannot reach the interior of the crystalline regions. In the accepted theory the endocellulase binds to the crystalline surface and cleaves the cellulose chain, forming new chain ends. Thereafter the exo component successively removes cellobiose units, which are then hydrolyzed to glucose by cellobiohydrolase. Hydrolysis by cellulases becomes less effective as the hydrolysis proceeds. This has been attributed to irreversible binding of the enzyme to the cellulose surface which blocks further reaction (Howell and Mangat 1978). Henrissat (1994) has suggested that in an endo–exo synergistic degradation of cellulose, the limiting enzyme for the production of soluble reducing sugars is the exo-enzyme. Small amounts of endo enzyme activity should provide a sufficient number of chain ends to saturate the exo-enzymes present. Therefore, the maximum amount of soluble reducing sugars should be observed with only a small amount of endo-

activity. Henrissat et al. (1985) worked with isolated cellulase fractions to show that with a mixture of CBHI and EGI a 1:1 mixture of the two enzymes was found to be most effective for digestion. However, the maximum activity was shifted to 95:5 between CHBII and EGI or EGII. Ooshima and co-workers (1986), who reported that Tween 20 enhanced sacchari?cation of highly crystalline Avicel by cellulase from Trichoderma viride, demonstrated that the surfactant effected an enrichment of endoglucanase in the liquid phase. Thus it appears that the surfactant, which reduces the surface tension of the solution, is inhibiting the non-productive attachment of the endoglucanase to the cellulose surface and permitting the saccharifying exoglucanase greater access to the cellulose chain ends, which results in enhanced sugar formation. In this study the sugars, undoubtedly a mixture of cellobiose and glucose, were calculated as glucose. Helle et al. (1993) showed that a surfactant must be present at the

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Figure 5. Relationship between crystallinity of cellulosic materials and % increase of glucose release. The incubation was carried out on 1 g of substrate, 1% (w/v) of cellulase containing 0.1% (w/v) Tween 20 in 20 mL of 50 mM acetate buffer at 50 °C and pH 4.8 for 48 h. Table 3. Tensile strength of various cellulosic yarns after cellulase treatment containing surfactant Method Concentration of surfactant a (%) Cupra rayon b Untreated Treated – 0 0.05 0.1 16.7±1.9 10.8±0.8 10.1±0.9 9.3±0.7
c

Tensile strength at break (g/tex)

Tencel 26.5±1.1 18.2±1.2 19±1.1 20.4±1.6

Cotton 18.2±1.5 4.3±1.3 4.7±1.4 4.8±1.2

a The incubation was carried out on 1 g of substrate, 1% (w/v) of cellulase containing Tween 20 in 20 mL of 50 mM acetate buffer at 50 °C and pH 4.8 for 48 h. b Cupra rayon is cuprammonium rayon. c Average of 9 individual determinations ± standard deviation.

beginning of the hydrolysis, demonstrating that the surfactant is not effective in desorbing the enzyme from the cellulose surface but only preventing its nonproductive sorption. Several authors had proposed mechanisms to explain the enhanced sacchari?cation of cellulose in the presence of surfactants (Castanon and Wilke 1981; Ooshima et al. 1986; Helle et al. 1993). Most are similar to that proposed for our observations with cellulosic ?bers of differing crystallinity.

Conclusions We have studied the enzyme characteristics of Trichoderma reesei cellulase and the enhancement of sacchari?cation by the cellulase of cellulosic materials

by the surfactant, Tween 20. The following can be concluded: (1) The surfactant, Tween 20, enhanced sugar formation from crystalline cellulosic materials such as Avicel, cotton and Tencel. (2) The maximum effect occurred at a surfactant concentration that is in close agreement with the critical micelle concentration for the surfactant. (3) The formation of reducing sugars was inversely related to the crystallinity of the celluloses, but the degree to which this sacchari?cation was enhanced by the presence of Tween 20 was directly related to their crystallinity. (4) Although inclusion of Tween 20 enhanced the sacchari?cation of the celluloses, it did not further reduce the tensile strength beyond that effected by the cellulase alone. (5) The enhanced sacchari?cation effected by the surfactant is attributed to inhibition of non-productive sorption of the endoglucanase on the cellulose sur-

89 face, which gives greater access to the cellulose chain ends by the exoglucanase.
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