Action of exo- and endo-type cellulases from Irpex lacteus on Valonia cellulose

JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 77, No. 5 , 4 9 6 - 5 0 2 . 1994

Action of Exo- and Endo-Type Cellulases from Irpex lacteus on Valonia Cellulose EIICHI HOSHINO, 1 MASAFUMI NOMURA, l MITSUO TAKAI, 2 MITSUO OKAZAKIfl KAZUTOSI NISIZAWA,4§ AND TAKAHISA KANDA 5. Household Products Research Laboratories, Kao Corporation, Minato, Wakayama, Wakayama 640,1 Department of Applied Chemistry, Faculty of Engineering, Hokkaido University, Sapporo, Hokkaido 060,2 Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, Tokida, Ueda, Nagano 386,3 Department of Fisheries, College of Agriculture and Veterinary Medicine, Nihon University, Setagaya-ku, Tokyo 154,4 and Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University, Wakasato, Nagano, Nagano 380,5 Japan Received 7 December 1993/Accepted9 February 1994 Ceilulolytic mode of action of the two highly purified exo- and endo-type ceilulases from Irpex lacteus on pure Valonia cellulose was investigated. Electron microscopy substantiated that both cellulases are adsorbed preferentially into the internal parts of microfibrils in the network structure of the cellulose at initial stages before enzymatic hydrolysis, and that the adsorption ratio of both cellulases onto the external surfaces of microfibrils increased with incubation time although this tendency was less remarkable with the exo-type cellulase than with the endo-type one. The exo-type ceilulase exhibited relatively high activity producing cellobiose throughout 12-h incubation, while the endo-type cellulase produced small amounts of cellooligosaccharides. The degree of polymerization was far more suppressed by the endo-type cellulase than by the exo-type one. Degradation by the cellulases in typical exo- and endo-fashions yielded quite different morphological patterns in the microfibrils. Exo-type cellulase loosened the network structure of microfibrils and made them slightly thinner, while endo-type cdlulase caused conspicuous swelling and dissolution of individual microfibrils.

action of Ex-1 and En-1 in the hydrolysis of amorphous and crystalline regions of cotton cellulose (11, 12). However, both regions of the cotton fiber could not be distinguished morphologically because of the highly developed and compact network structure of cotton microfibrils (11, 13, 14). A study was conducted to clarify the actions of Ex-1 and En-1 on microfibril regions using Valonia cellulose which seemed to have a microfibril structure with high crystallinity useful for the present investigation (15, 16).

The enzymatic hydrolysis of native cellulose has been assumed to occur first at internal glucosidic bonds within an intact glucan chain by random attack of endo-type cellulase [1,4-(1,3 ; 1,4)-/~-v-glucan 4-glucanohydrolase, EC 3.2.1.4] to create a new nonreducing chain end for saccharification by exo-type cellulase [1,4-/3-D-glucan cellobiohydrolase, EC 3.2.1.91] (1-3). The mechanism, however, has not been fully elucidated (4, 5) because of complex interaction between cellulases and substrates. In addition, the complex fine structure of native cellulose may further complicate elucidation of the degradation mechanism. The widely accepted mechanism has not always been substantiated by direct experimental evidences. In elucidating the mode of action of each cellulase, therefore, it is needed first to investigate precisely morphological changes accompanying the degradation process by a cellulase system. The degree of crystallinity of cellulose is one of the main physical parameters influencing the rate of enzymatic hydrolysis, with cellulase action depending largely on the crystalline and amorphous regions of native cellulose (6). In the ceUulase system of Irpex lacteus, two types of cellulase exist as main components (named Ex-1 and En-1), being clearly different in substrate specificity (7, 8). In previous papers, the crystallinity of cellulosic substrate has been found to greatly affect the mode of action of Ex-1 and En-1 (9, 10). Furthermore, some examinations have been carried out to compare the modes of

MATERIALS AND METHODS

Enzyme source Exo-type cellulase (Ex-1) and endotype cellulase (En-1) used in this work were obtained in the same manner as that reported previously (7, 8) starting from Driselase, a commercial enzyme preparation from L lacteus (Kyowa Hakko, Tokyo). The molecular weights of Ex-1 and En-1 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis were approximately 49,000 and 36,000, respectively. Substrates Valonia cellulose was prepared from air-dried Valonia macrophysa by boiling with 1% NaOH for 5 h and then washing successively with 1% acetic acid, distilled water and acetone. Enzyme assays The reaction mixture consisted of 0.75 wt% of Valonia cellulose, 0.05 M sodium acetate buffer (pH 5.0), and enzyme (16.9 ~M Ex-1 or En-1) in a total volume of 2ml. It was incubated at 30°C with mechanical shaking (50 strokes/min) for a given period, and centrifuged. Reducing sugar produced in the supernatant was determined by the method of Somogyi

* Corresponding author. § Present address: 10-4, Kouyama-3, Nerima-ku, Tokyo 176. 496

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ACTION OF IRPEX CELLULASESON VALONIA CELLULOSE 497

(17) and Nelson (18). Thin-layer chromatography (TLC) Reaction products released from the substrate were identified by TLC. Aliquots (40/21) of hydrolysis products in the incubation mixture were subjected to TLC with silica gel 60 (0.25 mm), E. Merck (Darmstadt, Germany). The solvent system of chloroform-methanol-water (90 : 65 : 15, v/v) was used and the spots were detected by heating the plate at 120°C for 10min after spraying with 50% sulfuric acid. Estimation of the degree of polymerization of cellulose Decrease in average degree of polymerization (DP) of residual substrate was estimated as follows: Residual cellulose collected on a glass filter was washed successively with distilled water, ethanol and acetone, and dried in a vacuum desiccator over phosphorus pentoxide. Approximately 10mg thereof was dissolved at 5°C in 10 ml Cadoxen solution which had been prepared according to the method of Donetzhuber (19). Viscosity of the cellulosic solution was measured at 30°C with an Ostwald-type viscometer. The D P value was calculated from the molecular weight obtained from the following Brown-Henley equation (20), which had been corrected for polydispersity on the basis of the Schultz-Zimm distribution equation; ~7:4.02 × 10 -4 ×/~W0'76 where ~ is the intrinsic viscosity and 33/w is the weightaverage molecular weight. Electron microscopic observation of Valonia cellulose The surface morphology of cellulolysis residues collected on a glass filter as described above was examined using a field-emission scanning electron microscope (FESEM, S-4000, Hitachi, Tokyo) at 3 or 5 kV accelerating voltage after critical-point drying. The distribution of adsorbed cellulases on the cross section of Valonia cellulose before commencement of enzymatic hydrolysis was observed by double-labeling immunoelectron microscopy based on the method of Geuze et al. (21). The reaction mixture consisted of 1.0 wt% of cellulosic substrate, 0.05M ammonium acetate buffer (pH 5.0), and the enzyme (20/~M Ex-1 or En-1) in a total volume of 0.5ml. After incubation at 5°C for an appropriate period with mechanical shaking (50 strokes/min), and with no enzymatic hydrolysis products being practically detectable under these conditions, the Valonia celluloses were fixed with 2% formaldehyde for 1 h and washed five times with 0.05 M ammonium ace-

tate buffer (pH 5.0). The cellulose specimens were then dehydrated with a graded series of ethanol (30-min incubation in each of 25, 50, 75, and 90% ethanol) at 5°C, followed by incubation in 100% ethanol at 5°C. The specimens were embedded in Durcupan epoxy resin (44600, Fluka Chemic AG, Switzerland) at 5°C, and the resin was polymerized at 37, 40, 50, and 60°C for 6 h each. An ultrathin cross section (60--100nm thick) prepared with a diamond knife (Diatome, Bienne, Switzerland) on an ultramicrotome (LKB 2188, Ultrotome Nova, LKB-Produkter AB, Microtomy Products, Bromma, Sweden) was soaked in phosphate-buffered saline (PBS), Dulbecco's PBS (Nissui Pharmaceutical, Tokyo), containing 0.8% bovine serum albumin (Sigma Chemical, St. Louis, MO, USA), 5% normal goat serum (CL 1200, Cedarlane Laboratories, Hornby, Canada), and 0.1% gelatin (AuroProbe One, GAR (1 nm), RPN 416, Amersham International plc., Amersham, England) at 20°C for 30 min to cover the parts without adsorbed enzyme. Then the purified anti-cellulase antibody (diluted 1 : 10 with PBS solution containing 0.8% bovine serum albumin, 1% normal goat serum and 0.1% gelatin) prepared from rabbit serum (11) was used to treat the ultrathin cross section at 30°C for 1 h. Goat anti-rabbit IgG antibody labeled with colloidal gold (R 1210, ULTRA Biotechnology MIC Medical, Liverpool, England) as the second antibody at a dilution ratio of 1 : 50 was reacted with the ultrathin cross section at 30°C for 1 h. After being stained with 0.1% phosphotungstic acid dissolved in 50% ethanol for 20 min followed by lead staining solution for 10 min, the distribution of colloidal gold in the ultrathin cross section was observed by transmission electron microscope (TEM, JEM-2000 FX, JEOL, Tokyo) at 80 kV accelerating voltage. RESULTS

Initial adsorption of Ex-1 and E n d on Valonia cdlulose microfibrils Localization of Ex-1 and En-1 adsorbed on the cross section of Valonia cellulose microfibrils was observed by double-labeling immunoelectron microscopy at an initial stage of enzymatic hydrolysis. Since clear visualization of the cross section was not achieved by the ordinary staining method using uranyl acetate and lead staining solution for the staining of cotton cellulose (11), staining was tried this time on the cross section of Valonia cellulose with various concentrations of phosphotungstic acid or phosphomolybdic acid

TABLE 1. The ratio of adsorbed enzymes on the internal parts and external surfaces of Valonia microfibrils obtained by counting the number of colloidal gold particles Ex-1 Incubation time (h) 0.5

En-1

Internal parts

Externalsurfaces

Percentage on internalparts (%)a

546 468 354 366 422 681 357 323 340 447

82 81 46 55 69 156 92 82 84 103

86.9 85.2 88.5 86.9 85.9 81.4 79.5 79.8 80.2 81.3

Internal parts

Externalsurfaces

Percentage on internalparts (%)~

229 460 183 178 240 292 296 409 267 240

57 123 54 50 63 134 133 187 127 112

80.1 78.9 77.2 78.1 79.2 68.5 69.0 68.6 67.8 68.2

a Percentage on internal parts (%)= [Internal parts/(Internal parts+External surfaces)] × 100

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FIG. 1. Double-labeling immunoelectron micrographs of Ex-I adsorbed on an ultrathin cross section of Valonia cellulose. Valonia cellulose was (a) incubated with Ex-1 for 0.5 h; (b) incubated with Ex-1 for 3 h; and (c) incubated without enzyme (control) at pH 5.0 and 5°C. The reaction mixture consisted of 1.0 wt% of Valonia cellulose and 20 ttM Ex-1. Black dots indicate the colloidal gold particles, and the cross section of each microfibril is indicated by white areas in these photographs. Arrowheads indicate some of the cross sections of microfibrils. Colloidal gold particles shown in (c) are due to nonspecific binding to Valonia cellulose. Bar, 0.1/~m. i n a d d i t i o n t o lead s t a i n i n g s o l u t i o n , a n d t h e l o c a l i z a t i o n o f t h e a d s o r p t i o n site o f e n z y m e s w a s s u c c e s s f u l l y visualized b y use o f s m a l l e r c o l l o i d a l g o l d p a r t i c l e s w i t h a size

J. FERMENT. BIOENG.,

FIG. 2. Double-labeling immunoelectron micrographs of En-l adsorbed on an ultrathin cross section of Va[onia cellulose. Valonia cellulose was (a) incubated with En-1 for 0.5 h; (b) incubated with En-I for 3 h; and (c) incubated without enzyme (control) at pH 5.0 and 5 °C. The reaction mixture consisted of 1.0 w t ~ of Valonia cellulose and 20/tM En-1. Arrowheads indicate the same as in Fig. 1. Bar, 0.1 pro. o f 5 n m i n s t e a d o f 10 n m d e s c r i b e d in a p r e v i o u s p a p e r (11). I n Figs. 1 a n d 2, a cross s e c t i o n o f e a c h m i c r o f i b r i l is v i s u a l i z e d as a clear w h i t e a r e a a n d it is clear t h a t b o t h E x - I a n d En-1 a r e m a i n l y a d s o r b e d i n t o t h e i n t e r n a l p a r t s o f m i c r o f i b r i l s w i t h o u t visible e n z y m a t i c h y d r o l -

VoL. 77, 1994

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ACTION OF IRPEX CELLULASES ON VALONIA CELLULOSE

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FIG. 3. Time course of reducing sugar production from Valonia cellulose by Ex-1 and En-1. Symbols: o , Ex-1; o , En-1. The reaction mixture consisted of 0.75 wt% of Valonia cellulose and 16.9/tM of Ex-1 or En-1. Reaction conditions: pH 5.0 and 30°C. ysis. The percentage o f a d s o r b e d enzymes on the internal parts o f microfibrils was obtained by counting the number o f colloidal gold particles on the internal parts and external surfaces o f the microfibrils (Table 1). Both cellulases showed a high affinity for crystalline microfibrils o f Valonia cellulose. The average values o f Ex-1 and En-1 for 0.5-h incubation period at 5°C obtained from several p h o t o g r a p h s were 86.7___1.2% and 78.7_+I.1%, respectively. However, these values decreased with incubation time (the values became 8 0 . 4 + 0 . 9 % for Ex-1 and 68.4_+0.4% for En-1 after 3-h incubation, which is the time required to attain a d s o r p t i o n equilibrium at 5°C), and the ratio o f a d s o r b e d enzymes relatively increased on the outer region o f microfibrils, especially with En-1. Saccharification activity of Ex-1 and En-1 Time courses o f the saccharification activities o f Ex-1 and En-1 for Valonia cellulose were investigated under the s t a n d a r d conditions o f p H 5.0 and 30°C. As shown in Fig. 3, Ex-1 p r o d u c e d larger a m o u n t s o f reducing sugars than En-1. The molecular activities o f Ex-1 and En-1 were 0.154 and 0.053 ( m o l - g l u c o s e / m i n . m o l - e n z y m e ) , respectively. Hydrolysis products by cellulases Hydrolysis p r o d -

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Reducing sugar as glucose (mM) FIG. 5. Decrease in DP of Valonia cellulose and simultaneous production of reducing sugar by Ex-I and En-1. Symbols: e , Ex-l; O, En-l. Reaction mixture consists of the same composition as that indicated in Fig. 3. After incubation with Ex-I or En-1 for an appropriate period, the DP value of residual substrate was measured and plotted against the reducing power produced in the supernatant. ucts formed from Valonia cellulose by Ex-1 or En-1 were identified by T L C at various incubation periods, and the results are shown in Fig. 4. The p r o d u c t was only cellobiose during 12-h incubation with Ex-1. On the other hand, traces o f cellobiose in addition to several kinds o f cellooligosaccharides were detected at later stages o f incubation with En-1. Degree of randomness in enzymatic hydrolysis The decrease in D P o f Valonia cellulose during enzymatic hydrolysis was investigated to estimate the randomness o f cellulase action, and expressed as the ratio o f decrease in D P to reducing sugar production. The intrinsic viscosity (7) o f cellulose was estimated b y extrapolation o f the linear plots o f C vs. ~sp/C and C vs. (In ~r)/C to obtain intercepts, where C is the concentration o f residual cellulose ( g / 0 , and ~sP and ~r are the specific and the relative viscosities, res e ~ i v e l y . On the basis o f the ~ values, the corresponding D P values were calculated for each incubation time. The degree o f randomness o f b o t h cellulases is shown in Fig. 5. The D P value o f Valonia cellulose is

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FIG. 4. Thin-layer chromatograms of the hydrolysis products formed from Valonia cellulose by Ex-1 and En-l. (a) Ex-1; (b) En-1. S, Standard; G~, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose. Reaction mixture consists of the same composition as that indicated in Fig. 3.

FIG. 6. Field-emission scanning electron micrograph of Valonia cellulose before enzymatic hydrolysis. Bar, 0.2 Fm.

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FIG. 7. Field-emission scanning electron micrographs of Valonia cellulose degraded by Ex-l. Valonia cellulose was (a) incubated with Ex-1 for 3 h; (b) incubated with Ex-I for 12 h; and (c) incubated with Ex-1 for 72 h at pH 5.0 and 30°C. Reaction mixture consists of the same composition as that indicated in Fig. 3. Bar, 0.2 ~m.

FIG. 8. Field-emission scanning electron micrographs of Valonia cellulose degraded by En-1. Valonia cellulose was (a) incubated with En-1 for 3 h; (b) incubated with En-1 for 12 h; and (c) incubated with En-I for 72 h at pH 5.0 and 30°C. Reaction mixture consists of the same composition as that indicated in Fig. 3. Bar, 0.2 pro.

markedly decreased by En-1 as compared with Ex-1. Therefore, it is clear that En-1 is more capable o f depolymerizing crystalline cellulose prior to the production o f reducing sugar than Ex-1. Morphological observation o f residual mierofibrils after enzymatic hydrolysis Field-emission scanning electron micrographs o f the surface feature o f the

microfibril structures before and after cellulolysis by both enzymes are shown in Figs. 6 to 8. Morphological changes o f the cellulose samples due to drying are minimized by means o f critical-point drying. Therefore, morphological differences in the microfibril surface were mainly due to treatment with Ex-1 and En-1. Ex-1 caused loosening o f the network structure o f micro-

ACTION OF IRPEX CELLULASES ON VALONIA CELLULOSE

VoL. 77, 1994

fibrils, and the microfibrils became slightly thinner than intact microfibrils being about 0.03/~m wide (Figs. 6 and 7). In contrast, En-1 treatment caused different severe morphological changes of microfibrils in comparison with Ex-1 treatment. Namely, pronounced swelling and dissolution of microfibrils occurred in a wide region and none of the micro fibrils were intact after a 72-h incubation (Fig. 8). In addition, morphological changes of the microfibril network layer below the surface also occurred by En-1 treatment as early as 3-h incubation. DISCUSSION It has been confirmed by electron microscopy that both exo- and endo-type cellulases from I. lacteus are strongly adsorbed into the internal parts of crystalline cellulose such as cotton fiber before enzymatic hydrolysis, and that the adsorption increases with incubation time (11). In our previous paper, it was also reported that both cellulases, especially Ex-1, show high affinities for crystalline cellulose and that their adsorption proceeds more smoothly with increasing crystallinity of cellulose while adsorption sites for enzymes per unit surface of the substrate are decreased in number (10). The present work using double-labeling immunoelectron microscopy indicates that the both cellulases showed strong adsorption into internal parts of crystalline microfibrils of Valonia cellulose at the initial stages of hydrolysis. Their adsorption extended at later stages onto the external surfaces of the microfibrils which seemed to be more amorphous. The adsorption process was less remarkable with Ex-1 than with En-1, and this may be due to the lower affinity of Ex-1 to amorphous cellulose than to crystalline one as compared with En-1 (10). The saccharification activity to Valonia cellulose of En-1 seemed quite low, however, it had a higher capacity for lowering D P of the cellulose than Ex-1, producing small amounts of several cellooligosaccharides at the later stages of hydrolysis. In contrast, Ex-1 actively produced cellobiose, which was possibly split off one by one from the nonreducing ends of the cellulose chain. These results from Valonia cellulose also agree well with findings obtained so far that Ex-1 and En-1 showed typical exo- and endo-type modes of action, respectively, for crystalline native cellulose such as cotton and cotton linter (9, 11, 12). It is further noted that the two cellulases were found by FESEM to cause considerably different morphological changes in Valonia cellulose. Ex-1 not only loosened the network structure of Valonia microfibrils but also made them slightly thinner. Since Ex-1 should degrade Valonia microfibrils in an exo-fashion, it seems difficult for Ex-1 to randomly attack inner cellulose molecules by penetration into compact microfibrils. It is possible that the degradation may take place initially at any of the terminal sites on the surface area and extend later all over the surface, making the microfibrils thinner as a whole. Chanzy et aL have similarly reported that the microfibrils of Valonia cellulose were eroded into narrower crystals and were subfibrillated into smaller elements by cellobiohydrolase I from Trichoderma reesei, which has some properties common to Ex-1, while their overall shape was maintained (22). In contrast, En-1 caused pronounced swelling and dissolution of microfibrils to an extent where there were no intact microfibril struc-

501

tures at the later stages of hydrolysis, the result being in agreement with our previous paper (15). It may be assumed that the lowered D P of Valonia cellulose by En-I treatment reflects such morphological changes of individual microfibrils as swelling and dissolution. However, the mechanism of thinning of microfibrils by the exofashion of Ex-1 remains unclear. Moreover, En-1 had a greater influence on the characteristic morphological changes of the microfibril network layer below the surface than Ex-1 at the earlier stages of hydrolysis. It is, therefore, suggested that there is a difference in the penetration rates of the two types of cellulase into the network structure of micro fibrils. REFERENCES 1. Nisizawa, K., Tomita, Y., Kanda, T., Suzuki, H., and Wakabayashi, K.: Substrate specificity of C~ and Cx cellulase components from fungi, p. 719-725. In Terui, G. (ed.), Fermentation technology today. Soc. Ferment. Technol., Osaka (1972). 2. Wood, T.M. and McCrae, S.I.: The purification and properties of the Cl component of Trichoderma koningii cellulase. Biochem. J., 128, 1183-1192 (1972). 3. International Union of Biochemistry and Molecular Biology, p. 347, p. 358. In Enzyme nomenclature. Academic Press, New York (1992). 4. Enari, T.-M. and Nikn-Paavola, M.-L.: Enzymatic hydrolysis of cellulose: is the current theory of the mechanisms of hydrolysis valid? CRC Crit. Rev. Biotechnol., 5, 67-87 (1987). 5. Okada, G.: Cellulase. Kagaku to Seibutsu, 23, 14-22 (1985). (in Japanese) 6. Fan, L.T., Lee, Y.-H., and Beardmore, D . H . : Major chemical and physical features of cellulosic materials as substrates for enzymatic hydrolysis. Adv. Biochem. Eng., 14, 101-117 (1980). 7. Kanda, T., Nakakubo, S., Wakabayashi, K., and Nisizawa, K.: Purification and properties of an exo-cellulase of Avicelase type from a wood-rotting fungus, Irpex lacteus (Polyporus tulipiferae). J. Biochem., 84, 1217-1226 (1978). 8. Kanda, T., Wakabayashi, K., and Nisizawa, K.: Purification and properties of a lower-molecular-weight endo-cellulase from Irpex lacteus (Polyporus tulipiferae). J. Biochem., 87, 16251634 (1980). 9. Hoshino, E., Sasaki, Y., Okazaki, M., Nisizawa, K., and Kanda, T.: Mode of action of exo- and endo-type cellulases from Irpex lacteus in the hydrolysis of cellulose with different crystallinities. J. Biochem., 114, 230--235 (1993). 10. Hoshino, E., Kanda, T., Sasaki, Y., and Nisizawa, K.: Adsorption mode of exo- and endo-cellulases from Irpex lacteus (Polyporus tulipiferae) on cellulose with different crystallinities. J. Biochem., 111, 600-605 (1992). 11. Hoshino, E., Sasaki, Y., Mori, K., Okazaki, M., Nisizawa, K., and Kanda, T.: Electron microscopic observation of cotton cellulose degradation by exo- and endo-type cellulases from Irpex lacteus. J. Biochem., 114, 236-245 (1993). 12. Hoshino, E., Kubota, Y., Okazaki, M., Nisizawa, K., and Kanda, T.: Hydrolysis of cotton cellulose by exo- and endotype cellulases from Irpex lacteus: differential scanning calorimetric study. J. Biochem., 115, 837-842 (1994). 13. Murata, M., Hoshino, E., Yokosuka, M., and Suzuki, A.: New detergent mechanism with use of novel alkaline cellulase. J. Am. Oil Chem. Soc., 68, 553-558 (1991). 14. deGruy, I. V., Carra, J. H., and Goynes, W. R.: The fine structure of cotton. An atlas of cotton microscopy, p. 118-121. In O'Connor, R.T. (ed.), Marcel Dekker Inc., New York (1973). 15. Takai, M., Hayashi, J., Nisizuwa, K., and Kanda, T.: Morphologic observation of cellulose microfibrils degraded by exo- and endocellulases. J. Appl. Polym. Sci.: Appl. Polym. Symp., 37, 345-361 (1983). 16. Kulshreshtha, A.K. and Dweitz, N.E.: Paracrystalline lattice

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disorder in cellulose. I. Reappraisal of the application of the two-phase hypothesis to the analysis of powder X-ray diffractograms of native and hydrolyzed cellulosic materials. J. Polym. Sci. Polym. Phys. Ed., 11, 487-497 (1973). 17. Somogyi, M.: Notes on sugar determination. J. Biol. Chem., 195, 19-23 (1952). 18. Nelson, N.: A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem., 153, 375-380 (1944). 19. Donetzhuher, A.: Zur herstellung des cellulose-16sungsmittels ))Cadoxen). Svensk Papp.-Tidn., 63, 447-448 (1960).

J. FERMENT. BIOEr~G., 20. Brown, W. and Wikstr6m, R.: A viscosity-molecular weight relationship for cellulose in Cadoxen and a hydrodynamic interpretation. Eur. Polymer J., 1, 1-10 (1965). 21. Geuze, H.J., Slot, J.W., van der Ley, P.A., and Scheffer, R. C. T.: Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections. J. Cell Biol., 89, 653-665 (1981). 22. Chanzy, H., Henrissat, B., Vuong, R., and Sehfilein, M.: The action of 1,4-~9-D-glucan cellobiohydrolase on Valonia cellulose microcrystals. An electron microscopic study. FEBS Lett., 153, ll3-11C 0983).