Biomacromolecules 2002, 3, 1359-1363
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Initial Characterization of Ethyl(hydroxyethyl) Cellulose Using Enzymic Degradation and Chromatographic Methods Sara Richardson,*,† Jon Lundqvist,‡ Bengt Wittgren,§ Folke Tjerneld,‡ and Lo Gorton† Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, Department of Biochemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and AstraZeneca R&D Mo¨lndal, SE-431 83 Mo¨lndal, Sweden Received July 15, 2002; Revised Manuscript Received August 14, 2002
Two different ethyl(hydroxyethyl) cellulose (EHEC) samples were characterized by size-exclusion chromatography (SEC) with multiangle light scattering (MALS) detection and high-performance anionexchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The aim of the study was to investigate the molar mass distribution and the heterogeneity of the substituent distribution, factors that are thought to affect the functional properties of EHEC. The presence of blocks of unsubstituted glucose units was studied by enzymic degradation of EHEC by two different endoglucanases from Trichoderma reesei. The SEC-MALS analysis of the hydrolysis products showed that both enzymes were strongly inhibited by the large number of substituents along the cellulose chain. However, as the weight-average molar mass was reduced from approximately 360 000 to 80 000 g/mol in one of the polymers and from 770 000 to 60 000 g/mol in the other polymer, it was suggested that both samples were composed of some unsubstituted regions where the enzymes got access to the glucosidic bonds. The amount of glucose released upon endoglucanase hydrolysis was determined by HPAEC-PAD, which gave information on the homogeneity of the substituent distribution. The production of unsubstituted glucose units indicated that one of the polymers had a more uneven distribution compared with the other. It was demonstrated that chemical characterization of EHEC is a complex task, which requires an analytical approach involving numerous different methods and techniques. Introduction Ethyl(hydroxyethyl) cellulose (EHEC) is a nonionic, water-soluble cellulose derivative produced by introduction of ethyl and ethylene oxide groups to the hydroxyl groups of the cellulose backbone. It is an important industrial product used mainly in water-based paints and building products as thickener, emulsifier, and dispersing agent.1 It is known that the chemical and physical properties of modified celluloses depend on, e.g., molar mass distribution, degree of substitution, and distribution of substituents in the polymer. Therefore, examination of any correlations between properties and the aforementioned factors is of importance, which in turn implies a demand for methods by which the molar mass and substituent distribution can be studied. Consequently, such methods need to be developed in order to understand the behavior of cellulose derivatives. The methods of choice for determination of the molar mass distribution of EHEC are size-exclusion chromatography (SEC)2,3 or field-flow fractionation (FFF),4 whereas methods or strategies for characterization of the substituent distribution have shown to be less straightforward. The substituent distribution in cellulose derivatives can be considered on * To whom correspondence may be addressed. Tel: +46-31-7761173. Fax: +46-31-7763798. E-mail:
[email protected]. † Department of Analytical Chemistry, Lund University. ‡ Department of Biochemistry, Lund University. § AstraZeneca R&D Mo ¨ lndal.
different structural levels, e.g., in the anhydroglucose unit (AGU) or along the polymer chain. The distribution on a monomer level (i.e., how the substituents are distributed at the 2-, 3- and 6-positions in the AGU) in EHEC has been investigated by means of carbon nuclear magnetic resonance (13C NMR) spectroscopy5 or standard methylation analysis.6 However, the distribution of substituents along the polymer chain is judged to be of more importance, as this is thought to correlate with the physical properties of the polymer.7,8 A heterogeneous distribution for example, with “blocks” of unsubstituted regions, could be one cause for the formation of aggregates, which may affect the properties of the polymer.9 Characterization of the heterogeneity of the substituent distribution, i.e., the average block length of unsubstituted regions, requires a selective degradation followed by analysis of the products released. Such selective degradation of cellulose derivatives has successfully been obtained using enzymic hydrolysis.10-13 Subsequent determination of the amount of unsubstituted products released from enzymic hydrolysis can give information on the length of unsubstituted regions and thus the heterogeneity of the substitution.14,15 Hitherto, the distribution on a polymer level in EHEC has been studied in only a few investigations.16,17 We herein report work on an analytical strategy for characterization of EHEC, where the molar mass distribution and the degree of enzymic degradation of the polymer have been investigated. Two different EHEC polymers were
10.1021/bm020081m CCC: $22.00 © 2002 American Chemical Society Published on Web 10/05/2002
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studied and compared. As an initial attempt to characterize the distribution of ethyl and ethylene oxide groups along the cellulose chain, EHEC was hydrolyzed with cellulose degrading enzymes. The intact polymers as well as the hydrolysis products were analyzed by SEC with multiangle light scattering (MALS) detection not only for examination of the molar mass distribution and the substituent distribution but also for elucidation of the enzyme action on the EHEC polymer. Furthermore, anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was employed for investigation of the presence of unsubstituted glucose in the hydrolysates as an additional tool for estimation of the heterogeneity of the substituent distribution.
Richardson et al.
Figure 1. Schematic illustration of a possible structural sequence of EHEC.
Experimental Section Chemicals. Two EHEC samples, EHM0 (ethyl hydroxy modified 0, i.e., not hydrophobically modified) with DSethyl ) 0.7 (degree of substitution, i.e., average number of hydroxyl groups in the monomer unit substituted with ethyl) and MSEO ) 1.8 (molar substitution, i.e., average number of moles of ethylene oxide groups per monomer unit) and E481-02506 with DSethyl ) 0.9 and MSEO ) 2.6, were gifts from Akzo Nobel Surface Chemistry (Stenungsund, Sweden). The DSethyl and MSEO values were given by the manufacturer. Before use, the modified cellulose samples were purified and desalted according to Thuresson et al.18 Glucose and cellobiose from Sigma Chemicals Co. (St. Louis, MO) were used as standards in the HPAEC-PAD experiments. Enzymes. Trichoderma reesei endoglucanase (EC 3.2.1.4) Cel7B (EG I)19 with a molecular mass of ∼50 kDa was purified according to Karlsson et al. (manuscript in preparation). Trichoderma reesei endoglucanase Cel12A (EG III),20 with a molecular mass of ∼24.5 kDa, was a kind gift from Dr. Michel Ward, Genencore, CA. Hydrolysis of EHEC. EHEC (0.05% w/v) was dissolved in 50 mM NaOAc buffer (pH 4.8) and kept at room temperature for 48 h under stirring. Enzymic hydrolysis of EHEC was accomplished by incubating the EHEC solution with 0.4 µmol of Cel7B/mg of EHEC and 0.8 µmol of Cel12A/mg of EHEC at 40 °C for 48 h at pH 4.8. Equipment. The molar mass distribution of intact EHEC and products obtained from hydrolysis of EHEC by Cel7B and Cel12A were determined by means of a size-exclusion chromatographic (SEC) system with multiangle light scattering (MALS) and refractive index (RI) detection. The separation column was a TSK-GEL GMPWXL 7.8 × 300 mm, particle size 13 µm, linear mixed bed size-exclusion column (Toso-Haas, Montgomeryville, PA). The pump was a Shimadzu LC10AD liquid chromatography pump (Shimadzu Corp, Tokyo, Japan). The degasser used was a Gastorr 154 (Gastorr, Japan). The mobile phase was an aqueous 50 mM NaOAc solution, and the flow rate of the mobile phase was held at 0.50 mL/min. The polymer sample was injected on the column by a Rheodyne 9125 injector (Rheodyne, Cotati, CA), equipped with a 100 µL sample loop. The injected amount of sample was 50 µg as the polymer concentration in the solution was held at 0.5 mg/mL. The light scattering photometer was a DAWN-DSP multiangle light scattering instrument (Wyatt Technology, Santa
Figure 2. The molar mass distribution of intact E481-02506, E48102506 hydrolyzed by Cel7B, and E481-02506 hydrolyzed by Cel12A, determined by SEC-MALS-RI.
Barbara, CA). Simultaneous concentration detection was performed using an Optilab DSP interferometric refractometer (Wyatt Technology). Both detectors used a wavelength of 633 nm. The recovery was obtained from the ratio of the mass eluted from the channel (determined by integration of the refractometer signal) to the mass injected. Determination of the amount of glucose liberated from enzymic hydrolysis of EHEC was achieved by HPAEC-PAD. The chromatographic system (Dionex 500; Dionex, Sunnyvale, CA) was the same as described in a previous work by Richardson et al.,21 except for the use of a CarboPac PA1 (2 × 250 mm) analytical column in the present study. Elution was performed using a gradient program with 150 mM NaOH (eluent A), 500 mM NaOAc prepared in 150 mM NaOH (eluent B), and water (eluent C). Between 0 and 5 min, the eluent composition was held constant at 90% A and 10% C. Between 5 and 20 min, eluent A decreased linearly from 90% to 20%, eluent B increased from 0% to 70%, and eluent C was held constant at 10%. At 20 min, the eluent composition was returned to 90% A and 10% C and held there until 30 min. The flow rate was 0.25 mL/min and the injection volume 20 µL. Identification and quantification of glucose in the enzymic hydrolysates were carried out using glucose standards. Blanks of enzyme and intact polymer were also analyzed. Results and Discussion SEC-MALS-RI. SEC in combination with MALS and refractive index (RI) detection is a very versatile technique for molar mass analysis, as the molar mass can be determined directly without any calibration standards.22-24 In this study, a SEC-MALS-RI system was used to characterize the molar mass and size of the intact and hydrolyzed EHEC polymers. A possible structural element of EHEC is depicted in Figure 1. The weight-average molar mass (Mw) of E481-02506 was determined to 772 000 g/mol and the polydispersity index Mw/Mn to 1.5 (Figure 2, Table 1). The Mw of EHM0 was significantly lower, approximately 359 000 g/mol. However,
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Characterization of Ethyl(hydroxyethyl) Cellulose Table 1. Weight-Average Molar Mass, Mw, Polydispersity Index, Mw/Mn, and Radius of Gyration, rG, Obtained from SEC-MALS Analysis of Intact EHM0 and E481-02506 and Hydrolysates of EHM0 and E481-02506 Obtained from Cel7B and Cel12A Hydrolysis EHM0
Mw (g/mol) Mw/Mn rGa (nm) a
E481-02506
intact
Cel7B
Cel12A
intact
Cel7B
Cel12A
359000 2.4 61
83000 1.5 39
102000 1.9 37
772000 1.5 77
57000 1.7 40
67000 1.9 38
Denotes the z-average of the radius of gyration.
Figure 3. The molar mass distribution of intact EHM0, EHM0 hydrolyzed by Cel7B, and EHM0 hydrolyzed by Cel12A, determined by SEC-MALS-RI.
the Mw/Mn index of EHM0 was higher compared with E48102506, approximately 2.4 (Figure 3, Table 1), which indicates a rather broad distribution. It is not unusual with a higher polydispersity index for low molar mass polymers, as the degradation process that produces these polymers often results in more polydisperse samples. Cellulose derivatives are known to have broad molar mass distribution, which put high demands on the actual characterization technique. A recent work4 employed the combination of flow field-flow fractionation (flow FFF) and MALS-RI for molar mass characterization of the particular EHM0 sample used in the present work. The Mw obtained in that study was 310 000 g/mol and the polydispersity 2.5, in good agreement with the SEC-MALS-RI results obtained here. However, the FFFMALS-RI results indicate that the EHEC sample also contains small amounts of a fraction having ultrahigh molar masses (∼108 g/mol). This fraction is not seen in the SECMALS-RI analysis, possibly due to adsorption to the column material. The origin of these components is not known, but it is possible that they contain parts of the cellulosic backbone that are less substituted,9 a hypothesis that requires careful examination of the substituent distribution. To acquire information on possible blocks of unsubstituted regions in EHEC, it is necessary to degrade the polymer into smaller fragments. This fragmentation can be achieved by cellulases, which constitute highly selective tools for use in the investigation of EHEC. The main advantage of using cellulases is that a selective degradation is obtained, as the enzymic hydrolysis is hindered by the substituents.25 Consequently, unsubstituted regions in the polymer are more readily attacked compared with low-substituted regions, whereas highly substituted regions are almost completely excluded from hydrolytic fragmentation. Characterization of
the products released from enzymic hydrolysis of EHEC can give information on the heterogeneity of the substituent distribution. If the polymer contains regions that are less substituted, the amount of unsubstituted glucose liberated after enzymic degradation should be pronounced. The cellulose hydrolyzing enzymes used in this work belong to the endoglucanase (EG) type that cleave the internal (1f4)-β-D-glucosidic linkages in the cellulose chain, producing smaller fragments of mono- and oligosaccharides.26 The SEC-MALS analysis of the hydrolysates showed that the molar masses of the products released from enzymic hydrolysis of EHM0 by Cel7B and Cel12A, 83 000 and 102 000 g/mol, respectively, were significantly lower than of the intact EHM0 polymer, 359 000 g/mol (see Table 1 and Figure 2). The E481-02506 polymer was degraded to a much higher extent, from 772 000 g/mol to 57 000 (Cel7B) and 67 000 g/mol (Cel12A), respectively (see Table 1 and Figure 3). The clear difference in molar mass distribution between intact and hydrolyzed EHEC (Figures 2 and 3) shows that both endoglucanases have capacity to degrade the EHEC polymers to some extent. However, as these enzymes are known to hydrolyze unmodified cellulose to low-molar mass products such as glucose, cellobiose, and cellotriose with molar masses in the range of a few hundreds of daltons,27 these results demonstrate the strongly reduced enzyme accessibility to the glucosidic linkages provided by the substitution groups. As a rough approximation, the endoglucanases hydrolyze on average only 4 glucosidic linkages in each cellulose chain of EHM0, whereas roughly 12-14 linkages per chain in E481-02506 are cleaved. This suggests that the major parts of these cellulose polymers are highly substituted. Furthermore, there is a pronounced difference between the degradation of EHM0 and E48102506, as both endoglucanases have the ability to hydrolyze a higher number of linkages in E481-02506. This finding indicates that the E481-02506 sample consists of more unor low-substituted regions, where the enzymes get access to the glucosidic bonds, compared with EHM0. Consequently, the former sample most likely has a more heterogeneous distribution of ethyl and ethylene oxide groups. Theoretically, if the two samples would have had a similar substituent distribution, EHM0 with the lower level of substitution should have been the more degraded sample. In this case, the results point toward the opposite, a fact that supports the suggestion of a more heterogeneous distribution of substituents in E481-02506. Additionally, there is a significant difference in molar mass distribution between the Cel7B- and Cel12A-hydrolysates (Figures 2 and 3). Obviously, Cel7B can adsorb more readily to the EHEC samples, as more glucosidic linkages are hydrolyzed and thus products with a lower average molar mass distribution are released from hydrolysis by this enzyme than by Cel12A. One explanation to this finding could be that Cel7B consists of two domains, one cellulose binding domain and one catalytic core, whereas Cel12A consists only of a catalytic core.28 The results obtained here are in accordance with a previous study on carboxymethyl cellulose (CMC), where it was shown that Cel7B was more efficient in the hydrolysis of CMC compared with Cel12A.29
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Table 2. The Relative Amount of Glucose Liberated from Enzymic Hydrolysis of EHM0 and E481-02506 by Cel7B and Cel12A glucose liberated (%)a sample
DSethyl
MSEO
Cel7B
Cel12A
EHM0 E481-02506
0.7 0.9
1.8 2.6
0.3 0.9
0.6 2.8
a The relative amount of glucose liberated is referred to unsubstituted anhydroglucose units.
Even though no detailed conclusions about the substitution pattern can be drawn from the SEC-MALS results, it is shown that methods for determination of molar mass distribution, such as SEC-MALS, are crucial in this type of analysis. Glucose Determination. To determine the amount of glucose and to monitor the mono- and oligosaccharides released from the endoglucanase hydrolysis of EHEC, the hydrolysates were subjected to analysis by anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). HPAEC-PAD is a widely used technique for separation and detection of mono- and oligosaccharides in enzymic hydrolysates,30-33 and has also been used for separation and detection of chemically substituted monosaccharides.12,34,35 Analysis of the amount of unsubstituted glucose produced after enzymic hydrolysis of modified polysaccharides is a fast and simple way to obtain information on the heterogeneity of the substitution pattern, as well as on the enzyme action. In the case of a polymer with a homogeneous distribution, the number of glucosidic bonds between two unmodified glucose units is less than that in a polymer with a more heterogeneous substituent distribution. Thus, there are fewer linkages available for enzymic hydrolysis and a lower amount of glucose is liberated upon enzymic degradation of a homogeneously substituted polymer compared with a more heterogeneously substituted polymer, which has larger regions of adjacent unsubstituted units where the enzyme can cleave the glucosidic linkages. In addition, there is a possibility that the extent of enzymic hydrolysis may be influenced by other structural features of the polymer, such as conformation and molar mass. However, this was not investigated in the present study. The amount of glucose produced from hydrolysis of EHM0 and E481-02506 by Cel7B and Cel12A, respectively, are shown in Table 2. According to the results obtained, the endoglucanase hydrolysis of E481-02506 liberates more glucose than does the hydrolysis of EHM0. A total of 2.8% of E481-02506 is liberated as glucose upon Cel12A hydrolysis, whereas only 0.6% of the EHM0 sample is converted into glucose (Table 2). These results suggest a more heterogeneous distribution in E481-02506, which is in agreement with the results obtained from the SEC-MALS analysis. Consideration of the DSethyl and MSEO values supports the conclusion of a more uneven distribution of substituents in E481-02506 compared with EHM0, as a similar distribution pattern of substituents in these two samples would imply a higher amount of unsubstituted glucose units released from EHM0, not the opposite as is the case in here. Consequently, E481-02506 consists of more regions along the cellulose chains where the DSethyl and MSEO
Figure 4. HPAEC-PAD chromatogram of Cel12A hydrolysate of E481-02506 (A), Cel12A hydrolysate of EHM0 (B), and blank of Cel12A (C).
deviate from the average to a higher extent compared with EHM0; i.e., the former sample has regions with either higher or lower DSethyl and MSEO values than expected from the total DSethyl and MSEO. Figure 4 shows the HPAEC-PAD chromatograms from the glucose analysis of the Cel12Ahydrolysates of E481-02506 and EHM0, respectively. Regarding the enzyme activity, it was shown that Cel12A is the more efficient enzyme in the production of glucose from these two polymers. Cel12A hydrolyses both EHEC samples in such way that a higher amount of glucose is released compared with hydrolysis by Cel7B. This result is somewhat surprising as the SEC-MALS analysis showed that Cel7B degraded the intact polymers to products with a lower average molar mass, with the conclusion that this enzyme degrades EHEC to a higher extent than does Cel12A. Similar results were obtained by Karlsson et al. in an investigation on CMC.29 However, in this case Cel12A seems to be more efficient in the production of glucose, although Cel7B is the more effective enzyme in the overall hydrolysis of EHEC. Conclusions An initial study of the chemical composition of two different EHEC polymers has been presented. The molar mass was investigated by SEC-MALS analysis, which revealed significant differences in molar mass distribution of the intact EHEC samples. The results obtained from enzymic hydrolysis with subsequent SEC-MALS analysis showed that E481-02506 was degraded to a higher extent compared with EHM0, indicating a more heterogeneous distribution of substituents in the former polymer. However, only on average 12-14 linkages per cellulose chain in E48102506 and approximately 4 linkages in EHM0 were cleaved, which demonstrates the strongly reduced enzyme accessibility to the glucosidic linkages provided by the substitution groups. Furthermore, the results from the SEC-MALS analysis showed that there were differences in the hydrolytic activity between two different endoglucanases. Analysis of the amount of glucose released from the enzymic hydrolysis of the EHEC polymers by HPAEC-PAD gave information on the homogeneity of the substituent distribution. According to the glucose determination, endoglucanase hydrolysis of EHM0 as well as of E481-02506 resulted in the production of unsubstituted glucose, indicating the presence of more or less widespread regions that are
Characterization of Ethyl(hydroxyethyl) Cellulose
unsubstituted. As the E481-02506 sample released a higher amount of glucose upon enzymic hydrolysis, it was concluded that this sample has a more heterogeneous substituent distribution compared with EHM0. The analytical approach described here can successfully be used for investigation of the presence of unsubstituted regions in EHEC, which is considered to be of importance as the degree of heterogeneity affects the functional properties. For a complete characterization of the substituent distribution, enzymic (endo- as well as exo-enzymes) and/ or acid degradation in combination with additional techniques such as NMR, MALDI-TOF MS, and GC-MS are required. Acknowledgment. This work was financially supported by the Centre for Amphiphilic Polymers (CAP), Lund, Sweden. References and Notes (1) Carlsson, A.; Lindman, B.; Nilsson, P.-G.; Karlsson, G. Polymer 1986, 27, 431. (2) Wittgren, B.; Porsch, B. Carbohydr. Polym. 2002, 49, 457. (3) Porsch, B.; Andersson, M.; Wittgren, B.; Wahlund, K.-G. J. Chromatogr., A 2002, 946, 69. (4) Andersson, M.; Wittgren, B.; Wahlund, K.-G. Anal. Chem. 2001, 73, 4852. (5) Zadorecki, P.; Hjertberg, T.; Arwidsson, M. Makromol. Chem. 1987, 188, 513. (6) Lindberg, B.; Lindquist, U.; Stenberg, O. Carbohydr. Res. 1988, 176, 137. (7) Arisz, P. W.; Kauw, H. J. J.; Boon, J. J. Carbohydr. Res. 1995, 271, 1. (8) Heinze, U.; Schaller, J.; Heinze, T.; Horner, S.; Saake, B.; Puls, J. Cellulose 2000, 7, 161. (9) Thuresson, K.; Lindman, B. Colloids Surf., A 1999, 159, 219. (10) Demeester, J.; Eigner, W.-D.; Huber, A.; Glatter, O. J. Wood Chem. Technol. 1988, 8, 135. (11) Gohdes, M.; Mischnick, P. Carbohydr. Res. 1998, 309, 109. (12) Horner, S.; Puls, J.; Saake, B.; Klohr, E.-A.; Thielking, H. Carbohydr. Polym. 1999, 40, 1. (13) Saake, B.; Horner, S.; Kruse, T.; Puls, J.; Liebert, T.; Heinze, T. Macromol. Chem. Phys. 2000, 201, 1996.
Biomacromolecules, Vol. 3, No. 6, 2002 1363 (14) Wirick, M. G. J. Polym. Sci. 1968, 6, 1705. (15) Mischnick, P.; Heinrich, J.; Gohdes, M.; Wilke, O.; Rogmann, N. Macromol. Chem. Phys. 2000, 201, 1985. (16) Brandt, L. In Ullmann’s Encyclopedia of Industrial Chemistry; Campbell, F. T., Pfefferkorn, R., Rounsaville, J. F., Eds.; VCH Verlagsgesellschaft: Weinheim, 1986; pp 461-488. (17) Do¨nges, R. Br. Polym. J. 1990, 23, 315. (18) Thuresson, K.; Karlstro¨m, G.; Lindman, B. J. Phys. Chem. 1995, 99, 3823. (19) Kleywegt, G. J.; Zou, J. Y.; Divne, C.; Davies, G. J.; Sinning, I.; Stahlberg, J.; Reinikainen, T.; Srisodsuk, M.; Teeri, T. T.; Jones, T. A. J. Mol. Biol. 1997, 272, 383. (20) Sandgren, M.; Shaw, A.; Ropp, T. H.; Wu, S.; Bott, R.; Cameron, A. D.; Stahlberg, J.; Mitchinson, C.; Jones, T. A. J. Mol. Biol. 2001, 308, 295. (21) Richardson, S.; Cohen, A.; Gorton, L. J. Chromatogr., A 2001, 917, 113. (22) Beri, R. G.; Walker, J.; Reese, E. T.; Rollings, J. E. Carbohydr. Res. 1993, 238, 11. (23) Roger, P.; Colonna, P. Carbohydr. Polym. 1993, 21, 83. (24) Wyatt, P. J. Anal. Chim. Acta 1993, 272, 1. (25) Saake, B.; Horner, S.; Puls, J. In Cellulose DeriVatiVes: Modification, Characterization and Nanostructures; Heinze, T., Glasser, G., Eds.; American Chemical Society: Washington, DC, 1998; pp 201-216. (26) Teeri, T. T.; Koivula, A. Carbohydr. Eur. 1995, 12, 28. (27) Karlsson, J.; Medve, J.; Tjerneld, F. Appl. Biochem. Biotechnol. 1999, 82, 243. (28) Karlsson, J. Fungal cellulasessStudy of hydrolytic properties of endoglucanases from Trichoderma reesei and Humicola insolens. Ph.D. Thesis, Lund University, Lund, Sweden, 2000. (29) Karlsson, J.; Momcilovic, D.; Wittgren, B.; Schulein, M.; Tjerneld, F.; Brinkmalm, G. Biopolymers 2002, 63, 32. (30) Torto, N.; Gorton, L.; Marko-Varga, G.; Emneu´s, J.; Åkerberg, C.; Zacchi, G.; Laurell, T. Biotechnol. Bioeng. 1997, 56, 546. (31) Richardson, S.; Nilsson, G. S.; Torto, N.; Laurell, T.; Gorton, L. Anal. Commun. 1999, 36, 189. (32) Zook, C. M.; LaCourse, W. R. Anal. Chem. 1998, 70, 801. (33) Cataldi, T. R. I.; Campa, C.; De Benedetto, G. E. Fresenius J. Anal. Chem. 2000, 368, 739. (34) Kragten, E. A.; Kamerling, J. P.; Vliegenthart, F. G. J. Chromatogr. 1992, 623, 49. (35) Heinrich, J.; Mischnick, P. J. Chromatogr., A 1996, 749, 41.
BM020081M
