Open Access Open Badges Research

How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

Gernot Jäger1, Michele Girfoglio2, Florian Dollo1, Roberto Rinaldi3, Hans Bongard3, Ulrich Commandeur2, Rainer Fischer24, Antje C Spiess5 and Jochen Büchs1*

Author Affiliations

1 AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany

2 Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany

3 Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr, Germany

4 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, D-52074 Aachen, Germany

5 AVT-Aachener Verfahrenstechnik, Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany

For all author emails, please log on.

Biotechnology for Biofuels 2011, 4:33  doi:10.1186/1754-6834-4-33

The electronic version of this article is the complete one and can be found online at:

Received:7 July 2011
Accepted:23 September 2011
Published:23 September 2011

© 2011 Jäger et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.


After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.


Recombinant swollenin can be easily produced with the robust yeast K. lactis. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.


Naturally occurring lignocellulose is a promising starting material for the sustainable production of platform chemicals and fuels [1-6]. The hydrolysis of its main component cellulose to glucose necessitates a cellulase system consisting of cellobiohydrolase (CBH, E.C., endoglucanase (EG, E.C. and β-glucosidase (E.C. [7-9]. Besides enzyme-related factors (for example, enzyme inactivation and product inhibition) [10], the enzymatic hydrolysis of cellulose is limited by its physical properties [11-14]. These properties, in particular, are the degree of polymerization, accessibility and crystallinity [15-18]. Cellulose accessibility, which is determined by cellulose particle size (external surface area) and porosity (internal surface area) [15,19], is the most important factor for hydrolysis [15,18,20-24]. This accessibility reflects the total surface area available for direct physical contact between cellulase and cellulose and, therefore, influences cellulase adsorption as well as the rate and extent of cellulose hydrolysis [21,25]. Furthermore, crystallinity is a relevant factor for cellulose hydrolysis, since it influences the reactivity of adsorbed cellulases [26]. Here, it should be noted that crystallinity may also affect cellulase adsorption [26,27] and, therefore, cellulose accessibility [15,21,28]. Up to now, the relationship between crystallinity and accessibility has not been clearly understood [15,29]. However, for high cellulose hydrolysis rates and yields, cellulose accessibility needs to be increased and, conversely, its crystallinity reduced [30,31]. To achieve this and accordingly improve subsequent hydrolysis, pretreatment techniques are essential [6,14,16,32].

Since pretreatment can be expensive, there is a prime motivation to screen and improve it [33-37]. Over time, many pretreatment technologies have been developed: physical (for example, milling or grinding), physicochemical (for example, steam explosion or ammonia fiber explosion), chemical (for example, acid or alkaline hydrolysis, organic solvents or ionic liquids), biological or electrical methods, or combinations of these methods [33,35]. Some of these techniques entail expensive equipment, harsh conditions and high energy input [33]. By contrast, in the past years, non-hydrolyzing proteins have been investigated that pretreat cellulose under mild conditions [17,20]. After regular lignocellulose pretreatment, these non-hydrolyzing proteins can be added during cellulose hydrolysis [38] or they can be utilized in a second pretreatment step in which cellulose is the substrate [17].

During this second pretreatment step, cellulose is incubated under mild conditions with non-hydrolyzing proteins that bind to the cellulose. As a result, cellulose microfibrils (diameter around 10 nm [39,40]) are dispersed and the thicker cellulose macrofibrils or fibers (diameter around 0.5 to 10 μm, consisting of microfibrils [39-41]) swell, thereby decreasing crystallinity and increasing accessibility [20,42-44]. This phenomenon was named amorphogenesis [20,42]. Furthermore, cellulose-binding proteins can lead to deagglomeration of cellulose agglomerates (diameter > 0.1 mm, consisting of cellulose fibers) [45,46], thereby separating cellulose fibers from each other and additionally increasing cellulose accessibility. Ultimately, amorphogenesis as well as deagglomeration promote cellulose hydrolysis [20].

Various authors have described hydrolysis-promoting effects when pretreating cellulose with single cellulose-binding domains [17], expansins from plants [38,47-49] or expansin-related proteins from Trichoderma reesei [50], Bacillus subtilis [51], Bjerkandera adusta [52] or Aspergillus fumigatus [46]. A prominent expansin-related protein is swollenin from the fungus T. reesei. In contrast to cellulases, the expression levels of swollenin in T. reesei are relatively low (1 mg/L) [50]. Thus, swollenin from T. reesei has been heterologously expressed in Saccharomyces cerevisiae [50], Aspergillus niger [50] and Aspergillus oryzae [53]. The expression levels in S. cerevisiae, however, are also low (25 μg/L) [50] and only A. oryzae produces swollenin in higher concentrations (50 mg/L) [53]. According to Saloheimo et al. [50], swollenin can disrupt the structure of cotton fiber or the cell wall of the algae Valonia macrophysa. Since swollenin shows a high sequence similarity to plant expansins [50], it may have a similar function and lead to the disruption of cellulosic networks within plant cell walls [20]. Thus, swollenin may have an important role in the enzymatic degradation of lignocellulose by T. reesei [54]. Up to now, however, there is no systematic and quantitative analysis of the effects of swollenin on cellulosic substrates and their hydrolysis.

First, this study presents an alternative way of producing recombinant swollenin in order to generate sufficient swollenin for industrial applications. Second, the main objective is to show how recombinant swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects their subsequent hydrolysis.

Results and discussion

Production and analysis of recombinant swollenin

Swollenin is a cellulase-related protein and consists of an N-terminal cellulose-binding domain connected by a linker region to an expansin-homologous domain [50]. The cDNA for swollenin from T. reesei was used as a template to clone a recombinant His-tagged swollenin (data not shown). After cloning, the recombinant swollenin was heterologously expressed by using the yeast K. lactis as expression host [55]. In addition, a non-transformed K. lactis wild type was cultivated as a reference. As shown by SDS-PAGE (Figure 1A), the supernatants of the wild type (lane 1) and the transformed clone (lane 2) showed only a few differences in protein secretion pattern. These differences could be explained by the influence of heterologous protein expression on the native secretome of K. lactis [56]. However, an intense protein band at about 80 kDa could be observed in the supernatant of the transformed clone which corresponds to the size of native swollenin from T. reesei (about 75 kDa, 49 kDa based on the primary sequence) [50]. Furthermore, this protein band was detected as a His-tagged protein by Western blot analysis (Figure 1B). In order to quantify the putative swollenin in the supernatant of K. lactis, the total protein concentration was determined and a densitometric analysis of the SDS-polyacrylamide gel (Figure 1A, lane 2) was conducted. The expression level of swollenin was approximately 20 to 30 mg/L, which is comparable with the results for other recombinant proteins expressed in K. lactis [55,56]. With respect to recombinant swollenin, lower or comparable expression levels were achieved by using S. cerevisiae (25 μg/L) [50] or A. oryzae (50 mg/L) [53] as expression hosts. Finally, this protein was purified by immobilized metal ion affinity chromatography. According to Figure 1A and 1B, the final fraction (lane 3) showed a protein band with high purity (around 75%).

thumbnailFigure 1. SDS-PAGE, Western blot and mass spectrometry of swollenin produced by Kluyveromyces lactis. (A) SDS-PAGE and (B) Western blot: (M) Molecular mass marker, (1) filtrated culture supernatant of K. lactis wild type, (2) filtrated culture supernatant of K. lactis expressing recombinant swollenin, (3) recombinant swollenin purified by immobilized metal affinity chromatography. 12% polyacrylamide gel, the same volume of the samples (15 μL) was loaded onto the particular slots; (C) Mass spectrometric results and primary sequence of recombinant swollenin. The protein band (around 80 kDa) was analyzed using a mass spectrometer and the Mascot database. The detected peptides are underlined and written in italic letters. The cellulose-binding domain [6-39], expansinA domain [243-401] and His-tag [476-483] are marked in grey. Potential areas for N-glycosylation and O-glycosylation are written in bold letters. The black arrows enclose the primary sequence of the native swollenin (CAB92328) without leader peptide.

To clearly identify the protein band at about 80 kDa (Figure 1A and 1B), its amino acid sequence was determined by using mass spectrometry [57] and the Mascot search engine [58]. Figure 1C shows the results of mass spectrometry and the expected amino acid sequence of the recombinant swollenin. As shown by a high Mascot score of 502 (Figure 1C), the protein at 80 kDa was clearly identified to be a variant of swollenin from T. reesei. Regarding the native swollenin sequence, a protein score of greater than 57 (homology threshold) indicates identity or extensive homology (P < 0.05). In addition, potential N- and O-glycosylation sites were detected by using the NetNGlyc 1.0 and NetOGlyc 3.1 servers [59] (Figure 1C). Here, it should be noted that the native swollenin contains almost no N-glycosylation [50]. Therefore, the difference between the calculated molecular mass of 49 kDa, based on the primary sequence of swollenin, and the observed molecular mass of 80 kDa (Figure 1A and 1B) may be explained by O-glycosylation and other post-translational modifications. Proofs are given as follows: (i) the linker region of cellulases or cellulase-related proteins is highly O-glycosylated [60]; (ii) swollenin contains potential O-glycosylation sites within the linker region (Figure 1C); (iii) no peptides of the linker region were identified by mass spectrometry, since glycosylation alters the mass/charge ratio of the peptides (Figure 1C).

Adsorption of swollenin

As the adsorption of proteins is a prerequisite for amorphogenesis [20], the adsorption isotherm of purified swollenin onto filter paper was determined. Preliminary adsorption kinetics showed that an incubation time of less than or equal 2 h was needed to reach equilibrium. Figure 2 illustrates that the adsorption of swollenin was a characteristic function of free swollenin concentration. After a sharp increase in adsorbed swollenin at low concentrations, a plateau was reached at higher concentrations (> 5 μmol/L). As denatured swollenin, boiled for 20 min, showed no adsorption (Figure 2), the adsorption was specific and required a functional protein structure. The Langmuir isotherm (Equation 1) provided a good fit (Figure 2, R2 = 0.91). Corresponding parameters - the maximum swollenin adsorption per g cellulose at equilibrium, Amax(swollenin); and the dissociation constant of swollenin, KD(swollenin) - are listed in the legend of Figure 2. Similar values of Amax and KD were found when analyzing the adsorption of purified cellulases onto filter paper ([61], CBH I: 0.17 μmol/g, 0.71 μmol/L; EG I: 0.17 μmol/g, 1.79 μmol/L). This may be attributed to the fact that swollenin exhibits a cellulose-binding domain with high homology to those of cellulases [62]. However, Amax was lower for swollenin than for single cellulases. According to Linder et al. [63], single amino acid substitutions of cellulose-binding domains can lead to adsorption differences. Furthermore, catalytic domains of cellulases are known to specifically adsorb onto cellulose independently of cellulose-binding domains [41]. In addition, the difference in Amax may be explained by the lower molecular mass of cellulases [64] and, therefore, a better access to internal binding sites as described for other proteins and materials [65,66].

thumbnailFigure 2. Adsorption isotherm of purified swollenin onto filter paper. The predicted Langmuir isotherm, according to Equation (1), is shown as a solid line (R2 = 0.91) and corresponding parameters (including standard deviations) are: Amax(swollenin) = 0.089 ± 0.006 μmol/g, KD(swollenin) = 0.707 ± 0.196 μmol/L. The initial swollenin concentration, added at the start of the incubation, is also shown for a better understanding of Figure 8; 20 g/L Whatman filter paper No.1 in 0.05 M sodium acetate buffer at pH 4.8, T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 2 h.

Pretreatment of filter paper with swollenin

To verify a potential effect of recombinant swollenin on cellulose, filter paper was pretreated with buffer, BSA or recombinant swollenin. Here, swollenin in an initial concentration of 20 mg per g cellulose was applied (> 80% saturation, Figure 2). It should be noted that all pretreatments were initiated with the same initial number (80) of filter paper agglomerates (initial diameter approximately 3 mm). As shown in Figure 3A, swollenin caused a deagglomeration of filter paper agglomerates (consisting of cellulose fibers). Since the cellulose fibers of a single agglomerate were separated by pretreatment with swollenin (Figure 3B), the number of bigger agglomerates obviously decreased (Figure 3A). This decrease in the number of bigger agglomerates (> 0.5 mm) was also quantified using image analysis (Figure 3A). During pretreatment, a shaken system with relatively low shear forces was applied. However, to exclude a sole mechanical effect on cellulose agglomerates due to shaking and to verify a specific effect of swollenin, filter paper was accordingly pretreated with buffer or the protein BSA (references). By contrast, the pretreatments with buffer or BSA showed much less deagglomeration (Figure 3A and 3B). Consequently, the deagglomeration was specifically caused by swollenin. As no reducing sugars were detected when using the sensitive p-hydroxy benzoic acid hydrazide assay after an incubation with swollenin for 48 h, the reduction in the number of large agglomerates was attributed to the aforementioned adsorption of swollenin onto filter paper (Figure 2) and the so-called non-hydrolytic deagglomeration [20].

thumbnailFigure 3. Photography and light microscopy of filter paper after pretreatment with swollenin. (A) Macroscopic pictures of pretreated filter paper in petri dishes and number of agglomerates. All pretreatments were initiated with the same initial number (80) of filter paper agglomerates (initial diameter approx. 3 mm). Number of agglomerates (> 0.5 mm) was measured by image analysis; (B) Light microscopy of pretreated filter paper. Eclipse E600 (Nikon); Pretreatment: 20 g/L cellulose in 0.05 M sodium acetate buffer at pH 4.8, 0.4 g/L BSA (approx. 6 μmol/L) or swollenin (approx. 5 μmol/L), T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 48 h.

As described by Saloheimo et al. [50], swollenin is also able to disrupt and swell cotton fibers. This phenomenon results from the dispersion of cellulose microfibrils and is called amorphogenesis [20,42]. In this current study, however, the swelling of cellulose fibers was not detected when Whatman filter paper No.1 - a different substrate - was used (Figure 3B). Reasons for this may be the different structure of filter paper than that of cotton used by Saloheimo et al. [50] or the low resolution of light microscopy. Therefore, scanning electron microscopy was applied to visualize the effect of swollenin on cellulose microfibrils (Figure 4A and 4B). After pretreatments with buffer or BSA, the microfibrils were not dispersed, thereby resulting in a smooth and uniform surface of the whole fiber. By contrast, swollenin caused the microfibrils to disperse, thereby creating a rough and amorphic surface on the cellulose fibers. Other authors found similar results via scanning electron microscopy after treating cellulose with cellulose-binding domains of cellulases [17,45,67]. However, the results of this current study indicate that recombinant swollenin from K. lactis may induce amorphogenesis of cellulosic substrates.

thumbnailFigure 4. Scanning electron microscopy of filter paper after pretreatment with swollenin. Pictures were taken at two different magnifications (A, B): see scale markers; Pretreatment: 20 g/L cellulose in 0.05 M sodium acetate buffer at pH 4.8, 0.4 g/L BSA (approx. 6 μmol/L) or swollenin (approx. 5 μmol/L), T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 48 h. Hitachi S-5500 (Hitachi).

The non-hydrolytic deagglomeration or amorphogenesis of cellulose was also described for single cellulose-binding domains of cellulases [17,45,67] and for other expansin-related proteins from B. subtilis [51], A. fumigatus [46] or B. adusta [52]. However, there is no detailed and quantitative analysis of different cellulosic substrates after pretreatment with non-hydrolyzing proteins, especially with regard to swollenin.

Effect of swollenin pretreatment on the physical properties of cellulosic substrates

To analyze in detail the effect of recombinant swollenin on cellulose, different cellulosic substrates were pretreated with buffer, BSA or recombinant swollenin. After pretreatment and removal of bound proteins, the physical properties of the pretreated cellulosic substrates were analyzed by laser diffraction, cellulase adsorption studies and crystallinity measurements.

As seen in Figure 5A-D, the cellulosic substrates showed broad and inhomogeneous particle-size distributions. Upon considering the same cellulosic substrate, the pretreatments with buffer or BSA led to no differences in particle-size distributions and in the resulting geometric mean particle sizes (Figure 5E-H). After swollenin pretreatment, however, the particle-size distributions shifted to lower values, and large cellulose agglomerates were predominantly deagglomerated to smaller particles. The bigger the initial particle size of the corresponding cellulosic substrate was, the greater the reduction in mean particle size by swollenin pretreatment was (filter paper > α-cellulose > Avicel). In the case of Sigmacell, all particle-size distributions were identical (Figure 5D), and the mean particle sizes did not change significantly due to pretreatment with swollenin (Figure 5H). This may be explained by the small initial particle size of Sigmacell and the absence of cellulose agglomerates.

thumbnailFigure 5. Particle size of cellulosic substrates after pretreatment with swollenin. (A, B, C, D) Volumetric particle-size distribution of pretreated cellulosic substrates: (A) Whatman filter paper No.1; (B) α-Cellulose; (C) Avicel PH101; (D) Sigmacell 101; (E, F, G, H) Geometric mean particle size of pretreated cellulosic substrates: (E) Whatman filter paper No.1; (F) α-Cellulose; (G) Avicel PH101; (H) Sigmacell 101. Errors are given as standard deviations; Pretreatment: 20 g/L cellulose in 0.05 M sodium acetate buffer at pH 4.8, 0.4 g/L BSA (approx. 6 μmol/L) or 0.4 g/L swollenin (approx. 5 μmol/L), T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 48 h. Particles (< 2 mm) were analyzed using the particle size analyzer LS13320 (Beckman Coulter).

Since cellulosic particle sizes (external surface areas) influence cellulose accessibility [15,19], they also affect the adsorption of cellulases [21,25] and they are an indication for the maximum cellulase adsorption [21]. To investigate if swollenin pretreatment actually affected cellulose accessibility, cellulase adsorption was analyzed after pretreatment with buffer or swollenin. According to various authors, the adsorption of total cellulase mixtures is not interpretable by simple Langmuir isotherms due to multicomponent cellulase adsorption [10,68]. Consequently, only the maximum cellulase adsorption per g cellulose Amax(cellulase) was determined by applying different incubation times and a total cellulase mixture at high concentrations. Since no further increase in cellulase adsorption was detected after 1.5 h (data not shown), adsorption equilibrium was verified. According to the literature, cellulase adsorption is rapid and adsorption equilibrium is usually reached within 0.5 to 1.5 h [41,64]. Saturation of all applied cellulosic substrates was reached when using the following cellulase/cellulose ratios: ≥ 100 mg/g (in the case of filter paper or α-cellulose), ≥ 150 mg/g (Avicel), ≥ 200 mg/g (Sigmacell). Table 1 summarizes the maximum cellulase adsorption per g cellulose (adsorption capacity) onto all applied cellulosic substrates after pretreatment with buffer or swollenin. In general, the determined Amax(cellulase) values are consistent with the adsorption data reported in the literature [10,41,69]. However, the pretreatment with swollenin caused a significant increase in maximum cellulase adsorption except for Sigmacell. The relative increase in cellulase adsorption between the pretreatment with swollenin and the pretreatment with buffer (filter paper > α-cellulose > Avicel > Sigmacell) showed a similar series as the relative reduction in mean particle size (filter paper > Avicel > α-cellulose > Sigmacell; Figure 5). Consequently, the increase in adsorption capacities of the swollenin-pretreated samples resulted primarily from the reduction in particle size and the corresponding increase in cellulose accessibility. However, in the case of α-cellulose, the increase in maximum cellulase adsorption was disproportionately higher. This can be explained by the effect of swollenin on other physical properties of cellulose, such as crystallinity, which may influence cellulase adsorption according to various authors [26,27]. Moreover, since all applied cellulosic substrates do not contain lignin, its influence on cellulose accessibility [31,70-72] could be neglected.

Table 1. Maximum cellulase adsorption onto cellulosic substrates after pretreatment with swollenin.

To additionally determine the influence of swollenin on the crystallinity of cellulose, the crystallinity index (CrI) of all pretreated cellulosic substrates was analyzed by X-ray diffraction (XRD) measurements (Figure 6A-D). A recrystallization of cellulose by incubation with aqueous solutions [73,74] was not observed, because the initial CrI of untreated substrates was higher than that of cellulosic substrates treated with buffer (data not shown). As illustrated by Figure 6, the pretreatment with buffer or BSA caused no differences in CrI; the CrI values were identical upon considering the same cellulosic substrate. By contrast, swollenin pretreatment specifically reduced the CrI as follows: filter paper (-10%), α-cellulose (-22%) and Avicel (-13%). However, in the case of Sigmacell, no effect of swollenin pretreatment on CrI was detected (Figure 6D) which can be explained by the low initial CrI and the amorphous structure of Sigmacell [75]. The strongest reduction in CrI was recorded in the case of α-cellulose (Figure 6B). Since α-cellulose is fibrous [64] and can consist of up to 22% xylan [76], it may be more sensitive to non-hydrolytic decrystallization [77]. However, the strong reduction in the CrI of α-cellulose explains the disproportionate increase in maximum cellulase adsorption onto α-cellulose (Table 1), since cellulase adsorption can increase with decreasing CrI [26]. As reported in the literature, similar reductions in crystallinity were found by using other non-hydrolyzing proteins: (i) the CrI of Avicel decreased by 9% to 12% after pretreatment with single cellulose-binding domains [17]; (ii) the CrI of filter paper decreased by 11.8% after pretreatment with Zea h, a protein from postharvest corn stover [48]. Up to now, however, the influence of swollenin on the CrI of different cellulosic substrates has not been quantified. Therefore, this study provides the first proof that swollenin does induce deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization) [20,42].

thumbnailFigure 6. Crystallinity index of cellulosic substrates after pretreatment with swollenin. (A) Whatman filter paper No.1; (B) α-Cellulose; (C) Avicel PH101; (D) Sigmacell 101. Errors are given as standard deviations; Pretreatment: 20 g/L cellulose in 0.05 M sodium acetate buffer at pH 4.8, 0.4 g/L BSA (approx. 6 μmol/L) or 0.4 g/L swollenin (approx. 5 μmol/L), T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 48 h. Powder XRD (STOE & Cie GmbH).

Hydrolysis of cellulosic substrates pretreated with swollenin

Upon using the same cellulase mixture, enzymatic hydrolysis rates are especially affected by the physical properties of the applied cellulose [10,14]. Since swollenin pretreatment affected cellulose particle size and maximum cellulase adsorption as well as crystallinity, the resulting effects on subsequent hydrolysis of all pretreated cellulosic substrates were analyzed by using rebuffered Celluclast®. As shown in Figure 7A-C, swollenin pretreatment significantly accelerated cellulose hydrolysis, and the saccharification after 72 h was increased. In contrast, the corresponding hydrolysis curves for buffer and BSA were almost the same by comparing the same cellulosic substrate. This is attributed to the fact that pretreatment with buffer and BSA had no significant effect on particle size (Figure 5), maximum cellulase adsorption (Table 1) or on CrI (Figure 6). In the case of filter paper (Figure 7A), the hydrolysis-accelerating effect of swollenin pretreatment was stronger than that for α-cellulose (Figure 7B) and Avicel (Figure 7C). This may be explained by the substantial decrease in mean particle size (Figure 5) and the strong increase in maximum cellulase adsorption (Table 1) for filter paper by swollenin pretreatment. Figure 7D shows that the hydrolysis curves of Sigmacell were almost the same, since swollenin pretreatment did not change the physical properties of Sigmacell.

thumbnailFigure 7. Hydrolysis of cellulosic substrates after pretreatment with swollenin. (A) Whatman filter paper No.1; (B) α-Cellulose; (C) Avicel PH101; (D) Sigmacell 101. Errors are given as standard deviations; Pretreatment: 20 g/L cellulose in 0.05 M sodium acetate buffer at pH 4.8, 0.4 g/L BSA (approx. 6 μmol/L) or 0.4 g/L swollenin (approx. 5 μmol/L), T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 48 h; Hydrolysis: 10 g/L pretreated cellulose in 0.05 M sodium acetate buffer at pH 4.8, 1 g/L rebuffered Celluclast®, T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm.

Furthermore, the relationship between the hydrolysis-accelerating effect and the amount of swollenin applied during pretreatment was investigated (Figure 8). Compared to the aforementioned experiments (Figure 7 and 8; 20 mg swollenin per g cellulose), less swollenin (5 mg per g cellulose) caused a less accelerated hydrolysis and the final concentration of reducing sugars was 0.85-fold smaller. However, when the amount of swollenin was decreased merely from 20 mg/g to 15 mg/g, the same reducing sugar concentration was detected after 72 h. Since maximum swollenin adsorption was reached at higher initial swollenin concentrations (> 60 mg/g for 95% saturation, Figure 2), these results show that even non-saturating swollenin concentrations of 15 to 20 mg/g are sufficient for a maximum hydrolysis-accelerating effect. This may be explained as follows: (i) not all accessible cellulose-binding sites must be occupied for a maximum hydrolysis-accelerating effect; (ii) swollenin reversibly binds to cellulose, thereby performing further deagglomeration and amorphogenesis at multiple cellulose-binding sites. The reversible adsorption onto cellulose-binding sites was already reported for cellulases containing cellulose-binding domains [78].

thumbnailFigure 8. Hydrolysis of filter paper after pretreatment with different swollenin concentrations. Errors are given as standard deviations; Pretreatment: 20 g/L Whatman filter paper No.1 in 0.05 M sodium acetate buffer at pH 4.8, different concentrations of swollenin, T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm, incubation time 48 h; Hydrolysis: 10 g/L pretreated cellulose in 0.05 M sodium acetate buffer at pH 4.8, 1 g/L rebuffered Celluclast®, T = 45°C, VL = 1 mL, n = 1000 rpm, d0 = 3 mm.

Finally, an empirical correlation for initial hydrolysis rates based on CrI and mean particle size was determined for the pretreated cellulosic substrates (Figure 9). In this investigation, the correlation showed that the swollenin-induced reduction in CrI and particle size resulted in high cellulose hydrolysis rates. Furthermore, Figure 9 illustrates the aforementioned differences in cellulose hydrolysis rates (Figure 7) for various substrates and pretreatments. In addition, it confirms the findings of other authors: (i) since smaller cellulose particle sizes lead to increased cellulase adsorption [25] (see previous section), hydrolysis rates increase with decreasing cellulose particle size [22-24]; (ii) since a reduction in CrI leads to increased cellulase adsorption and higher reactivity of adsorbed cellulases, hydrolysis rates correlate inversely with the CrI of the applied cellulose [24,26]. It should be noted that Figure 9 shows an empirical correlation for the conducted hydrolysis experiments. By applying other concentrations or types of cellulases and cellulosic substrates, different physical properties of the substrate (for example, porosity, [79]) might predominate.

thumbnailFigure 9. Influence of crystallinity and mean particle size on hydrolysis of cellulosic substrates. Data points for cellulosic substrates were obtained from Figure 5 (mean particle size), Figure 6 (crystallinity index) and Figure 7 (initial hydrolysis rate from 0 to 6 h). TableCurve 3D was used to determine an empirical surface fit (R2 = 0.93) based on a non-linear Gaussian cumulative function.


Recombinant swollenin was easily produced with the yeast K. lactis and purified by affinity chromatography. Additionally, the adsorption of swollenin onto cellulose was quantified for the first time, and its adsorption parameters were comparable to those of individual cellulases. The pretreatment with swollenin caused a significant decrease in particle size as well as in crystallinity of the cellulosic substrates, thereby substantially increasing maximum cellulase adsorption. Moreover, pretreatment of the cellulosic substrates with swollenin - even in non-saturating concentrations - significantly accelerated the hydrolysis. By correlating particle size and crystallinity with initial hydrolysis rates, it could be shown that high initial hydrolysis rates resulted from the swollenin-induced reduction in particle size and crystallinity. Consequently, this study shows an efficient means to produce recombinant swollenin with the robust yeast K. lactis. Moreover, this study shows that swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects cellulosic substrates and their hydrolysis.

A pretreatment of cellulosic substrates has been presented here which is simply based on the incubation of recombinant swollenin under mild conditions. Since the enzymatic hydrolysis of cellulose is a rate-limiting processing step in biorefineries [41], this pretreatment could significantly improve hydrolysis rates. To exclude possible side effects between swollenin and cellulase, swollenin pretreatment was performed as a separate step within this study. In future studies, swollenin should be directly added during cellulose hydrolysis. Since standard assays are missing for deagglomeration, amorphogenesis and for the comparison of different non-hydrolyzing proteins, this study may serve as an initial means to establish such assays.


Cellulosic substrates and cellulases

The cellulosic substrates Whatman filter paper No.1, α-cellulose, Avicel PH101 and Sigmacell 101 were purchased from Sigma-Aldrich (MO, USA). Physical properties and product information have been summarized by various authors [10,64]. Agglomerates of Whatman filter paper No.1 were prepared by using a hole-punch and quartering the resulting filter paper discs. The final filter paper agglomerates had an average diameter of approximately 3 mm. The cellulase preparation Celluclast® 1.5 L (Novozymes, Bagsværd, DK) - a filtrated culture supernatant of T. reesei [80] - was used for the hydrolysis of the pretreated cellulosic substrates. According to various authors, Celluclast® contains CBHs (Cel7A and Cel6A), EGs (for example, Cel7B and Cel5A) as well as β-glucosidases [81,82]. To remove salts, sugars and other interfering components, Celluclast® was previously rebuffered with an Äkta FPLC (GE Healthcare, Little Chalfont, UK). Celluclast® was loaded on Sephadex G-25 Fine (2.6 cm × 10 cm, GE Healthcare), and 0.05 M sodium acetate (pH 4.8) was used as a running buffer at 110 cm/h. Sephadex G-25 Fine exhibits an exclusion limit of 1-5 kDa which is comparable to the molecular mass cut-off of dialysis membranes for protein desalting. Since cellulases have a molecular mass of > 25 kDa [62,83,84], the mixture of cellulases was not changed during rebuffering. Chromatography was conducted at room temperature, and the automatically collected fractions were directly cooled at 4°C. To determine specific filter paper activities, different dilutions of Celluclast® and the rebuffered Celluclast® - applied for all hydrolysis experiments - were tested according to Ghose [85]. Here, the following specific filter paper activities (per g protein) were measured: 201 U/g (Celluclast®) and 279 U/g (rebuffered Celluclast®).

Genetic engineering for recombinant swollenin

The below-mentioned cloning procedure was designed for secreted protein expression according to the K. lactis Protein Expression Kit (New England Biolabs, MA, USA). The cDNA of the swollenin-coding region was synthesized by reverse-transcription PCR using mRNA isolated from T. reesei QM9414 (swo1 gene [GenBank: AJ245918], protein sequence [GenBank: CAB92328]) and reverse transcriptase (M-MLV, Promega, WI, USA) according to the manufacturer's protocol. Specific primers were applied to synthesize a cDNA starting from the 19th codon of the swollenin-coding region and, therefore, missing the secretion signal sequence of T. reesei [50]. By using the aforementioned primers, SalI and SpeI restriction sites were added upstream and downstream of the swollenin-coding region, respectively. The amplified cDNA was cloned into the pCR2.1-TOPO vector (Invitrogen, CA, USA) according to the manufacturer's protocol. After DNA sequencing and isolation of a correct clone, the DNA was excised from pCR2.1-TOPO and cloned into the pKLAC1-H vector using XhoI and SpeI restriction enzymes (New England Biolabs, MA, USA) according to the manufacturer's protocol. The pKLAC1-H is a modified version of the integrative pKLAC1 vector (New England Biolabs; [GenBank: AY968582]). The pKLAC1 - developed by Colussi and Taron [55] - exhibits the α-mating factor signal sequence and can be used for the expression and secretion of recombinant proteins in K. lactis [55]. The pKLAC1-H was constructed by including an additional SpeI restriction site directly followed by a His-tag coding sequence (6xHis) between the XhoI and AvrII restriction sites of pKLAC1. The DNA sequence of the final pKLAC1-H construct (containing the DNA coding for recombinant swollenin) is shown in Additional file 1. Moreover, the final amino acid sequence of recombinant swollenin (without the α-mating factor signal sequence) is given in Figure 1C.

Additional file 1. DNA sequence of pKLAC1-H construct (containing the DNA coding for recombinant swollenin).

Format: PDF Size: 49KB Download file

This file can be viewed with: Adobe Acrobat ReaderOpen Data

Expression and purification of recombinant swollenin

All below-mentioned transformation, selection and precultivation procedures - developed by Colussi and Taron [55] - were performed according to the manufacturer's protocol (K. lactis Protein Expression Kit, New England Biolabs). After cloning, K. lactis GG799 cells were transformed with pKLAC1-H (containing the DNA coding for recombinant swollenin), and transformed clones were selected (acetamide selection). One clone was precultivated in YPGal (yeast extract, peptone and galactose) medium, consisting of 20 g/L galactose, 20 g/L peptone and 10 g/L yeast extract - all media components were purchased from Carl Roth (Karlsruhe, Germany). After inoculation with 2.5 mL of the preculture, the main culture was cultivated in triplicates in 2 L Erlenmeyer flasks with YPGal medium under the following constant conditions: temperature T = 30°C, total filling volume VL = 250 mL, shaking diameter d0 = 50 mm, shaking frequency n = 200 rpm. Additionally, a non-transformed K. lactis wild type was cultivated as a reference. After incubation for 72 h, the main cultures were centrifuged (6000 g, 20 min, 4°C), and the pooled supernatants of the triplicates were treated with endoglycosidase Hf by using 20 U per μg protein for 12 h [50] according to the manufacturer's protocol without denaturation (New England Biolabs). Afterwards, the protein solution was concentrated 100-fold at 4°C using a Vivacell 100 ultrafiltration system with a molecular mass cut-off of 10 kDa (Sartorius Stedim Biotech, Göttingen, Germany). For affinity chromatography, the recombinant swollenin was previously rebuffered using Sephadex G-25 Fine (2.6 cm × 10 cm, GE Healthcare) at 110 cm/h with a running buffer (pH 7.4) consisting of 0.05 M sodium dihydrogen phosphate, 0.3 M sodium chloride and 0.01 M imidazole. The rebuffered sample was loaded on Ni Sepharose 6 Fast Flow (1.6 cm × 10 cm; GE Healthcare) at 120 cm/h. The bound swollenin was eluted with the aforementioned running buffer, containing 0.25 M imidazole.

SDS-PAGE and Western blot analysis

SDS-PAGE and Western blot analysis were applied to analyze the purity and to identify the recombinant swollenin. Novex 12% polyacrylamide Tris-Glycine gels (Invitrogen), and samples were prepared according to the manufacturer's protocol. The Plus Prestained Protein Ladder (Fermentas, Burlington, CA, USA) was used as a molecular mass marker. Finally, the proteins were stained with Coomassie Brilliant Blue and analyzed densitometrically [86] using the scanner Perfection V700 (Epson, Suwa, Japan). The molecular mass and purity of swollenin was determined using the software TotalLab TL100 (Nonlinear Dynamics, Newcastle, UK). For Western blot analysis, gels were blotted onto a nitrocellulose membrane (Whatman, Springfield Mill, UK) according to the manufacturer's protocol (Invitrogen). The membranes were blocked at room temperature with 50 g/L skim milk dissolved in phosphate buffered saline containing 0.5 g/L Tween-20 (PBST) for 30 min. To detect the recombinant swollenin, the membranes were incubated at room temperature for 1.5 h with a rabbit polyclonal antibody against His-tag (Dianova, Hamburg, Germany) diluted 1:10,000 in PBST. After the membrane was washed thrice with PBST, it was incubated with alkaline phosphatase conjugated goat anti-rabbit IgG (Dianova) diluted 1:5,000 in PBST at room temperature for 1 h. Finally, bound antibodies were visualized by incubating the membrane for 5 min with nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) diluted 1:100 in phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl2, pH 9.6).

Measurement of protein concentration

Protein concentrations were analyzed with the bicinchoninic acid assay [87] using the BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) and BSA as a standard. Depending on the protein concentration of the samples, the standard procedure (working range: 0.02 to 2 g/L) or the enhanced procedure (working range: 0.005 to 0.25 g/L) was performed according to the manufacturer's protocol. The absorbance at 562 nm was measured with a Synergy 4 microtiter plate reader (BioTek Instruments, VT, USA). To quantify swollenin in the culture supernatant of K. lactis, the bicinchoninic acid assay was combined with the aforementioned SDS-PAGE (including densitometric analysis). Here, total protein concentrations were determined and multiplied with the ratio of swollenin to total protein (purity).

Mass spectrometry and glycosylation analysis

Mass spectrometry was applied to identify the expressed and purified recombinant swollenin. The protein band (approximately 80 kDa) was excised from the SDS-polyacrylamide gel, washed in water, reduced with dithiothreitol, alkylated with iodoacetamide, and digested with trypsin [88]. Peptide analysis was carried out using a nanoHPLC (Dionex, Germering, Germany) coupled to an ESI-QUAD-TOF-2 mass spectrometer (Waters Micromass, Eschborn, Germany) as previously described [89]. The Mascot algorithm (Matrix Science, London, UK) was used to correlate the mass spectrometry data with amino acid sequences in the Swissprot database. Thereby, the sequences of the analyzed peptides could be identified, and, ultimately, protein matches could be determined. The Mascot score is derived from the ions scores of the detected peptides matching the peptides in the database and reflects a non-probabilistic basis for ranking protein hits [90]. By using this database, the peptide mass tolerance was set at ± 0.3 Da. Additionally, the following modifications to the amino acids in brackets were allowed: carbamidomethyl (C), carboxymethyl (C), oxidation (M), propionamide (C). Moreover, potential areas for N-glycosylation and O-glycosylation were identified by using the NetNGlyc 1.0 and NetOGlyc 3.1 servers webcite[59].

Adsorption experiments

Adsorption experiments were performed in 0.05 M sodium acetate buffer (pH 4.8) using 20 g/L untreated filter paper and various concentrations (0.05 to 1.25 g/L) of purified swollenin. Solutions with filter paper and solutions with swollenin were preincubated separately at 45°C for 10 min, and experiments were started by mixing both solutions. The final mixtures were incubated in 2 mL Eppendorf tubes on a thermomixer MHR23 (simultaneous shaking and temperature control; HLC Biotech, Bovenden, Germany) under the following constant conditions for 2 h: T = 45°C, VL = 1 mL, d0 = 3 mm, n = 1000 rpm. The shaking frequency was chosen to ensure the complete suspension of cellulose particles [64,91]. Three different blanks were incubated similarly: (i) without swollenin, (ii) without filter paper, or (iii) without filter paper and without swollenin. The incubation was stopped by centrifugation (8000 g, 1 min), and the supernatants were immediately analyzed for unbound swollenin by using the bicinchoninic acid assay. The adsorbed swollenin concentration was calculated as the difference between initial (blanks) and unbound swollenin concentration. Adsorption isotherm parameters were determined using the Langmuir isotherm [92]:

<a onClick="popup('','MathML',630,470);return false;" target="_blank" href="">View MathML</a>


in which A denotes the amount of adsorbed protein per g cellulose (μmol/g), Amax, the maximum protein adsorption per g cellulose at equilibrium (μmol/g), E, the free protein concentration (μmol/L), and KD, the dissociation constant (μmol/L). Within the literature [61], the association constant KA (L/μmol) is sometimes used instead of the dissociation constant KD.

To analyze the effect of swollenin pretreatment (see below) on cellulase adsorption [44,93], the maximum cellulase adsorption was also determined by incubating various concentrations (0.7 to 2.5 g/L) of rebuffered Celluclast® with 10 g/L pretreated cellulosic substrates. Here, all incubations were conducted under the aforementioned conditions for 1 h, 1.5 h and 2 h.

Pretreatment with swollenin

Pretreatment experiments were performed with 20 g/L cellulosic substrates and various concentrations of swollenin in 0.05 M sodium acetate buffer (pH 4.8). The mixtures were incubated as triplicates in 2 mL Eppendorf tubes on a thermomixer under the following constant conditions: T = 45°C, VL = 1 mL, d0 = 3 mm, n = 1000 rpm. To exclude a sole mechanical effect on cellulosic substrates due to shaking and to verify a specific effect of swollenin, blanks without swollenin (buffer) or with 0.4 g/L BSA instead of swollenin were incubated similarly. To detect a possible hydrolytic activity of recombinant swollenin, the sensitive p-hydroxy benzoic acid hydrazide assay [94] was applied by using glucose as a standard. After incubation for 48 h, the supernatants of the pretreatment solution were analyzed and the absorbancies were measured at 410 nm in a Synergy 4 microtiter plate reader. Subsequently, all cellulosic samples were washed to remove adsorbed proteins. Therefore, the mixtures were centrifuged (14,000 × g, 10 min, 4°C), and the cellulosic pellets were washed four times with 800 μL 0.05 M citrate buffer (pH 10) [95], and once with 800 μL distilled water. Finally, the triplicates were pooled. According to Zhu et al. [95], citrate buffer (pH 10) is an appropriate washing solution, and a single washing step with 0.05 M citrate buffer (pH 10) leads to a desorption efficiency of 61% in case of fungal cellulases and Avicel. Since no acids or bases are formed during the washing procedure, the weak buffer capacity of citrate buffer at pH 10 can be neglected. In this study, the washing procedure was conducted four times to ensure a high desorption of swollenin. The measurements of protein concentration in the washing supernatants - by applying the aforementioned bicinchoninic acid assay (working range starting from 0.005 g/L) - showed that swollenin desorbed almost completely. Already after three washing steps, a total swollenin desorption efficiency of > 90% was achieved.

Photography and microscopy

Photography and microscopy were applied to visualize the effect of swollenin pretreatment on filter paper. After pretreatment with buffer, BSA or swollenin, the different filter paper solutions were transferred into petri dishes, the particles were evenly distributed and images were taken with an Exilim EX-FH100 camera (Casio, Tokyo, Japan). Afterwards, the number of filter paper agglomerates (> 0.5 mm) was determined by image analysis using the software UTHSCSA ImageTool 3.0 (freeware) and a ruler as a reference. Light microscopic pictures were taken with an Eclipse E600 (Nikon, Tokyo, Japan). Additionally, scanning electron microscopy was performed using a Hitachi S-5500 (Hitachi, Tokyo, Japan) and a field emission of 5 kV. All washed filter paper samples were covered with a layer of carbon (3 nm) and, subsequently, with a layer of PtPd (3 nm, 80% to 20%). The images were taken by using secondary electrons.

Laser diffraction and X-ray diffraction

The particle-size distributions of all pretreated cellulosic substrates were measured by laser diffraction [96] using a LS13320 (Beckman Coulter, CA, USA). In the case of filter paper, particles with an average diameter of greater than 0.75 mm were manually removed before laser diffraction to exclude a disturbance of measurement signals. The geometric mean particle size was calculated using the software LS 5.01 (Beckman Coulter). Moreover, the CrI was determined by powder XRD. XRD patterns were obtained using a STOE STADI P transmission diffractometer (STOE & Cie GmbH, Darmstadt, Germany) in Debye-Scherrer geometry (Curadiation, λ = 1.54060 Å) with a primary monochromator and a position-sensitive detector. Thereby, XRD patterns were collected with a diffraction angle 2θ from 10° to 30° (increments of 0.01°) and a counting time of 6 s per increment. The sample was adhered to a polyester foil (biaxially-oriented polyethylene terephthalate) by using a dilute solution of glue. After drying the sample in open-air, the sample was covered with a second polyester foil. This set was then fixed in a sample holder. To improve statistics and level out sample orientation effects, the sample was rotated at around 2 Hz during XRD measurement. The CrI was calculated using the peak height method [28] and the corresponding equation:

<a onClick="popup('','MathML',630,470);return false;" target="_blank" href="">View MathML</a>


where I002 is the maximum intensity of the crystalline plane (002) reflection (2θ = 22.5°) and IAM is the intensity of the scattering for the amorphous component at about 18° in cellulose-I [97]. Here, it should be noted that there are several methods for calculating CrI from XRD data and these methods can provide significantly different results [28,70]. Although the applied peak height method produces CrI values that are higher than those of other methods, it is still the most commonly used method and ranks CrI values in the same order as the other methods [28].

Hydrolysis experiments and dinitrosalicylic acid assay

Hydrolysis experiments with 10 g/L pretreated cellulosic substrate and 1 g/L rebuffered Celluclast® were conducted in 0.05 M sodium acetate buffer (pH 4.8). The mixtures were incubated as triplicates in 2 mL Eppendorf tubes on a thermomixer under the following constant conditions: T = 45°C, total filling VL = 1 mL, d0 = 3 mm, n = 1000 rpm. In general, attention has to be paid to cellulase inactivation, which would reduce the final yield of cellulose hydrolysis [98]. In this current study, however, a shaken system with relatively low shear forces was applied. According to Engel et al. [99], rebuffered Celluclast® is stable under the applied incubation conditions, so that cellulase inactivation could be neglected. The shaking frequency was chosen to ensure the complete suspension of cellulose particles [64,91]. Thus, mass transfer limitations are excluded, and the whole cellulose particle surface becomes accessible to the cellulases, thereby optimizing cellulase adsorption and activity [64]. Three different blanks were incubated similarly: (i) without cellulase, (ii) without substrate, or (iii) without substrate and without cellulase. The dinitrosalicylic acid assay [100] was applied to quantify the reducing sugars released during hydrolysis by using glucose as a standard. After defined time intervals, samples were taken, and the hydrolysis was stopped (10 min, 100°C). According to Wood and Bhat [101], low reducing sugar concentrations were quantified by adding 1.25 g/L glucose to the samples. The absorbancies were measured at 540 nm in a Synergy 4 microtiter plate reader. Since the dinitrosalicylic acid assay exhibits a lower sensitivity towards cellobiose than glucose, reducing sugar concentrations may be underestimated when glucose is used as a standard and β-glucosidase is not in excess [102]. However, under the applied hydrolysis conditions, cellobiose did not accumulate (the highest cellobiose to glucose ratio was measured in the case of Sigmacell after 10 h at 0.12) and, therefore, this underestimation was minimal and the addition of β-glucosidase was not needed. Initial hydrolysis rates (g/(L*h)) were calculated by applying a linear fit to the reducing sugar concentration data from 0 to 6 h.

Computational methods

Parameters (including standard deviations) of the adsorption model were calculated by nonlinear, least squares regression analysis using MATLAB R2010 (The MathWorks, Natick, USA). TableCurve 3D 4.0 (Systat Software, San Jose, CA, USA) was used to empirically correlate CrI and mean particle size with initial hydrolysis rates via the non-linear Gaussian cumulative function:

<a onClick="popup('','MathML',630,470);return false;" target="_blank" href="">View MathML</a>


in which a, b, c, d, e, f and g denote the various fitting parameters of the non-linear Gaussian cumulative function (-).

List of abbreviations

a: non-linear Gaussian cumulative function parameter (-); A: adsorbed protein per g cellulose (μmol/g); Amax: maximum protein adsorption per g cellulose at equilibrium (μmol/g or mg/g); b: non-linear Gaussian cumulative function parameter (-); BSA: bovine serum albumin; c: non-linear Gaussian cumulative function parameter (-); CBH: cellobiohydrolase; CrI: crystallinity index (%); d: non-linear Gaussian cumulative function parameter (-); d0: shaking diameter (mm); e: non-linear Gaussian cumulative function parameter (-); E: free protein concentration (μmol/L); EG: endoglucanase; f: non-linear Gaussian cumulative function parameter (-); g: non-linear Gaussian cumulative function parameter (-); I002: maximum intensity of the crystalline plane (002) reflection (1/s); IAM: XRD scattering for the amorphous component at 18o in cellulose-I (1/s); KA: association constant (L/μmol) KD: dissociation constant (μmol/L); λ: wavelength (Å); n: shaking frequency (rpm); NBT/BCIP: nitro blue tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate; P: probability for significant scores (protein matching) (-); PBST: phosphate buffered saline containing Tween-20; R2: coefficient of determination (-); T: temperature (°C); θ: diffraction angle (°); VL: filling volume (mL); XRD: X-ray diffraction; YPGal: medium containing yeast extract, peptone and galactose.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

GJ designed and carried out experiments, analyzed results and wrote the manuscript. MG carried out the cloning of swollenin. FD carried out the pretreatment with swollenin (incl. analysis) and subsequent hydrolysis. RR carried out the measurements of CrI. HB carried out scanning electron microscopy. UC and AS reviewed the manuscript. RF and JB coordinated the study and reviewed the manuscript. All authors read and approved the final manuscript.


This work was performed as part of the Cluster of Excellence "Tailor-Made Fuels from Biomass", which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities.


  1. Huber GW, Iborra S, Corma A: Synthesis of transportation fuels from biomass: chemistry, catalysts, and engineering.

    Chem Rev 2006, 106:4044-4098. PubMed Abstract | Publisher Full Text OpenURL

  2. Fukuda H, Kondo A, Tamalampudi S: Bioenergy: Sustainable fuels from biomass by yeast and fungal whole-cell biocatalysts.

    Biochem Eng J 2009, 44:2-12. Publisher Full Text OpenURL

  3. Okano K, Tanaka T, Ogino C, Fukuda H, Kondo A: Biotechnological production of enantiomeric pure lactic acid from renewable resources: recent achievements, perspectives, and limits.

    Appl Microbiol Biotechnol 2010, 85:413-423. PubMed Abstract | Publisher Full Text OpenURL

  4. Pristavka AA, Salovarova VP, Zacchi G, Berezin IV, Rabinovich ML: Enzyme recovery in high-solids enzymatic hydrolysis of steam-pretreated willow: Requirements for the enzyme composition.

    Appl Biochem Microbiol 2000, 36:237-244. Publisher Full Text OpenURL

  5. Klosowski G, Mikulski D, Czuprynski B, Kotarska K: Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase, proteolytic enzymes and enzymes degrading non-starch polysaccharides.

    J Biosci Bioeng 2010, 109:466-471. PubMed Abstract | Publisher Full Text OpenURL

  6. Quiroz-Castaneda RE, Perez-Mejia N, Martinez-Anaya C, Acosta-Urdapilleta L, Folch-Mallol J: Evaluation of different lignocellulosic substrates for the production of cellulases and xylanases by the basidiomycete fungi Bjerkandera adusta and Pycnoporus sanguineus.

    Biodegradation 2011, 22:565-572. PubMed Abstract | Publisher Full Text OpenURL

  7. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD: Biomass recalcitrance: Engineering plants and enzymes for biofuels production.

    Science 2007, 315:804-807. PubMed Abstract | Publisher Full Text OpenURL

  8. Quiroz-Castaneda RE, Balcazar-Lopez E, Dantan-Gonzalez E, Martinez A, Folch-Mallol J, Martinez-Anaya C: Characterization of cellulolytic activities of Bjerkandera adusta and Pycnoporus sanguineus on solid wheat straw medium.

    Electron J Biotechnol 2009, 12:1-8. OpenURL

  9. Schröter K, Flaschel E, Puhler A, Becker A: Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production.

    Appl Microbiol Biotechnol 2001, 55:727-733. PubMed Abstract | Publisher Full Text OpenURL

  10. Zhang YH, Lynd LR: Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems.

    Biotechnol Bioeng 2004, 88:797-824. PubMed Abstract | Publisher Full Text OpenURL

  11. Desai SG, Converse AO: Substrate reactivity as a function of the extent of reaction in the enzymatic hydrolysis of lignocellulose.

    Biotechnol Bioeng 1997, 56:650-655. PubMed Abstract | Publisher Full Text OpenURL

  12. Wang LS, Zhang YZ, Gao PJ, Shi DX, Liu HW, Gao HJ: Changes in the structural properties and rate of hydrolysis of cotton fibers during extended enzymatic hydrolysis.

    Biotechnol Bioeng 2006, 93:443-456. PubMed Abstract | Publisher Full Text OpenURL

  13. Zhang S, Wolfgang DE, Wilson DB: Substrate heterogeneity causes the nonlinear kinetics of insoluble cellulose hydrolysis.

    Biotechnol Bioeng 1999, 66:35-41. PubMed Abstract | Publisher Full Text OpenURL

  14. Kumar R, Wyman CE: Does change in accessibility with conversion depend on both the substrate and pretreatment technology?

    Bioresour Technol 2009, 100:4193-4202. PubMed Abstract | Publisher Full Text OpenURL

  15. Chandra RP, Bura R, Mabee WE, Berlin A, Pan X, Saddler JN: Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics?

    Adv Biochem Engin/Biotechnol 2007, 108:67-93. Publisher Full Text OpenURL

  16. Alvira P, Tomas-Pejo E, Ballesteros M, Negro MJ: Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review.

    Bioresour Technol 2010, 101:4851-4861. PubMed Abstract | Publisher Full Text OpenURL

  17. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS: Biological pretreatment of cellulose: Enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases.

    Bioresour Technol 2011, 102:2910-2915. PubMed Abstract | Publisher Full Text OpenURL

  18. Mansfield SD, Mooney C, Saddler JN: Substrate and enzyme characteristics that limit cellulose hydrolysis.

    Biotechnol Progr 1999, 15:804-816. Publisher Full Text OpenURL

  19. Chandra R, Ewanick S, Hsieh C, Saddler JN: The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, part 1: A modified Simons' staining technique.

    Biotechnol Progr 2008, 24:1178-1185. Publisher Full Text OpenURL

  20. Arantes V, Saddler J: Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis.

    Biotechnol Biofuels 2010, 3:1-11. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  21. Arantes V, Saddler J: Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates.

    Biotechnol Biofuels 2011, 4:3. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  22. Dasari RK, Berson RE: The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries.

    Appl Biochem Biotechnol 2007, 137:289-299. PubMed Abstract | Publisher Full Text OpenURL

  23. Yeh AI, Huang YC, Chen SH: Effect of particle size on the rate of enzymatic hydrolysis of cellulose.

    Carbohydr Polym 2010, 79:192-199. Publisher Full Text OpenURL

  24. Jäger G, Wulfhorst H, Zeithammel EU, Elinidou E, Spiess AC, Büchs J: Screening of cellulases for biofuel production: Online monitoring of the enzymatic hydrolysis of insoluble cellulose using high-throughput scattered light detection.

    Biotechnol J 2011, 6:74-85. PubMed Abstract | Publisher Full Text OpenURL

  25. Kim DW, Yang JH, Jeong YK: Adsorption of cellulase from Trichoderma viride on microcrystalline cellulose.

    Appl Microbiol Biotechnol 1988, 28:148-154. Publisher Full Text OpenURL

  26. Hall M, Bansal P, Lee JH, Realff MJ, Bommarius AS: Cellulose crystallinity - a key predictor of the enzymatic hydrolysis rate.

    FEBS J 2010, 277:1571-1582. PubMed Abstract | Publisher Full Text OpenURL

  27. Ooshima H, Sakata M, Harano Y: Adsorption of cellulase from Trichoderma viride on cellulose.

    Biotechnol Bioeng 1983, 25:3103-3114. PubMed Abstract | Publisher Full Text OpenURL

  28. Park S, Baker JO, Himmel ME, Parilla PA, Johnson DK: Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance.

    Biotechnol Biofuels 2010, 3:10. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  29. Ramos LP, Nazhad MM, Saddler JN: Effect of enzymatic-hydrolysis on the morphology and fine-structure of pretreated cellulosic residues.

    Enzyme Microb Technol 1993, 15:821-831. Publisher Full Text OpenURL

  30. Jeoh T, Ishizawa CI, Davis MF, Himmel ME, Adney WS, Johnson DK: Cellulase digestibility of pretreated biomass is limited by cellulose accessibility.

    Biotechnol Bioeng 2007, 98:112-122. PubMed Abstract | Publisher Full Text OpenURL

  31. Rollin JA, Zhu Z, Sathitsuksanoh N, Zhang YHP: Increasing cellulose accessibility is more important than removing lignin: A comparison of cellulose solvent-based lignocellulose fractionation and soaking in aqueous ammonia.

    Biotechnol Bioeng 2011, 108:22-30. PubMed Abstract | Publisher Full Text OpenURL

  32. Wyman CE: What is (and is not) vital to advancing cellulosic ethanol.

    Trends Biotechnol 2007, 25:153-157. PubMed Abstract | Publisher Full Text OpenURL

  33. Kumar P, Barrett DM, Delwiche MJ, Stroeve P: Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production.

    Ind Eng Chem Res 2009, 48:3713-3729. Publisher Full Text OpenURL

  34. Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M: Features of promising technologies for pretreatment of lignocellulosic biomass.

    Bioresour Technol 2005, 96:673-686. PubMed Abstract | Publisher Full Text OpenURL

  35. Galbe M, Zacchi G: Pretreatment of lignocellulosic materials for efficient bioethanol production.

    Adv Biochem Eng/Biotechnol 2007, 108:41-65. Publisher Full Text OpenURL

  36. Selig MJ, Tucker MP, Sykes RW, Reichel KL, Brunecky R, Himmel ME, Davis MF, Decker SR: Lignocellulose recalcitrance screening by integrated high-throughput hydrothermal pretreatment and enzymatic saccharification.

    Ind Biotechnol 2010, 6:104-111. Publisher Full Text OpenURL

  37. Decker S, Brunecky R, Tucker M, Himmel M, Selig M: High-throughput screening techniques for biomass conversion.

    Bioenerg Res 2009, 2:179-192. Publisher Full Text OpenURL

  38. Baker J, King M, Adney W, Decker S, Vinzant T, Lantz S, Nieves R, Thomas S, Li L-C, Cosgrove D, Himmel M: Investigation of the cell-wall loosening protein expansin as a possible additive in the enzymatic saccharification of lignocellulosic biomass.

    Appl Biochem Biotechnol 2000, 84-86:217-223. PubMed Abstract | Publisher Full Text OpenURL

  39. Zhao H, Kwak JH, Conrad Zhang Z, Brown HM, Arey BW, Holladay JE: Studying cellulose fiber structure by SEM, XRD, NMR and acid hydrolysis.

    Carbohydr Polym 2007, 68:235-241. Publisher Full Text OpenURL

  40. Paiva AT, Sequeira SM, Evtuguin DV, Eds: Nanoscale structure of cellulosic materials: challenges and opportunities for AFM.


  41. Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS: Microbial cellulose utilization: fundamentals and biotechnology.

    Microbiol Mol Biol Rev 2002, 66:506-577. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  42. Coughlan MP: The properties of fungal and bacterial cellulases with comment on their production and application.

    Biotechnol Genet Eng Rev 1985, 3:39-109. Publisher Full Text OpenURL

  43. Klyosov AA: Trends in biochemistry and enzymology of cellulose degradation.

    Biochemistry 1990, 29:10577-10585. PubMed Abstract | Publisher Full Text OpenURL

  44. Rabinovich ML, Vanviet N, Klyosov AA: Adsorption of cellulolytic enzymes on cellulose and kinetics of the action of adsorbed enzymes. Two types of interaction of the enzymes with an insoluble substrate.

    Biochemistry Moscow 1982, 47:369-377. OpenURL

  45. Din N, Gilkes NR, Tekant B, Miller RC, Warren RAJ, Kilburn DG: Non-hydrolytic disruption of cellulose fibres by the binding domain of a bacterial cellulase.

    Nat Biotechnol 1991, 9:1096-1099. Publisher Full Text OpenURL

  46. Chen XA, Ishida N, Todaka N, Nakamura R, Maruyama JI, Takahashi H, Kitamoto K: Promotion of efficient saccharification of crystalline cellulose by Aspergillus fumigatus Swo1.

    Appl Environ Microbiol 2010, 76:2556-2561. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  47. Cosgrove DJ: Loosening of plant cell walls by expansins.

    Nature 2000, 407:321-326. PubMed Abstract | Publisher Full Text OpenURL

  48. Han YJ, Chen HZ: Synergism between corn stover protein and cellulase.

    Enzyme Microb Technol 2007, 41:638-645. Publisher Full Text OpenURL

  49. Wei W, Yang C, Luo J, Lu C, Wu Y, Yuan S: Synergism between cucumber [alpha]-expansin, fungal endoglucanase and pectin lyase.

    J Plant Physiol 2010, 167:1204-1210. PubMed Abstract | Publisher Full Text OpenURL

  50. Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssonen E, Bhatia A, Ward M, Penttila M: Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.

    Eur J Biochem 2002, 269:4202-4211. PubMed Abstract | Publisher Full Text OpenURL

  51. Kim ES, Lee HJ, Bang WG, Choi IG, Kim KH: Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose.

    Biotechnol Bioeng 2009, 102:1342-1353. PubMed Abstract | Publisher Full Text OpenURL

  52. Quiroz-Castaneda R, Martinez-Anaya C, Cuervo-Soto L, Segovia L, Folch-Mallol J: Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.

    Microb Cell Fact 2011, 10:8. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  53. Wang M, Cai J, Huang L, Lv Z, Zhang Y, Xu Z: High-level expression and efficient purification of bioactive swollenin in Aspergillus oryzae.

    Appl Biochem Biotechnol 2010, 162:2027-2036. PubMed Abstract | Publisher Full Text OpenURL

  54. Banerjee G, Car S, Scott-Craig JS, Borrusch MS, Aslam N, Walton JD: Synthetic enzyme mixtures for biomass deconstruction: Production and optimization of a core set.

    Biotechnol Bioeng 2010, 106:707-720. PubMed Abstract | Publisher Full Text OpenURL

  55. Colussi PA, Taron CH: Kluyveromyces lactis LAC4 promoter variants that lack function in bacteria but retain full function in K. lactis.

    Appl Environ Microbiol 2005, 71:7092-7098. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  56. Lodi T, Neglia B, Donnini C: Secretion of human serum albumin by Kluyveromyces lactis overexpressing KlPDI1 and KlERO1.

    Appl Environ Microbiol 2005, 71:4359-4363. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  57. Schuchardt S, Sickmann A: Protein identification using mass spectrometry: a method overview.

    EXS 2007, 97:141-170. PubMed Abstract OpenURL

  58. Grosse-Coosmann F, Boehm AM, Sickmann A: Efficient analysis and extraction of MS/MS result data from Mascot result files.

    BMC Bioinf 2005, 6:290. BioMed Central Full Text OpenURL

  59. Julenius K, Molgaard A, Gupta R, Brunak S: Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites.

    Glycobiology 2005, 15:153-164. PubMed Abstract | Publisher Full Text OpenURL

  60. Stals I, Sandra K, Geysens S, Contreras R, Van Beeumen J, Claeyssens M: Factors influencing glycosylation of Trichoderma reesei cellulases. I: Postsecretorial changes of the O- and N-glycosylation pattern of Cel7A.

    Glycobiology 2004, 14:713-724. PubMed Abstract | Publisher Full Text OpenURL

  61. Nidetzky B, Steiner W, Claeyssens M: Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis.

    Biochem J 1994, 303:817-823. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  62. Ouyang J, Yan M, Kong D, Xu L: A complete protein pattern of cellulase and hemicellulase genes in the filamentous fungus Trichoderma reesei.

    Biotechnol J 2006, 1:1266-1274. PubMed Abstract | Publisher Full Text OpenURL

  63. Linder M, Lindeberg G, Reinikainen T, Teeri TT, Pettersson G: The difference in affinity between 2 fungal cellulose-binding domains is dominated by a single amino-acid substitution.

    FEBS Lett 1995, 372:96-98. PubMed Abstract | Publisher Full Text OpenURL

  64. Jäger G, Wu Z, Garschhammer K, Engel P, Klement T, Rinaldi R, Spiess A, Büchs J: Practical screening of purified cellobiohydrolases and endoglucanases with alpha-cellulose and specification of hydrodynamics.

    Biotechnol Biofuels 2010, 3:1-12. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  65. Hunter AK, Carta G: Protein adsorption on novel acrylamido-based polymeric ion-exchangers. IV. Effects of protein size on adsorption capacity and rate.

    J Chromatogr A 2002, 971:105-116. PubMed Abstract | Publisher Full Text OpenURL

  66. Oberholzer MR, Lenhoff AM: Protein adsorption isotherms through colloidal energetics.

    Langmuir 1999, 15:3905-3914. Publisher Full Text OpenURL

  67. Gao P-J, Chen G-J, Wang T-H, Zhang Y-S, Liu J: Non-hydrolytic disruption of crystalline structure of cellulose by cellulose binding domain and linker sequence of cellobiohydrolase I from Penicillium janthinellum.

    Acta Biochim Biophys Sin 2001, 33:13-18. PubMed Abstract | Publisher Full Text OpenURL

  68. Beldman G, Voragen AGJ, Rombouts FM, Searlevanleeuwen MF, Pilnik W: Adsorption and kinetic behavior of purified endoglucanases and exoglucanases from Trichoderma viride.

    Biotechnol Bioeng 1987, 30:251-257. PubMed Abstract | Publisher Full Text OpenURL

  69. Hong J, Ye XH, Zhang YHP: Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications.

    Langmuir 2007, 23:12535-12540. PubMed Abstract | Publisher Full Text OpenURL

  70. Sathitsuksanoh N, Zhu Z, Wi S, Percival Zhang YH: Cellulose solvent-based biomass pretreatment breaks highly ordered hydrogen bonds in cellulose fibers of switchgrass.

    Biotechnol Bioeng 2011, 108:521-529. PubMed Abstract | Publisher Full Text OpenURL

  71. Selig MJ, Vinzant TB, Himmel ME, Decker SR: The effect of lignin removal by alkaline peroxide pretreatment on the susceptibility of corn stover to purified cellulolytic and xylanolytic enzymes.

    Appl Biochem Biotechnol 2009, 155:397-406. PubMed Abstract | Publisher Full Text OpenURL

  72. Selig MJ, Viamajala S, Decker SR, Tucker MP, Himmel ME, Vinzant TB: Deposition of lignin droplets produced during dilute acid pretreatment of maize stems retards enzymatic hydrolysis of cellulose.

    Biotechnol Progr 2007, 23:1333-1339. Publisher Full Text OpenURL

  73. Wormald P, Wickholm K, Larsson PT, Iversen T: Conversions between ordered and disordered cellulose. Effects of mechanical treatment followed by cyclic wetting and drying.

    Cellulose 1996, 3:141-152. Publisher Full Text OpenURL

  74. Ouajai S, Shanks RA: Solvent and enzyme induced recrystallization of mechanically degraded hemp cellulose.

    Cellulose 2006, 13:31-44. Publisher Full Text OpenURL

  75. Dourado F, Mota M, Pala H, Gama FM: Effect of cellulase adsorption on the surface and interfacial properties of cellulose.

    Cellulose 1999, 6:265-282. Publisher Full Text OpenURL

  76. Gupta R, Lee YY: Mechanism of cellulase reaction on pure cellulosic substrates.

    Biotechnol Bioeng 2009, 102:1570-1581. PubMed Abstract | Publisher Full Text OpenURL

  77. Whitney SEC, Gidley MJ, McQueen-Mason SJ: Probing expansin action using cellulose/hemicellulose composites.

    Plant J 2000, 22:327-334. PubMed Abstract | Publisher Full Text OpenURL

  78. Linder M, Teeri TT: The cellulose-binding domain of the major cellobiohydrolase of Trichoderma reesei exhibits true reversibility and a high exchange rate on crystalline cellulose.

    PNAS 1996, 93:12251-12255. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  79. Huang R, Su R, Qi W, He Z: Understanding the key factors for enzymatic conversion of pretreated lignocellulose by partial least square analysis.

    Biotechnol Progr 2010, 26:384-392. OpenURL

  80. Henrissat B, Driguez H, Viet C, Schulein M: Synergism of cellulases from Trichoderma reesei in the degradation of cellulose.

    Nat Biotechnol 1985, 3:722-726. Publisher Full Text OpenURL

  81. Nagendran S, Hallen-Adams HE, Paper JM, Aslam N, Walton JD: Reduced genomic potential for secreted plant cell-wall-degrading enzymes in the ectomycorrhizal fungus Amanita bisporigera, based on the secretome of Trichoderma reesei.

    Fungal Genet Biol 2009, 46:427-435. PubMed Abstract | Publisher Full Text OpenURL

  82. Rosgaard L, Pedersen S, Langston J, Akerhielm D, Cherry JR, Meyer AS: Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated barley straw substrates.

    Biotechnol Progr 2007, 23:1270-1276. Publisher Full Text OpenURL

  83. Herpoel-Gimbert I, Margeot A, Dolla A, Jan G, Molle D, Lignon S, Mathis H, Sigoillot J-C, Monot F, Asther M: Comparative secretome analyses of two Trichoderma reesei RUT-C30 and CL847 hypersecretory strains.

    Biotechnol Biofuels 2008, 1:18. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  84. Kubicek CP: The cellulase proteins of Trichoderma reesei: structure, multiplicity, mode of action and regulation of formation.

    Adv Biochem Eng/Biotechnol 1992, 45:1-27. Publisher Full Text OpenURL

  85. Ghose TK: Measurement of cellulase activities.

    Pure Appl Chem 1987, 59:257-268. Publisher Full Text OpenURL

  86. Tan HY, Ng TW, Liew OW: Effects of light spectrum in flatbed scanner densitometry of stained polyacrylamide gels.

    Biotechniques 2007, 42:474-478. PubMed Abstract | Publisher Full Text OpenURL

  87. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC: Measurement of protein using bicinchoninic acid.

    Anal Biochem 1985, 150:76-85. PubMed Abstract | Publisher Full Text OpenURL

  88. Winters MS, Day RA: Detecting protein-protein interactions in the intact cell of Bacillus subtilis (ATCC 6633).

    J Bacteriol 2003, 185:4268-4275. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  89. Tur MK, Neef I, Jost E, Galm O, Jager G, Stocker M, Ribbert M, Osieka R, Klinge U, Barth S: Targeted restoration of down-regulated DAPK2 tumor suppressor activity induces apoptosis in Hodgkin lymphoma cells.

    J Immunother 2009, 32:431-441. PubMed Abstract | Publisher Full Text OpenURL

  90. Ramos-Fernandez A, Paradela A, Navajas R, Albar JP: Generalized method for probability-based peptide and protein identification from tandem mass spectrometry data and sequence database searching.

    Mol Cell Proteomics 2008, 7:1748-1754. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  91. Kato Y, Hiraoka S, Tada Y, Nomura T: Performance of a shaking vessel with current pole.

    Biochem Eng J 2001, 7:143-151. PubMed Abstract | Publisher Full Text OpenURL

  92. Bansal P, Hall M, Realff MJ, Lee JH, Bommarius AS: Modeling cellulase kinetics on lignocellulosic substrates.

    Biotechnol Adv 2009, 27:833-848. PubMed Abstract | Publisher Full Text OpenURL

  93. Yuldashev B, Rakhimov M, Rabinovich ML: The comparative study of cellulase behaviour on the surface of cellulose and lignocellulose during enzymatic hydrolysis.

    Appl Biochem Microbiol 1993, 29:58-68. OpenURL

  94. Lever M: New reaction for colorimetric determination of carbohydrates.

    Anal Biochem 1972, 47:273-279. PubMed Abstract | Publisher Full Text OpenURL

  95. Zhu ZG, Sathitsuksanoh N, Zhang YHP: Direct quantitative determination of adsorbed cellulase on lignocellulosic biomass with its application to study cellulase desorption for potential recycling.

    Analyst 2009, 134:2267-2272. PubMed Abstract | Publisher Full Text OpenURL

  96. Bowen P: Particle size distribution measurement from millimeters to nanometers, and from rods to platelets.

    J Disper Sci Technol 2002, 23:631-662. Publisher Full Text OpenURL

  97. Cao Y, Tan H: Study on crystal structures of enzyme-hydrolyzed cellulosic materials by X-ray diffraction.

    Enzyme Microb Technol 2005, 36:314-317. Publisher Full Text OpenURL

  98. Reese ET: Shear inactivation of cellulases of Trichoderma reesei.

    Enzyme Microb Technol 1980, 2:239-240. Publisher Full Text OpenURL

  99. Engel P, Mladenov R, Wulfhorst H, Jäger G, Spiess AC: Point by point analysis: how ionic liquid affects the enzymatic hydrolysis of native and modified cellulose.

    Green Chem 2010, 12:1959-1966. Publisher Full Text OpenURL

  100. Miller GL: Use of dinitrosalicylic acid reagent for determination of reducing sugar.

    Anal Chem 1959, 31:426-428. Publisher Full Text OpenURL

  101. Wood TM, Bhat KM: Methods for measuring cellulase activities.

    Method Enzymol 1988, 160:87-112. OpenURL

  102. Zhang YHP, Himmel ME, Mielenz JR: Outlook for cellulase improvement: Screening and selection strategies.

    Biotechnol Adv 2006, 24:452-481. PubMed Abstract | Publisher Full Text OpenURL