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Holzforschung, Vol. 60, pp. 398401, 2006 Copyright by Walter de Gruyter Berlin New York. DOI 10.1515/HF.2006.062

Effect of acetyl groups on enzymatic hydrolysis of cellulosic substrates

Xuejun Pan*, Neil Gilkes and Jack N. Saddler

Department of Wood Science, University of British Columbia, Vancouver, BC, Canada
*Corresponding author. Department of Wood Science, University of British Columbia, 2424 Main Mall, Vancouver, BC, Canada V6T 1Z4 E-mail: xuepan@forestry.ubc.ca

Abstract
Evidence showed that acetyl groups introduced during acetic acid delignification was a primary cause of the poor enzymatic digestibility of acetic acid pulp. The inhibition by acetyl groups could be removed by saponification. Acetyl groups might inhibit the enzymes by interfering with the productive binding (hydrogen bonds) between cellulose and the catalytic domain of cellulases, by affecting the binding of CBD to cellulose, or by increasing the diameter of the cellulose chain. Keywords: acetic acid pulp; acetyl group; cellulose acetate; enzymatic hydrolysis; lignocellulosics.

that the enzymatic hydrolysability of acetic acid pulp was not negatively correlated with lignin or xylan content. Previous research (Pan et al. 2005) indicated that an acetic acid pulp had much poorer enzymatic digestibility than the substrate from an organosolv ethanol process at a comparable lignin content. The objective of the present paper was to investigate the cause of poor enzymatic digestibility of acetylated cellulosic substrates. Acetic acid pulp from Douglas fir was compared with substrates prepared using steam explosion and an organosolv ethanol process. Cellulose acetate was used as a model to elucidate the impact of acetyl groups on the enzymatic hydrolysis of cellulose.

Experimental
Cellulose acetate (CA) was purchased from Eastman Chemical Company (Kingsport, TN, USA). Partially deacetylated CA (PDCA) and completely deacetylated CA (DCA) were prepared from CA by controlled saponification using varied NaOH doses and temperatures. The acetyl content of CA, PDCA, and DCA was 40.4%, 12.5%, and 0.0% (w/w), respectively. Acetic acid pulp (AcP) was prepared from Douglas fir using an atmospheric acetic acid (AcOH) process under the conditions of 95% (v/v) AcOH, 0.25% HCl in AcOH as the catalyst (w/v), a 5-h reaction time and an AcOH/wood ratio of 5:1 (v/w), as previously described (Pan and Sano 1999). Steam-exploded Douglas fir (SEDF) was prepared using a laboratory-scale 1-l steam gun, as previously described (Pan et al. 2004). Organosolv ethanol pulp (EP) was prepared from mixed softwood (spruce/pine/fir, 40:40:20) as previously reported (Pan et al. 2005). Enzymatic hydrolysis was conducted according to a previously described procedure (Pan et al. 2005). Cellulase (Celluclast) and b-glucosidase (Novozym 188) were generously provided by Novozymes (Franklinton, NC, USA). Hydrolysis data reported are the average of duplicate experiments. Concentrations of monosaccharides were determined using a DX-500 HPLC system (Dionex, Sunnyvale, CA, USA) as previously described (Pan et al. 2004). Klason lignin determination was carried out according to Tappi standard T222 om-98. Acidsoluble lignin was determined using a UV spectrometer at 205 nm (Dence 1992). The acetyl content of CA, PDCA, DCA and AcP was determined using gas chromatography (GC) as previously described (Pan and Sano 2005). The GC conditions were: Hewlett Packard 5890-II system; Stabliwax-DA capillary column; carrier gas N2 at a flow rate of 3.5 ml miny1; injection volume 2 ml; injection temperature 2008C; FID detector at 2508C; and oven temperature 1808C for 17 min. The content of acetyl groups was calculated based on the acetic acid detected.

Introduction
Enzymatic hydrolysis of natural lignocellulosic materials is a very slow process because cellulose hydrolysis is hindered by structural parameters of the substrate, such as lignin and hemicellulose content, surface area, and cellulose crystallinity (Mansfield et al. 1999). To enhance enzymatic hydrolysis, the natural substrate must therefore be pretreated to break down cell walls, remove lignin and hemicellulose, increase the surface area, and reduce the crystallinity. Typical pretreatments include steam explosion (Galbe and Zacchi 2002), dilute acid pretreatment (Gorhmann et al. 1985; Allen et al. 2001), organosolv processes (Holtzapple and Humphrey 1984; Pan et al. 2005), and others (Wyman et al. 2005). Acetic acid is one of the organic solvents used in organosolv processes for biomass pretreatment and fractionation. Acetic acid pulping has been extensively investigated for the production of easy-bleaching pulp for paper, high-value lignin and hemicellulose sugars from wood and agricultural residues (Davis et al. 1986; Sano et al. 1989; Shukry et al. 1992; Nimz and Scho ne 1993; Pan and Sano 1999). In particular, atmospheric aceticacid pulping (Sano et al. 1989; Pan and Sano 1999) appears to be more promising because of the low operation temperature and pressure, which does not require the high-pressure reactor that other organosolv processes do. In addition to being used for paper manufacture, acetic acid pulp has also been evaluated as a substrate for bioconversion. Vazquez et al. (2000) found

Results and discussion

The chemical composition of the substrates used in this study is given in Table 1, including SEDF, organosolv EP from mixed softwood (spruce/pine/fir), AcP from Douglas fir and DAcP (deacetylated AcP). The results show that

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Acetyl groups and enzymatic hydrolysis of cellulose 399

Table 1 Chemical composition of the substrates used. Component Acetyl (%) Total lignin (%) Klason lignin (%) Acid-soluble lignin (%) Carbohydrate (%) Arabinose Galactose Glucose Xylose Mannose ND, not detected. Douglas fir wood Trace 29.8 29.4 0.4 0.8 4.2 51.8 3.7 13.3 Steam-exploded Douglas fir ND 42.8 41.7 1.1 0.3 0.5 61.4 5.8 2.1 Acetic acid pulp 6.2 18.7 18.2 0.5 ND ND 83.7 1.4 3.2 Deacetylated acetic acid pulp ND 17.1 16.7 0.4 ND ND 86.9 1.6 3.7 Organosolv ethanol pulp ND 18.4 17.9 0.5 ND ND 89.0 0.9 0.9

steam explosion significantly removed hemicellulose sugars, in particular galactose, mannose and arabinose. Since no delignification occurred during steam explosion, lignin was enriched to 42.8% in SEDF owing to the removal of hemicellulose. On the other hand, acetic acid extensively delignified Douglas fir and dissolved more hemicellulose than steam explosion. As a reference, EP had a comparable composition to AcP, except for less xylose and mannose. Acetyl groups (6.2%) were detected in AcP, which were introduced mainly by partial acetylation of cellulose during acetic acid pulping (Sato et al. 2003). The acetyl groups in AcP could be removed by saponification under mild conditions using 1% NaOH at 508C for 2 h. Deacetylation removed all acetyl groups, but did not significantly affect other components, as shown in Table 1 (DAcP). Research indicated that dilute alkali such as that used in deacetylation of AcP (1% NaOH) does not affect the crystalline structure of cellulose (Fengel et al. 1995).

The enzymatic hydrolysability (cellulose-to-glucose conversion yield) of different substrates is shown in Figure 1. These results show that AcP had a very poor digestibility (Figure 1a), and only ;10% of the cellulose was hydrolysed to glucose after 72-h incubation. In contrast, EP, with a comparable composition to AcP, showed excellent hydrolysability; and a cellulose-to-glucose conversion of over 90% was achieved within 48 h. The digestibility of AcP was even poorer than that of SEDF, which had a lignin content of up to 42.8%. In general, a substrate with more lignin is supposed to have poorer digestibility owing to the resistance of the lignin to enzyme action (Mansfield et al. 1999; Chang and Holtzapple 2000). The results imply that a factor other than lignin was responsible for the poor digestibility of AcP. When AcP was carefully deacetylated under mild conditions, the hydrolysability of DAcP was significantly improved, with the 72-h conversion of cellulose to glucose increased to 60% from 10%. It is believed that this

Figure 1 Enzymatic hydrolysis of different substrates. (a) Acetic acid pulp from Douglas fir (AcP), deacetylated AcP (DAcP), steamexploded Douglas fir (SEDF) and organosolv ethanol pulp from mixed softwood (EP); refer to Table 1 for chemical composition of the substrates. (b) Cellulose acetate (CA), partially deacetylated CA (PDCA) and completely deacetylated CA (DCA); refer to the experimental section for the acetyl contents. Enzymatic hydrolysis conditions: cellulase loading of 20 filter paper units gy1 cellulose, b-glucosidase loading of 40 IU gy1 cellulose, 2% (w/v) consistency of cellulose in 50 mM acetate buffer, pH 4.8, 458C, and shaker speed, 150 rpm.

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400 X. Pan et al.

improvement is predominantly due to the removal of acetyl groups, since the composition and structure of the substrate did not significantly change during deacetylation, as discussed above. To demonstrate the influence of acetyl groups on cellulose hydrolysis, cellulose acetates were used as models, including cellulose acetate (CA), partially deacetylated CA (PDCA) and fully deacetylated CA (DCA). Their enzymatic digestibility is presented in Figure 1b. With an acetyl content of 40.4% wDS (degree of substitution) 2.7x, CA was completely indigestible, with -2% hydrolysed after 72-h incubation. When the acetyl groups were reduced to 12.5% (DS 0.8) by partial deacetylation, PDCA showed significantly enhanced hydrolysis. Furthermore, when the acetyl groups were completely removed, DCA was completely hydrolysed within 24 h at a very high speed. In addition, it was observed that the hydrolysis residue of PDCA had higher acetyl content (27.4%, DS 1.8) than the original PDCA (12.5%, DS 0.8), implying that less substituted glucose units were preferentially hydrolysed. This evidence indicates that acetyl groups are a critical inhibitor of the enzymatic hydrolysis of cellulose. It was reported that the biodegradability of xylan, starch and cellulose acetates was correlated to the DS of acetyl groups (Glasser et al. 1995; Samios et al. 1997). The effect of acetyl groups on hydrolysis can be explained by the interaction between enzymes and cellulose. The cellulases from the fungus Trichoderma reesei used in this research consist of cellobiohydrolases (CBH I and II), endoglucanases (EG IIV) and b-glucosidase (Henriksson et al. 1996). The cellulases are two-domain enzymes, consisting of a large catalytic domain and a small cellulose-binding domain (CBD) linked by a heavily glycosylated peptide (Tomme et al. 1988). The function of CBD is to bind the cellulases to cellulose. The threedimensional structures show that the catalytic domain of in length in which the CBH forms a tunnel of ;50 A active site is enclosed, while EG has its active site in a deep but open groove rather than in a tunnel (Divne et al. 1994; Henriksson et al. 1996). It is inside this tunnel or groove that the cellulose chains are progressively cleaved by the active sites. For example, when a cellulose chain enters the tunnel of CHB I with the reducing end ahead, the 10 binding sites (amino acid residues) located inside the tunnel bind the cellulose chain in a productive binding mode through hydrogen bonds between the hydroxyl groups of the cellulose and the amino acid residues. This productive binding guides, conforms and positions the cellulose chain inside the tunnel in such a way that the glycosidic bond to be cleaved is facing the active site, resulting in cleavage of a cellobiose unit from the chain (Divne et al. 1998). When the hydroxyl groups of cellulose were substituted by acetyl groups, as occurs during acetic acid pulping, the productive binding mode described above could not be formed through hydrogen bonds. This might be the main cause of the poor hydrolysability of acetylated cellulose. Another explanation is that the acetyl groups change the hydrophobicity of cellulose and, therefore, negatively affect the binding of CBD to cellulose. In addition, the increased dimension (diameter) of the cellulose

chain on the introduction of acetyl groups probably means that the chain is too big (thick) to fit through the tunnel or groove of the enzymes. To support this hypothesis, the diameters of cellulose, cellulose 2,6-diacetate and cellulose triacetate chains were estimated using CS Chem3D Pro software (version 5.0, CambridgeSoft Cor, poration, Cambridge, MA, USA) as 7.3, 10.4 and 11.9 A respectively. These values may be different from the real diameters of cellulose and cellulose acetates, but at least this calculation demonstrates that the introduction of acetyl groups significantly increases the diameter of the cellulose chain. It was reported that the cellulose diam (Baker et al. 1997; Zhang et eter is in the range 510 A al. 1997). These proposed explanations of the effects of acetyl groups on cellulose hydrolysis require further investigation and confirmation.

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