Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin
Introduction
Lignocellulosic biomass is the most abundant organic material on earth and it could be a suitable low-cost feedstock for production of fuel ethanol and other chemicals in the future. Softwoods are a potential source of raw material in the northern countries when available as waste from the forest industry. Unfortunately, softwoods have turned out to be the most difficult raw materials for enzymatic hydrolysis. Understanding the underlying mechanism of the enzymatic hydrolysis of lignocellulose is of great importance in order to enhance the hydrolysis rate.
A range of hydrolytic enzymes is needed to degrade crystalline cellulose efficiently. The cellulase system of Trichoderma reesei is one of the most efficient for cellulose hydrolysis. It contains at least two cellobiohydrolases (CBH I and CBH II; EC 3.2.1.91), five endoglucanases (EG I–V; EC 3.2.1.4) and two β-glucosidases (EC 3.2.1.21) (Nevalainen and Penttilä, 1995). All T. reesei cellulases, except EG III, as well as many other cellulases from other micro-organism have a two domain structure consisting of a catalytic domain (CD) and a cellulose binding domain (CBD), which are bound together by a flexible linker.
The role of CBD in the total hydrolysis of cellulose has been difficult to define. It has been recognized that CBD increases the enzyme concentration on the surface of the solid substrate, and it might also help to solubilise single glucan chains off the cellulose surface (Linder and Teeri, 1997). Separate CDs can still hydrolyze even insoluble substrates. Separate CDs, which are most probably formed via proteolytic action, are also found in culture filtrates produced by T. reesei (Suurnäkki et al., 2000). It has been suggested that the catalytic domain and the intact protein may have different binding sites on the cellulose substrate. The CBD may also promote non-productive binding at higher enzyme concentrations (Linder and Teeri, 1997).
The mechanism of enzyme adsorption on pure cellulose has been widely studied. The process of enzyme adsorption is of key importance to fundamental knowledge of enzymatic hydrolysis of cellulose. Enzymes can bind to the solid surface either specifically or non-specifically. The interactions are usually noncovalent, i.e. by hydrogen bonding, electrostatic or hydrophobic interactions (Brash and Horbett, 1995). Other adsorbed proteins, as well as low-molecular weight ions in the interfacial region, may affect the adsorption. Electrostatic forces contribute to the binding, but they do not dominate protein adsorption in all conditions (Norde and Haynes, 1995). Surface properties have an enormous effect on the mechanism, rate and extent of adsorption. The hydrophilicity of surface has generally been regarded as a very important factor: the more hydrophobic the surface is the higher the extent of adsorption. All the major T. reesei cellulases have hydrophobic amino acids exposed on the surface (Reinikainen et al., 1995). The hydrophobic residues on the surface of the enzyme may also lead to binding to the hydrophobic surface of lignin.
Lignocellulosic materials have a very heterogeneous structure, consisting of cellulose, hemicelluloses and lignin. Despite of extensive research work, it is still not completely understood which substrate characteristics have the highest impact on the hydrolysis rate of cellulose in wood. The important characteristics include accessibility, degree of crystallinity and polymerization, as well as lignin distribution (Mansfield et al., 1999). The presence of lignin definitely plays an important role especially in the enzymatic degradation of softwood. It has been shown that the softwood derived substrates are more resistant to lignin removal and enzymatic hydrolysis than hardwood and annual plant substrates. The residual softwood lignin can act as a steric hindrance to cellulolytic enzymes, thus preventing the effective binding to cellulose (Mooney et al., 1999, Ramos et al., 1992).
Recycling of cellulases after the enzymatic hydrolysis of lignocellulose has proved out to be difficult due to low enzyme recoveries. Several authors have suggested that cellulases adsorb to the lignin fraction of lignocellulose (Chernoglazov et al., 1988, Converse et al., 1990, Hogan and Mes-Hartree, 1990, Ooshima et al., 1990, Sutcliffe and Saddler, 1986). There is, however still a limited knowledge on the adsorption of individual cellulase components on different lignocellulosic materials, derived from forestry sources.
In this work, the role of lignin on the adsorption of CBH I and EG II and their catalytic domains was studied using in vitro tritiated enzymes as tracers to determine the free protein concentration. CBH I is the main constituent of the T. reesei cellulase system, comprising about 60% of the cellulases secreted by the fungus. EG II represents endoglucanase activity in this study. It comprises about 10% of the cellulases of T. reesei. Adsorption of whole enzymes and their catalytic domains on steam pretreated softwood, on a lignin-rich hydrolysis residue and on an alkali-extracted lignin was studied.
Section snippets
Purification and tritiation of enzymes
CBH I, CBH II and EG II of T. reesei were purified as described by Rahkamo et al. (1996) and their CDs as described by Suurnäkki et al. (2000). CBH I and its CD were tritiated in vitro as described previously (Palonen et al., 1999). In this work, EG II and EG II CD were labeled using a similar method. Proteins were concentrated to 2–3 g l−1 and the buffer was exchanged to 200 mM HEPES buffer (pH 8.5). Tritium enriched NaBH4 (50 mCi; 3.6 μmol; TRK45; Amersham) dissolved in the same buffer and 80 μl
Lignocellulosic materials
Table 1 summarizes the Klason lignin and protein contents of the materials. The lignin content of SPS (38%) is higher that that of untreated wood (28%) due to the hemicellulose solubilization in the pretreatment process. The lignin extraction from SPS by NaOH gave a lignin with a very viscous and sticky appearance (alkali-lignin). The CEL-lignin was prepared by enzymatic hydrolysis using an excess of cellulases. The high Klason lignin content of CEL-lignin (91%) indicates a nearly total
Discussion
Steam pretreatment is an efficient and widely used method to enhance the enzymatic accessibility of lignocellulosic materials. Hemicelluloses are hydrolyzed and solubilized during the steam pretreatment, leaving most of the lignin together with cellulose in the solid fraction. There are several indications that lignin restricts the enzymatic hydrolysis of cellulose either by physical entrapping of cellulose or by adsorbing cellulolytic enzymes.
In this study, we were able to determine accurately
Acknowledgements
HP acknowledges financial support from the Nordic Energy Research Program and Emil Aaltonen foundation. Professor Liisa Viikari is thanked for useful discussions. Dr. Markus Linder and Dr. Tapani Reinikainen are thanked for the critical reading of the manuscript.
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Present address: Department of Applied Chemistry and Microbiology, University of Helsinki, P.O. Box 27, FIN-00014 Helsinki, Finland.