The most abundant polysaccharide in the biosphere, cellulose, consists of D-glucose units linked together in linear chains via β-1,4 glycosidic bonds. These chains can vary in length and often consist of many thousands of units. Cellulose chains form numerous intra- and intermolecular hydrogen bonds, which result in the formation of insoluble cellulose microfibrils. This crystalline cellulose is a recalcitrant material with a natural half-life of over five million years.
In order to access this important renewable carbon source, microorganisms, such as bacteria and fungi, produce a cocktail of enzymes to break down crystalline cellulose into glucose. Three general classes of cellulase enzymes act synergistically to hydrolyze the crystalline cellulose into the simple energy source glucose. Endo-β-1,4-glucanases (EC 3.2.1.4) randomly hydrolyze amorphous regions of crystalline cellulose generating oligosaccharides of various lengths and consequently new chain ends. Cellobiohydrolases (or exo-β-1,4-cellobiohydrolase, EC 3.2.1.91) hydrolyze processively cellobiose units from one end of the cellulose chain. Finally, β-1,4-glucosidases (EC 3.2.1.21) hydrolyse cellobiose into glucose.
Most cellobiohydrolases and endo-β-1,4-glucanases are multidomain proteins consisting of a catalytic core domain and a cellulose-binding domain separated by a flexible linker region. The cellulose-binding domain promotes adsorption of the enzyme to regions of the cellulosic substrate (Tomme, P., et al. 1988. Eur. J. Biochem 170:575-581; Lehtio J., et al. 2003 Proc. Natl. Acad. Sci. USA. 100:484-489), while the catalytic core domain is responsible for the cleavage of cellulose. The linker region may ensure an optimal interdomain distance between the core domain and the cellulose-binding domain (Srisodsuk M., et al. 1993. J. Biol. Chem. 268:20756-20761).
The catalytic domains are classified into the glycoside hydrolase families based on amino acid sequence similarities whereby a family comprises enzymes having similar fold and hydrolytic mechanisms but may differ in their substrate specificity. Trichoderma reesei contains known cellulase genes for two cellobiohydrolases, i.e., Cel7A (also known as CBH1, which is a member of Family 7) and Cel6A (CBH2), at least eight endo-β-1,4-glucanases, i.e., Cel7B (EG1), Cel5A (EG2), Cel12A (EG3), Cel61A (EG4), Cel45A (EG5), Cel74A (EG6), Cel61B (EG7), and Cel5B (EG8), and at least seven β-1,4-glucosidase, i.e., Cel3A (BG1), CellA (BG2), Cel3B (BG3), Cel3C (BG4), CellB (BG5), Cel3D, and Cel3E (Foreman, P. K., et al. 2003. J. Biol. Chem. 278:31988-31997).
T. reesei Cel6A (or TrCel6A) is one of the two major cellobiohydrolases secreted by this fungus and has been shown to be efficient in the enzymatic hydrolysis of crystalline cellulose. TrCel6A is a member of glycoside hydrolase Family 6, which comprises enzymes that hydrolyse β-1,4 glycosidic bonds with inversion of anomeric configuration and includes cellobiohydrolases as well as endo-β-1,4-glucanases. The three dimensional structures of TrCel6A (Rouvinen J., et al. 1990. Science 249:380-386. Erratum in: Science 1990 249:1359), Thermobifida fusca endo-β-1,4-glucanase Cel6A (TfCel6A, Spezio M., et al. 1993. Biochemistry. 32:9906-9916), Humicola insolens cellobiohydrolase Cel6A (HiCel6A, Varrot, A., et al. 1999 Biochem. J. 337:297-304), Humicola insolens endo-β-1,4-glucanase Cel6B (HiCel6B, Davies, G. J., et al. 2000. Biochem. J. 348:201-207), and Mycobacterium tuberculosis H37Rv Cel6A (MtCel6A, Varrot. A., et al. 2005. J. Biol. Chem. 280:20181-20184) are known.
Applications of cellulase enzymes in industrial processes are numerous and have proven commercially useful within the textile industry for denim finishing and cotton softening; in the household and industrial detergents for color brightening, softening, and soil removal; in the pulp and paper industries for smoothing fiber, enhancing drainage, and de-inking; in the food industry for extracting and clarifying juice from fruits and vegetables, and for mashing; in the animal feed industry to improve their nutritional quality; and also, in the conversion of plant fibers into glucose that are fermented and distilled to make low CO2 cellulose ethanol to reduce fossil fuel consumption, which is an emerging industry around the world (e.g. Gray K. A., et al. 2006. Curr. Opin. Chem. Biol. 10:141-146).
In order to obtain enzyme variants with improved stability properties, three strategies have generally been used within the art: 1) isolation of thermophilic enzymes from extremophiles, residing in severe environments such as extreme heat or cold, high salt concentrations or high or low pH conditions (e.g. U.S. Pat. No. 5,677,151 U.S. Pat. Appl. No. 20060053514); 2) protein engineering by rational design or site-directed mutagenesis, which relies on sequence homology and structural alignment within a family of proteins to identify potentially beneficial mutations using the principles of protein stability known in the art (reviewed in: Eijsink, V. G., et al. 2004. J. Biotechnol. 113:105-20.); and 3) directed evolution involving the construction of a mutant library with selection or screening to identify improved variants and involves a process of iterative cycles of producing variants with the desired properties (recently reviewed in: Eijsink V G, et al. 2005. Biomol. Eng. 22:21-30). This approach requires no structural or mechanistic information and can uncover unexpected beneficial mutations. Combining the above strategies has proven to be an efficient way to identify improved enzymes (Chica R. A., et al. 2005. Curr. Opin. Biotechnol. 16:378-384).
Using rational design, Zhang et al. (Zhan S et al., 2000. Eur. J. Biochem. 267:3101-15), introduced a new disulfide bond across the N- and C-terminal loops from TfCel6B using two double mutations, and four glycine residue mutations were chosen to improve thermostability. None of the mutations increased thermostability of this cellobiohydrolase and most mutations reduced thermostability by 5-10° C. Surprisingly, the double mutation N233C-D506C showed a decrease of 10° C. for the T50 (Zhang S et al., 2000. Eur. J. Biochem. 267:3101-15), or a slight increase of about 2° C. for the T50 (Ai, Y. C. and Wilson, D. B. 2002. Enzyme Microb. Technol. 30:804-808). Wohlfahrt (Wohlfahrt, G., et al. 2003. Biochemistry. 42:10095-10103) disclosed an increase in the thermostability of TrCel6A, at an alkaline pH range, by replacing carboxyl-carboxylate pairs into amide-carboxylate pairs. A single mutant, E107Q, and a triple mutant, E107Q/D170N/D366N, have an improved Tm above pH 7 but a lower Tm at pH 5, which is the optimal pH of the wild-type TrCel6A. These mutations are found in, or close to, the N- and C-terminal loops. Hughes et al (Hughes, S. R., et al. 2006. Proteome Sci. 4:10-23) disclose a directed evolution strategy to screen mutagenized clones of the Orpinomyces PC-2 cellulase F (OPC2Cel6F) with targeted variations in the last four codons for improved activity at lower pH, and identified two mutants having improved activity at lower pH and improved thermostability.
The above reports describing rational design of Family 6 cellulases suggest that the introduction of hydrogen or disulfide bonds into the C-terminal loops is not a good strategy to increase the thermostability at optimal hydrolysis conditions. Furthermore, stabilizing the exo-loop of the T. reesei Family 7 cellobiohydrolase Cel7A, which forms the roof of the active site tunnel, by introducing a disulfide bond with mutation D241C/D249C showed no improvement in thermostability (von Ossowski I., et al. 2003. J. Mol. Biol. 333:817-829).
TrCel6A variants with improved thermostability are described in US Patent Publication No. 20060205042. Mutations were identified based alignment of TrCel6A amino acid sequence with those of eight Family 6 members using structural information and a modeling program. This alignment served as basis for the determination of a so-called consensus sequence. Those mutations that, according to the 3D-structure model of TrCel6A, fit into the structure without disturbance and were likely to improve the thermostability of the enzyme were selected as replacement for improved thermostability of TrCel6A. Among those identified as improving the thermostability of TrCel6A was the mutation of the serine at position 413 to a tyrosin (S413Y). This mutation increased the retention of enzymatic activity after a 1 hour pre-incubation at 61° C. from 20-23% for the parental TrCel6A to 39-43% for TrCel6A-S413Y; however, after a 1 hour pre-incubation at 65° C., the parent TrCel6A retained 5-9% of its activity while TrCel6A-S413Y retained 6-8% of its activity. The melting temperature, or Tm, improved by 0.2-0.3° C., from 66.5° C. for the parental TrCel6A to 66.7-66.8° C. for TrCel6A-S413Y.
Despite knowledge of the mechanisms of and desirable attributes for cellulases in the above and related industrial applications, the development of thermostable cellulases with improved stability, catalytic properties, or both improved stability and catalytic properties, would be advantageous. Although thermophilic and thermostable enzymes may be found in nature, the difficulty in achieving cost-effective large-scale production of these enzymes has limited their penetration into markets for industrial use. Therefore, a need exists for improved stable cellulases which can be economically produced at a high-level of expression by industrial micro-organisms such as T. reesei. 