Cellulase enzymes are widely used to improve the appearance and softness of cellulose-containing fabrics. One common application of cellulase enzymes is for treating denim fabrics so as to impart to them a “stone-washed” appearance. Such a process is known in the industry as “bio-stoning”. Cellulase enzymes have largely replaced stones for generating the soft, faded denim that is desired by consumers. A second widespread application of cellulase enzymes is to remove cotton fuzz and loose surface fibers in or on the fabric. This process, known as “depilling” or “biopolishing”, smooths the surface of the fabric, which in turn improves its softness and appearance. Cellulase treatment also aids in the prevention of subsequent formation of fiber pills that make the garments appear worn.
Fungi such as Trichoderma secrete a number of different cellulase enzymes (also referred to herein as an “enzyme mixture”) that are individually known as components. The more prevalent of these enzyme components include cellobiohydrolase (CBH), endoglucanase (EG), and beta-glucosidase enzymes. Cellulase enzyme components typically comprise a cellulose binding domain (CBD) and a catalytic domain. A region between these two domains known as a “linker” serves as a flexible spacer between the CBD and the catalytic domain.
The cellobiohydrolase (CBH) and endoglucanase (EG) components can be further divided into glycosyl hydrolase families (Davies and Henrissat, 1995), some of which have been identified as contributing to improvements in the look and feel of the fabric. Trichoderma reesei is a widely studied and industrially important fungus for the production of cellulases. It produces at least six genetically different cellulases: two cellobiohydrolases (Cel7A and Cel6A, formerly known as CBH I and II, respectively) and at least four endoglucanases (Cel7B, Cel5A, Cel12A and Cel45A, formerly known as EGI, EGII, EGIII and EGV, respectively).
Efforts have been made to improve the properties of cellulase mixtures for textile applications by varying the relative proportions of the cellobiohydrolase and endoglucanase components in a secreted enzyme mixture relative to the natural mixture. For instance, WO 92/17574 discloses an approach that involves adjusting the amounts of EG type components relative to CBH I type components (Cel7A) so that the protein weight ratio is greater than 5:1. Cotton-containing fabrics treated with such compositions exhibited decreased strength loss during textile treatment compared to fabrics containing greater amounts of CBHI type (Cel7A) components.
Improvements in depilling and bio-stoning have also been achieved by elevating the content of single components in the enzyme mixture. U.S. Pat. No. 5,858,767 discloses Trichoderma cellulase preparations enriched in the CBHII cellobiohydrolase (Cel6A) in an otherwise normal background cellulase composition. Such compositions were found to improve the appearance of fabrics in depilling applications. U.S. Pat. No. 5,874,293 discloses cellulase mixtures enriched in EGII endoglucanase (Cel5A) that show improvements in bio-stoning applications. EP 866 165 discloses enzyme compositions enriched in EGII (Cel5A) with improvements in depilling applications.
However, despite these efforts, there is a continuous need for improved cellulases and compositions thereof that are more efficient in fabric treatment and in other fields where cellulases have been traditionally used. In particular, there is a continuous need for more catalytically efficient cellulases to improve process economics. Such a need could be met by improving the specific activity of components in the enzyme mixture. By providing for a more active cellulase, less enzyme may be required, which in turn could significantly reduce processing costs.
Researchers have modified Family 5 cellulases (also referred to herein as “Cel5”) by protein engineering with the aim of improving their activity for the efficient conversion of cellulose to glucose during the production of ethanol from biomass. In Acidothermus cellulolyticus Cel5A (SEQ ID NO:13; AcCel5A), a Y245G mutation increased the activity of the enzyme on dilute-acid pretreated yellow poplar sawdust (Baker et al., 2005). Increased activity was mainly driven by a decrease in inhibition by cellobiose.
Variants of Bacillus subtilis (strain BME-15) Cel5A (SEQ ID NO:14; BsCel5A) carrying multiple catalytic domains and CBD mutations exhibited increased specific activity of up to 2.68 fold using carboxymethyl cellulose (CMC) as a substrate (Lin et al., 2008). However, the activity level reached by the best mutant was 4.88 U/mg, whereas Trichoderma EGII activity on the same substrate was reported to be 39.9 U/mg (Xiao et al., 2002).
In addition, the effect of genetic modification on the activity of Trichoderma reesei Cel5A (TrCel5A; SEQ ID NO:1) at pH values higher than its optimal range has been examined. Commercially available endoglucanases from Trichoderma reesei have optimum activity in the pH range of 4-6. The goal of such studies is to increase the activity of the enzyme at higher pH values so that it can be utilized in industrial processes that operate at neutral or alkaline conditions.
Mutation N321T in the mature TrCel5A cellulase (without the secretion signal) was identified to increase the optimal pH of the enzyme by 0.6 to 0.8 pH units over the wild-type enzyme via directed evolution (Wang et al., 2005). Site-saturation of this position showed that an N to R substitution resulted in the highest shift in optimum pH with an increase of 1.4 pH units (Qin et al., 2008a). However, the specific activity of this variant was greatly decreased compared to wild-type. After subsequent error-prone PCR and DNA shuffling steps, a variant Q139R/L218H/W276R/N342T (equivalent to Q118R/L197H/W255R/N321T in SEQ ID:1) was isolated with an optimal pH increase of 1.4 units without significant loss in specific activity (Qin et al., 2008b).
Studies on a Family 5 alkalophilic cellulase NK1 from Bacillus cellulosilyticus (SEQ ID NO:15; BcNK1) (formerly known as Bacillus sp. N-4) showed that the C-terminal portion of the catalytic domain is critical for the alkalophilicity of enzyme, especially residues S287 and A296 (Nakamura et al., 1991; Park et al., 1993). Mutating these residues for the equivalent residues in Bacillus subtilis neutral cellulase (BSC) made the Bacillus cellulosilyticus NK1 pH profile very similar to the Bacillus subtilis pH profile. Among these two mutations, S287N caused a greater effect on the pH profile than A296S.
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