Family 6 cellulase with decreased inactivation by lignin

A modified Trichoderma reesei Family 6 (TrCel6A) cellulase enzyme comprising amino acid substitutions at a couple of positions selected from the group consisting of 129, 322, 363 and 410 of SEQ ID NO: 1 is supplied. Genetic constructs and genetically modified microbes comprising nucleic sequences encoding the modified TrCel6a cellulase are also supplied. The modified TrCel6A cellulase of this creation display at least a 15 percent decline in inactivation by lignin relative to some parental TrCel6A cellulase where the modified TrCel6A is based. These kinds of cellulases find use in various applications in business requiring enzymatic hydrolysis of cellulose in the presence of lignin, e.g., the hydrolysis of pretreated lignocellulosic feedstocks for the production of fermentable sugars, sugar alcohols and fuel alcohols.


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More than half of organic carbon in the world is found in the cell walls of plants. Plant cell walls comprise three main substances: cellulose, hemicellulose, and lignin. Together these chemicals are called”lignocellulose,” and they representa potential source of sugars and other organic molecules such as fermentation to ethanol or other high-value products.

The conversion of lignocellulosic biomass to ethanol has become an integral characteristic of emerging energy policies due to the environmentally favorable and sustainable nature of cellulosic ethanol. There are lots of technologies being developed forcellulose conversion. Of interest here is a method by which lignocellulosic biomass is subjected to a pretreatment that raises its susceptibility to hydrolytic enzymes, followed by enzymatic hydrolysis to sugars and also the cessation of these sugarsto ethanol or alternative naturally-occurring natural molecules (e.g. butanol). Frequent pretreatment methods include dilute acid vapor explosion (U.S. Pat. No. 4,461,648), ammonia freeze explosion (AFEX; Holtzapple et al., 1991), and organosolv extraction (U.S. Pat. No. 4,409,032). Hydrolysis and fermentation systems may be either separate (sequential hydrolysis and fermentation; SHF) or coincident (simultaneous saccharification and fermentation; SSF). In most instances, the hemicellulose and cellulose are brokendown to sugars that may be fermented, whereas the lignin becomes split and may be utilized either as a solid fuel or as a supply for other organic molecules.

The choice of enzymes for conversion of pretreated lignocellulosic biomass to sugars is highly dependent on the pretreatment method. Dilute acid steam burst leads to considerable chemical hydrolysis of the hemicellulose, thereby makingenzymes for the conversion of hemicellulose to sugars less related to this process. By comparison, AFEX and organosolv extraction both leave hemicellulose and cellulose largely intact. Organosolv extraction, unlike dilute acid steam burst or AFEXremoves a significant portion of the lignin from substrate. In most instances, the primary goal for enzymatic hydrolysis is the cellulose, which is converted into sugars with a combo of cellulase enzymes.

There are two principle kinds of cellulase enzymes: endoglucanases, which divides glycosidic bonds in the center of cellulose chains, and in doing this, produce new series ends, and cellobiohydrolases, which divides short oligosaccharides from theends of cellulose chains. Glucosidases digest short oligosaccharides to monosaccharides. These three enzyme components thus behave responsibly to create an efficient cellulolytic enzyme system. Many cellulases have a similar modular structure,which consists of a catalytic domain, linker peptide and a carbohydrate-binding module (CBM).

Altered cellulase enzymes and methods for alteration are extensively described. By way of instance, variants of Trichoderma reesei Cel7A and Cel6A to improve thermostability are reported (U.S. Pat. No. 7,375,197; WO 2005/028636; U.S.Publication No. 2007/0173431; U.S. Publication No. 2008/167214; WO 2006/074005; U.S. Publication No. 2006/0205042; U.S. Pat. No. 7,348,168; WO 2008/025164). Specifically, substitution of the serine at position 413 at T. reesei Cel6A with a proline,or substitution of the amino acid at the equivalent to position 413 with a proline in other Family 6 cellulases confers increased thermostability (WO 2008/025164). Mutations at the equivalent of places 103, 136, 186, 365 and 410 within the catalyticdomain of T. reesei Cel6A and other Family 6 cellulases are demonstrated to lead to decreased inhibition by sugar (U.S. Patent Publication No: 2009/0186381). Variants with resistance to proteases and to surfactants for detergent formulations have beencreated for textile applications (WO 99/01544; WO 94/07998; and U.S. Pat. No. 6,114,296).

The unwanted effects of lignin on cellulase enzyme systems are well recorded. Removal of lignin from hardwood (aspen) was shown to boost sugar yield by enzymatic hydrolysis (Kong et al., 1992). Similarly, removal of lignin from softwood(Douglas fir) was shown to improve enzymatic hydrolysis of the cellulose, an effect attributed to improved accessibility of the enzymes to the cellulose (Mooney et al., 1998). Other groups have shown that cellulases purified from Trichodermareesei bind to isolated lignin (Chemoglazov et al., 1988) and also have theorized on the function of the different binding domains from the enzyme-lignin interaction (Palonen et al., 2004). Binding to lignin and inactivation of Trichoderma reesei cellulases hasbeen discovered when lignin is inserted back to a pristine cellulose system (Escoffier et al., 1991). Only in one case was lignin reported to have no significant effect on cellulases (Meunier-Goddik and Penner, 1999). Other reports suggest that somehemicellulases could be resistant to, and even triggered by, lignin and lignin breakdown products (Kaya et al., 2000). Therefore, it’s usually recognized that lignin is a critical limitation to enzymatic hydrolysis of cellulose.

CBMs are reportedly involved in lignin binding. As an instance, removal of this CBM from Trichoderma Cel7A essentially eliminates binding to alkali extracted lignin and also to residual lignin prepared by enzyme hydrolysis (Palonen et al., 2004).

Catalytic domain names are also allegedly involved with binding lignin. Cel7B from Humicola sp., that doesn’t have a CBM, is bound extensively by lignin (Berlin et al., 2005b). Similarly Trichoderma Cel5A core, devoid of a CBM, doesn’t bindenzymic lignin and binds alkali extracted lignin to a lesser degree than does the full size protein (Palonen et al., 2004).

The development of lignin resistant cellulases with maintained cellulose binding affinity and indigenous cellulolytic activity represents a large hurdle from the commercialization of cellulose conversion to soluble sugars such as glucose for theproduction of ethanol and other products. A number of methods are suggested to reduce the negative impact of lignin on the cellulase system. Non-specific binding proteins (e.g. bovine serum albumin; BSA) are shown to block interactionsbetween cellulases and lignin surfaces (Yang and Wyman, 2006; US24185542 A1; US26088922 A1; WO05024037 A2, A3; WO09429474 A1). Other chemical blocking agents and surfactants are shown to possess a similar effect (Tu et al., 2007; U.S. Pat. No.7,354,743). While it’s been suggested to seek out and identify lignin-resistant variants of cellulase enzymes (Berlin et al., 2005a), no powerful work in this direction was previously documented.

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