# Patent

## Title
Modified beta-glucosidases with improved stability

## Abstract

Provided are modified beta-glucosidase enzymes, derived from the Trichoderma reesei Cel3A beta-glucosidase, that exhibit improved stability at low pH, low pH and high aeration, low pH and high agitation, or low pH and elevated temperature. Also provided are genetic constructs comprising nucleotide sequences encoding for modified beta-glucosidase enzymes, methods for the production of modified beta-glucosidase enzymes from host strains and the use of the modified beta-glucosidase enzymes in the hydrolysis of cellulose.

## Background
TECHNICAL FIELD
This invention relates to a modified beta-glucosidase of Trichoderma reesei. More specifically, the invention relates to a modified Trichoderma reesei beta-glucosidase with improved stability. The present invention also relates to a genetic construct comprising nucleotide sequences encoding a modified beta-glucosidase, methods for the production of a modified beta-glucosidase from host strains and the use of a modified beta-glucosidase in the hydrolysis of cellulose and in the production of compounds such as those used in the medical and food industries.
BACKGROUND OF THE INVENTION
Beta-glucosidases comprise members of Glycosyl Hydrolase Families 1 and 3 whose primary enzymatic function is the hydrolysis of the beta-glycosidic bonds linking carbohydrate residues in cellobiose or soluble cellodextrins. Some beta-glucosidases are specific for cellobiose or aryl glucosides, most of those characterized have broad specificity and can hydrolyse a broad range of substrates (Bhatia et al., 2002). Under certain conditions, these enzymes can also catalyze the synthesis of glycosidic linkages through the reverse reaction or through transglycosylation (Sinnott, 1990). Both the hydrolytic and synthetic capabilities of this enzyme class can be employed in biotechnological applications.
One major application of the hydrolytic activity of beta-glucosidase is the alleviation of product inhibition of cellulase systems. Cellobiose, a major product of cellulose hydrolysis, strongly inhibits the activity of cellobiohydrolases (EC 3.2.1.91). The inclusion of sufficient beta-glucosidase to hydrolyse cellobiose to glucose, which is less inhibitory, results in significant gains in activity at higher degrees of substrate conversion (U.S. Pat. No. 6,015,703).
Numerous other biotechnological applications of beta-glucosidase have been reviewed by Bhatia et al. (2002). Examples of applications that depend on its hydrolytic activity include the release of medically important compounds from flavanoid and isoflavanoid glucosides and the liberation of fragrant compounds in fruit juices and wines (U.S. Pat. No. 6,087,131). A representative use of the synthetic activity is the production of alkyl-glucosides for use as a detergent (Ducret et al., 2002).
The activity of glycosyl hydrolases, including beta-glucosidases, is dependent on specific protonation states of the catalytic amino acids (glutamate or aspartate) in the active site of the enzyme, which are influenced by pH. For example, beta-glucosidase I from Trichoderma reesei is most active in the pH range 5.0-5.5 and dramatically less so under more acidic or basic conditions (Woodward and Arnold, 1981).
The pH dependencies of the activity and stability of a protein are not necessarily related. The destabilizing effects of acidic or alkaline conditions result from protonation or deprotonation of amino acid sidechains which may not be catalytic or even in close proximity to the active site. pH-dependent denaturation primarily results from the differing pKa values of specific amino acid sidechains in the native and denatured states, which introduce pH dependence to the free energy difference between states. Additionally, proteins can become highly charged at extremes of pH and experience increased intramolecular electrostatic repulsion (Fersht, 1998).
An engineered enzyme with an altered pH optimum will not necessarily be stable at the new pH for extended periods. Several glycoside hydrolases have undergone mutagenesis to alter their activity or stability at pH values important for industrial applications. For a starch processing application, an alpha-amylase from Bacillus licheniformis was engineered to include two amino acid replacements, MIST and N188S, which increased its low pH (5.2) activity to 140% of wild-type (U.S. Pat. No. 5,958,739). The addition of a third mutation, H133Y, further increased the activity to more than 150% of the double mutant. Replacement of a loop in a Bacillus endoglucanase, identified with a rational design approach, shifted its pH optimum for a textiles application. With an Ala-Gly-Ala replacement, the pH optimum was shifted up by more than 1 pH unit (U.S. Publication No. 2005/0287656). A further example is the incorporation of amino acid replacements A162P and W62E into Ce145 endoglucanase from Humicola insolens. These mutations increased activity at alkaline conditions (pH 10) to 124-144% of that of the wild-type (U.S. Pat. No. 5,792,641). As a final example, the activity of a recombinant Trichoderma xylanase at pH above 5.5 was significantly improved by incorporation of various combinations of the replacements N10H, N11D, Y27M, and N29L (U.S. Pat. No. 5,866,408).
There are few reports of engineering beta-glucosidases though mutagenesis to modulate properties of these enzymes. For example, the thermostability of a quadruple mutant A16T/G142S/H226Q/D703G of an Aspergillus beta-glucosidase was increased such that it retained  ̃50% of its activity after one hour of incubation at 65° C. vs. 0-5% for wild-type variants. This enzyme was constructed using a combination of random mutagenesis, site-saturation and shuffling (U.S. Publication No. 2004/013401). Amino acid substitutions at one or more of positions 43, 101, 260 and 543 of Trichoderma reesei beta-glucosidase I resulted in modified beta-glucosidase with increased catalytic efficiency (U.S. Provisional Application No. 61/182,275). The following mutations were found to be particularly advantageous for increasing the catalytic efficiency of Trichoderma reesei beta-glucosidase I: V43I, V43C, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.
It is also noted that enzymes with altered pH stability profiles do not necessarily require altered pH optima to be of utility. For example, the pH of a cell culture used to express an enzyme may be different than the intended working pH of the enzyme in its application as a biocatalyst. If the stability of the enzyme is compromised at the expression pH, the overall yield of enzymatic activity will be reduced. This has been observed with Trichoderma reesei beta-glucosidase expressed in Saccharomyces cerevisiae (Cummings and Fowler, 1996). An unbuffered expression medium was observed to become more acidic over time, dropping from pH 6.0 to 2.0-3.0; this pH drop was correlated to a sharp decline in beta-glucosidase activity.
Instability can be further exacerbated by hydrodynamic shear arising from mixing of the cell culture, particularly in the presence of gas-liquid interfaces such as those produced by aeration (Weijers and Van't Riet, 1992; Elias and Joshi, 1998). Some enzymes may be further inactivated by shear stresses present during post-production processes such as ultrafiltration or in their final applications.
Shear inactivation of glycosyl hydrolases has been reported in the literature: Jones and Lee (1988) described the inactivation of a T. reesei cellulase mixture in a reactor system incorporating a high speed impeller, but only in the presence of air; Sachse et al. (1990) reported a higher specific activity of T. reesei cellulase produced in a low-shear vs. a conventional stirred reactor; Reese (1980) also reported the inactivation of T. reesei cellulase by shaking during hydrolysis and observed that the effect could be ameliorated by the use of surfactants; finally, Gunjikar et al. (2001) reported deactivation of exoglucanases, endoglucanases and beta-glucosidase in mixed reactors, the magnitude of which was proportionate to the mixing energy applied.
SUMMARY OF THE INVENTION
This invention relates to a modified beta-glucosidase of Trichoderma reesei. More specifically, the invention relates to a modified Trichoderma reesei beta-glucosidase with improved stability.
It is an object of the present invention to present variants of beta-glucosidase with improved stability.
The present invention provides a modified beta-glucosidase with improved stability in aqueous solution at low pH, at low pH with high agitation, low pH with high agitation, or low pH and elevated temperature. Beta-glucosidases of the present invention find utility in industrial processes requiring maintenance of activity under conditions of low pH and high aeration or high agitation, such as microbial fermentation, or low pH and elevated temperature, such as in the production of fermentable sugars by the enzymatic hydrolysis of cellulosic feedstocks.
This invention relates specifically to a modified beta-glucosidase of Trichoderma reesei produced by substitution of the amino acid at one or more of positions 66, 72, 101, 235, 248, 369 and 386 in the beta-glucosidase I or TrCel3A sequence (SEQ ID NO: 100). The inventors discovered that substitution of the native amino acid at one or more of these positions results in at least a 2-fold improvement, for example, from about 2-fold to about 500-fold improvement, in the stability of the beta-glucosidase in aqueous solution at low pH, at low pH and elevated temperature, at low pH with high agitation, or low pH with high aeration.
The modified TrCel3A beta-glucosidase may be derived from a parental TrCel3A beta-glucosidase that is otherwise identical to the modified TrCel3A beta-glucosidase except for the substitution of the naturally occurring amino acid at one or more of positions 66, 72, 101, 235, 248, 369, and 386. Furthermore, the modified TrCel3A beta-glucosidase may contain additional amino acid substitutions at positions other than at positions 66, 72, 101, 235, 248, 369, and 386, provided that these additional substitutions are also present in the corresponding parental TrCel3A. The modified TrCel3A beta-glucosidase may contain additional amino acid substitutions at one or more of positions 43, 96, 260 and 543.
The modified TrCel3A beta-glucosidase of the present invention exhibits from about 80% to about 99.9% amino acid sequence identity to native TrCel3A of SEQ ID NO: 1 or 100. For example, the modified TrCel3A exhibits from about 90% to 99.9% amino acid identity to the native TrCel3A of SEQ ID NO: 1 or 100 or from about 95% to 99.9% amino acid identity to the native TrCel3A of SEQ ID NO: 1 or 100.
Further, the modified TrCel3A beta-glucosidase, as defined above, exhibits at least a 2-fold improvement, for example, from about a 2-fold to about a 500-fold improvement, in stability in an aqueous solution (a) from about pH 2 to about 4.5, (b) from about pH 2 to about 4.5 aerated at a superficial gas velocity of from about 0.1 to about 100 cm/s, or from about 0.5 to 5 vvm (c) from about pH 2 to about 4.5 with agitation by shaking from about 300 to about 1000 rpm, (d) from about pH 2 to about 4.5 with agitation by impeller stifling with a tip speed of from about 0.5 to about 10 m/s; (e) from about pH 2 to about 4.5 in a bioreactor agitated at from about 0.2 to about 15 hp/100 gallons; or (f) from about pH 2 to about 4.5 at a temperature between 30° C. and 60° C.
The present invention also relates to a modified TrCel3A comprising an amino acid sequence as defined by
V661 SEQ ID NO: 2 or SEQ ID NO: 101;
SEQ ID NO: 3 or SEQ ID NO: 102;
SEQ ID NO: 5 or SEQ ID NO: 104;
SEQ ID NO: 6 or SEQ ID NO: 105;
SEQ ID NO: 7 or SEQ ID NO: 106;
SEQ ID NO: 8 or SEQ ID NO: 107;
SEQ ID NO: 9 or SEQ ID NO: 108;
SEQ ID NO: 10 or SEQ ID NO: 109;
SEQ ID NO: 11 or SEQ ID NO: 110;
SEQ ID NO: 12 or SEQ ID NO: 111;
SEQ ID NO: 13 or SEQ ID NO: 112;
SEQ ID NO: 14 or SEQ ID NO: 113;
SEQ ID NO: 15 or SEQ ID NO: 114;
SEQ ID NO: 16 or SEQ ID NO: 115;
SEQ ID NO: 4 or SEQ ID NO: 103; or
SEQ ID NO: 55 or SEQ ID NO: 116.
The invention also relates to a genetic construct for directing expression and secretion of the modified TrCel3A from a host microbe including, but not limited to, strains of Trichoderma reesei. 
The genetic construct of the present invention comprise a nucleic acid sequence encoding a modified TrCel3A that is from about 80% to about 99.9% identical to SEQ ID NO: 1 or 100 and that comprises an amino acid substitution at one or more of positions 66, 72, 101, 235, 248, 369 and 386, which nucleic acid sequence is operably linked to nucleic acid sequences regulating its expression and secretion from a host microbe. For example, the nucleic acid sequences regulating the expression and secretion of the modified TrCel3A beta-glucosidase may be derived from the host microbe used for expression of the modified TrCel3A beta-glucosidase. The host microbe may be a yeast, such as Saccharomyces cerevisiae, or a filamentous fungus, such as Trichoderma reesei. 
The invention also relates to a genetic construct as defined above, wherein the modified TrCel3A beta-glucosidase encoded by the genetic construct further comprises additional amino acid substitutions at positions other than 66, 72, 101, 235, 248, 369 and 386. The modified TrCel3A beta-glucosidase encoded by the genetic construct may contain additional amino acid substitutions at one or more of positions 43, 96 260 and 543.
The invention also relates to a genetically modified microbe comprising a genetic construct encoding the modified TrCel3A beta-glucosidase the genetically modified microbe being capable of expression and secretion of a modified TrCel3A beta-glucosidase exhibiting from about 80% to about 99.9% amino acid sequence identity to SEQ ID NO: 1 or 100 and comprising an amino acid substitution at one or more of positions 66, 72, 101, 235, 248, 369 and 386. The genetically modified microbe may be capable of expression and secretion of a modified TrCel3A beta-glucosidase further comprising additional amino acid substitutions at positions other than 66, 72, 101, 235, 248, 369 and 386. The modified TrCel3A beta-glucosidase expressed and secreted by the genetically modified microbe may contain additional amino acid substitutions at one or more of positions 43, 96, 260 and 543. The genetically modified microbe may be a yeast or filamentous fungus. For example, the genetically modified microbe may be a species of Saccharomyces, Pichia, Hansenula, Trichoderma, Hypocrea, Aspergillus, Fuscarium, Humicola or Neursopora. 
The present invention also relates to the use of a modified TrCel3A beta-glucosidase, as defined above, in a hydrolysis reaction containing a cellulosic substrate and a cellulase mixture comprising the modified TrCel3A beta-glucosidase.
The invention also relates to a process of producing a modified TrCel3A beta-glucosidase, as defined above, including transformation of a yeast or fungal host with a genetic construct comprising a nucleic acid sequence encoding the modified TrCel3A beta-glucosidase, selection of recombinant yeast or fungal strains expressing the modified TrCel3A beta-glucosidase, culturing the selected recombinant strains in submerged liquid fermentations under conditions that induce the expression of the modified TrCel3A beta-glucosidase and recovering the modified TrCel3A beta-glucosidase by separation of the culture filtrate from the host microbe.
The modified TrCel3A beta-glucosidase of the present invention finds use in a variety of applications in industry that require stability at low pH, a combination of low pH and high aeration, a combination of low pH and high agitation or a combination of low pH and elevated temperature. For example, modified Trichoderma reesei TrCel3A beta-glucosidase, as described herein, may be used in industrial processes in which lignocellulosic substrates are converted to fermentable sugars for the production of ethanol or other products, or microbial fermentation processes.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts plasmid vector YEp352/PGK91-1/αss6H-TrCel3A directing the expression and secretion of native and modified TrCel3A from recombinant Saccharomyces cerevisiae. 
FIG. 2 depicts plasmid vector pc/xCel3A-AT003-pyr4 directing the expression and secretion modified TrCel3A from recombinant Trichoderma reesei. 
FIG. 3 shows the inactivation of wild-type TrCel3A as a function of pH and temperature. (A) Plot of residual beta-glucosidase activity vs incubation time in aqueous solution with mild shaking (200 rpm) at pH 3.0, 4.0 or 5.0 at 30° C. or at 50° C. (B) Plot of residual beta-glucosidase activity vs incubation time in aqueous solution at 30° C. and pH 3.0 or pH 5.0 in unbaffled flasks with mild shaking (200 rpm), in baffled flasks with shaking at 200 rpm (“shaking”), or in unbaffled flasks with mechanical stifling (“stirring”).
FIG. 4 is a scatter plot of residual enzyme activity following pre-incubation at pH 3.5 versus pH 5.0. The data relate to the screening of one 96-well culture plate containing parental and modified TrCel3A beta-glucosidases. The parental TrCel3A data was fit by linear regression in which the y-intercept was fixed to zero.
FIG. 5 shows the inactivation of parental and modified TrCel3A beta-glucosidases at pH 3.0 and 30° C. shaken at 400 rpm in baffled flasks for 0 to 30 hours. The concentration of TrCel3A in these assays ranged from 4.8-10.8 μg/mL and the residual activity at each time interval was measured as described in Example 1.
FIG. 6 is a plot of the inactivation constants, tau (in h), versus the relative activity of parental and modified TrCel3A beta-glucosidases. The activity of the wildtype is set to 1.0 and all variants are compared to this value. Data represent the mean of triplicates, except for N248K which was assayed as a singleton, and the error bars represent +/− one standard deviation.
FIG. 7 depicts the inactivation of parental and modified TrCel3A beta-glucosidases at pH 3.0 and 30° C. shaken at 400 rpm in baffled flasks for 0 to 100 hours. The concentration of TrCel3A in these assays ranged from 4.8-10.8 μg/mL and the residual activity at each time interval was measured as described in Example 1.
FIG. 8 depicts the inactivation of modified TrCel3A beta-glucosidases at pH 3.0 and 30° C. shaken at 400 rpm in baffled flasks for 0 to 300 hours. The concentration of modified TrCel3A in these assays ranged from 4.8-10.8 μg/mL and the residual activity at each time interval was measured as described in Example 1.
FIG. 9 depicts the relative stability of parental and modified beta-glucosidase. The specific cellobiase activity (measured at pH 5.0) of the parental TrCel3A and aggregate modified TrCel3A-AT003 beta-glucosidases in cellulase mixtures produced in pilot Trichoderma reesei fermentations conducted for 165 hours at 28° C. and at either pH 3.0 and pH 5.0 expressed as a ratio. A ratio of 1.0 indicates that a beta-glucosidase is equally stable at pH 3.0 and pH 5.0; lower values indicate reduced stability at pH 3.0 vs. pH 5.0. Error bars represent one standard deviation.
FIG. 10 depicts the stability of parental and modified beta-glucosidase under conditions which could be used for cellulose hydrolysis. Parental and modified beta-glucosidase, within a cellulase mixture, was incubated at 50 or 60° C. at pH 3.0, 3.5, 4.0 or 5.0. Samples were taken from the enzyme at the times indicated and tested in a pNPGase assay. A model of first-order exponential decay was fit to the data to determine the mean life-time of each enzyme under each set of conditions. All data were normalized to the best fit value of initial activity as determined by the model and inactivation curves are displayed with respect to this initial activity.
FIG. 11 shows an alignment of the amino acid sequences of 45 fungal Family 3 beta-glucosidases, including TrCel3A, a consensus Family 3 beta-glucosidase sequence, and the % sequence identity of each amino acid sequence to that of TrCel3A. A graphical representation of the frequency of occurrence of the amino acid at each position of the consensus Family 3 beta-glucosidase sequence of FIG. 11 among the 45 fungal Family 3 beta-glucosidases is shown underneath the aligned sequences.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to modified beta-glucosidase. More specifically, the invention relates to modified beta-glucosidase I of Trichoderma reesei (hereinafter TrCel3A) with increased stability in aqueous solutions at low pH, at low pH with high aeration, low pH with high agitation, or low pH and elevated temperature relative to the parental TrCel3A from which it is derived. The present invention also relates to genetic constructs comprising nucleotide sequences encoding for modified TrCel3A, methods for the production of the modified TrCel3A from host strains and the use of the modified TrCel3A to alleviate product inhibition of cellulases in the hydrolysis of cellulose. The present invention also relates to the use of the modified TrCel3A to catalyze the production of other chemical compounds, including but not limited to those from the medical or food industries, either through hydrolysis, reverse hydrolysis or transglycosylation.
The following description is of a preferred embodiment by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.
Modified Beta-glucosidases
The term “beta-glucosidases” refers to enzyme classified in EC3.2.1.21 and that transfer a glycosyl group between oxygen nucleophiles, generally resulting in the hydrolysis of a beta-glucosidic bond linking carbohydrate residues in aryl, amino-, alkyl-beta-D-glucosides, cyanogenic-glucosides, short chain oligosaccharides and disaccharides. In oligosaccharides containing more than two glucosides, beta-glucosidase activity decreases as chain length increases. Beta-glucosidases hydrolyze beta-1,4-glucosidic bonds via a double displacement reaction, resulting in a net retention of anomeric configuration. Two acidic amino acids, aspartic (D) and/or glutamic (E) acid, are directly involved in substrate catalysis. One of these residues acts as a nucleophile and forms an enzyme-glycosyl intermediate. The other acidic residue acts as an acid-base catalyst. In the Trichoderma reesei beta-glucosidase 1, herein referred to as TrCel3A whose amino acid sequence is presented in SEQ ID NO: 100, the aspartic acid at position 236 serves as the nucleophile and the glutamic acid at position 447 is the acid-base catalyst.
Beta-glucosidases are a subset of beta-glycosidases belonging to glycosyl hydrolase (GH) Families 1 and 3, using the classification system developed by Henrissat and coworkers (Henrissat, B. (1991); Henrissat, B. and Bairoch, A. (1996)). There are currently 115 GH families that have been identified using this classification system, which are listed in the database of Carbohydrate Active Enzymes (CAZy) Family 1 comprises beta-glucosidases from archaebacteria, plants and animals. Beta-glucosidases from some bacteria, mold and yeast belong to Family 3. For the purpose of this invention, a “beta-glucosidase” is therefore defined as any protein that is classified in EC 3.2.1.21 and categorized as a Family 3 glycosyl hydrolase according to the CAZy system.
The three dimensional structure of beta-D-glucan exohydrolase, a Family 3 glycosyl hydrolase, was described by Varghese et al. (1999). The structure was of a two domain globular protein comprising a N-terminal (alpha/beta)8 TIM-barrel domain and a C-terminal a six-stranded beta-sandwich, which contains a beta-sheet of five parallel beta-strands and one antiparallel beta-strand, with three alpha-helices on either side of the sheet. This structure is likely shared by other Family 3 enzymes.
As shown in FIG. 11, the primary amino acid sequence of Family 3 beta-glucosidases show a high degree of similarity. Multiple alignment across 45 Family 3 beta-glucosidase amino acid sequences shows that the most naturally occurring Family 3 beta-glucosidases of fungal origin show from about 40% to about 100% amino acid sequence identity to the amino acid sequence of TrCel3A (FIG. 11). In particular, there are several regions of very high amino acid sequence conservation within the Family 3 beta-glucosidases including, for example, from amino acids 225-256 and 439-459, containing the catalytic amino acids D236 and E447, respectively.
By “TrCel3A” it is meant the Family 3 glycosyl hydrolase produced by Trichoderma reesei defined by the amino acid sequence in SEQ ID NO: 100. TrCel3A is also known as Trichoderma reesei β-glucosidase or BGL1. By “native” or ild-type” TrCel3A, it is meant the TrCel3A of SEQ ID NO: 100 without any amino acid substitutions. By “modified TrCel3A”, it is meant a TrCel3A which comprises one or more amino acid substitutions, introduced by genetic engineering techniques, selected from the group consisting of V66X (i.e., Val at position 66 is replaced by any amino acid X), S72X, V101X, T235X, N248X, N369X, A386X. Genetic engineering techniques for altering amino acid sequences include, but are not limited to, site-directed mutagenesis, cassette mutagenesis, random mutagenesis, synthetic oligonucleotide construction, cloning and other genetic engineering techniques as would be known by those of skill in the art (Eijsink V G, et al. 2005.). It will be understood that a modified TrCel3A may be derived from wild-type TrCel3A or from a TrCel3A that already contains other amino acid substitutions. Modified TrCel3A beta-glucosidases of the present invention include those comprising amino acid substitutions at any one of V66X, S72X, V101X, T235X, N248X, N369X and A386X, at any two of V66X, S72X, V101X, T235X, N248X, N369X and A386X, at any three of V66X, S72X, V101X, T235X, N248X, N369X and A386X, at any four of V66X, S72X, V101X, T235X, N248X, N369X and A386X, at any five of V66X, S72X, V101X, T235X, N248X, N369X and A386X, at any six of V66X, S72X, V101X, T235X, N248X, N369X and A386X, or at all seven of V66X, S72X, V101X, T235X, N248X, N369X and A386X.
It will be understood that the modified TrCel3A beta-glucosidase may be derived from wild-type TrCel3A beta-glucosidase or from a TrCel3A beta-glucosidase that contains other amino acid substitutions. For example, the modified TrCel3A beta-glucosidase may contain amino acid substitution at one or more of positions 43, 96 260 and 543. Alternatively, after production of a modified TrCel3A beta-glucosidase comprising mutations at one or more of positions 66, 72, 101, 235, 248, 369 and 386, it may be subsequently further modified to contain additional amino acid substitutions, including but not limited to those set forth above.
As used herein in respect of modified TrCel3A beta-glucosidase amino acid sequences, “derived from” refers to the isolation of a target nucleic acid sequence element encoding the desired modified TrCel3A beta-glucosidase using genetic material or nucleic acid or amino acid sequence information specific to the corresponding parental TrCel3A beta-glucosidase. As is known by one of skill in the art, such material or sequence information can be used to generate a nucleic acid sequence encoding the desired modified TrCel3A beta-glucosidase using one or more molecular biology techniques including, but not limited to, cloning, sub-cloning, amplification by PCR, in vitro synthesis, and the like.
In a first embodiment of the invention, the modified TrCel3A, as defined above, exhibits from about 80% to about 99.9% amino acid sequence identity to SEQ ID NO: 1 or 100, or any amount therebetween. For example, the modified TrCel3A may exhibit from about 90% to about 99.9% amino acid sequence identity to SEQ ID NO: 1 or 100 or from about 95% to about 99.9% amino acid sequence identity to SEQ ID NO: 1 or 100. Methods to align amino acid sequences are well known and available to those of skill in the art and include BLAST (Basic Local Alignment Search Tool, see Altschul et al., J. Mol. Biol. 215:403-410, 1990) which is useful for aligning two sequences and CLUSTALW for alignment of two or more sequences. Sequence identity may also be determined by manual alignment and visual inspection.
In other embodiments of the invention, the modified TrCel3A exhibits from about 80% to about 99.9% amino acid sequence identity to SEQ ID NO: 1 or 100 and at least a 2-fold improvement in stability in an aqueous solution a) from about pH 2 to about 4.5, (b) from about pH 2 to about 4.5 aerated at a superficial gas velocity of from about 0.1 to about 100 cm/s, or (c) from about pH 2 to about 4.5 with agitation by shaking from about 300 to about 1000 rpm, (d) from about pH 2 to about 4.5 with agitation by impeller stirring with a tip speed of from about 0.5 to about 10 m/s, (e) from about pH 2 to about 4.5 in a bioreactor agitated at from about 0.2 to about 15 hp/100 gallons, or (f) from about pH 2 to about 4.5 at a temperature between 30° C. and 60° C.
By “parental TrCel3A”, it is meant a TrCel3A that that does not contain a substitution of its original amino acid(s) at positions 66, 72, 101, 235, 248, 369 or 386 and that is otherwise identical to the modified TrCel3A. As such, the parental TrCel3A may contain amino acid substitutions at as many as 116 (i.e, 20% of 582 amino acids) other positions that have been introduced by genetic engineering or other techniques. For example, the parental TrCel3A may comprise amino acid substitutions at one or more of positions 43, 96, 260 and 543.
In order to assist one of skill in the art regarding those amino acid positions of the TrCel3A beta-glucosidase at which amino acid substitutions (other than V66X, S72X, V101X, T235X, N248X, N369X and A386X) may be made and produce an active beta-glucosidase, an alignment of 45 Family 3 beta-glucosidases derived from fungal sources along with a consensus beta-glucosidase sequence consisting of the amino acids that naturally occur with the highest frequency at each position is provided in FIG. 11 along with a graph showing the frequency of occurrence of each amino acid of the consensus sequence at each position. Using the information provided in FIG. 11, one of skill in the art would recognize regions of low sequence conservation to other Family 3 beta-glucosidases. Non-limiting examples of such regions include, for example, the regions between positions 1-20, 303-323 and 403-414 and select amino acid positions within these regions.
As described in more detail herein, several modified TrCel3A beta-glucosidases have been prepared that exhibit increased stability under conditions of low pH and agitation. A list of several mutants, which is not to be considered limiting in any manner, is presented in Table 1.







TABLE 1







TrCel3A beta-glucosidases with improved stability










New mutant TrCel6A-S413P
SEQ ID NO:













TrCel3A-V66I
2



TrCel3A-S72N
3



TrCel3A-V101M
4



TrCel3A-T235S
5



TrCel3A-N248K
6



TrCel3A-N369K
7



TrCel3A-A386T
8



TrCel3A-V66I-S72N
9



TrCel3A-S72N-F96L-V101M
10



TrCel3A-S72N-F96L-V101M-N369K
11



TrCel3A-S72N-V101M-N369K-A386T
12



TrCel3A-V66I-S72N-V101M-N369K-A386T
13



TrCel3A-S72N-F96L-V101M-N369K-A386T
14



TrCel3A-V66I-S72N-F96L-V101M-N369K-
15



A386T




TrCel3A-S72E-F96L-V101M-N369K-A386T
16



TrCel3A-S72N-F96L-V101M-N369P-A386T
55



TrCel3A-V66I full
101



TrCel3A-S72N full
102



TrCel3A-V101M full
104



TrCel3A-T235S full
105



TrCel3A-N248K full
106



TrCel3A-N369K full
107



TrCel3A-A386T full
108



TrCel3A-V661-S72N full
109



TrCel3A-S72N-F96L-V101M full
110



TrCel3A-S72N-F96L-V101M-N369K full
111



TrCel3A-S72N-V101M-N369K-A386T full
112



TrCel3A-V66I-S72N-V101M-N369K-
113



A386T full




TrCel3A-S72N-F96L-V101M-N369K-
114



A386T full




TrCel3A-V66I-S72N-F96L-V101M-N369K-
115



A386T full




TrCel3A-S72E-F96L-V101M-N369K-
103



A386T full




TrCel3A-S72N-F96L-V101M-N369P-
116



A386T full









Modified TrCel3A beta-glucosidases with Improved Stability.

Functional inactivation of enzymes is measured by determination of the inactivation rate constant, ki, a parameter with units of inverse time which determines the instantaneous rate of decrease of enzyme activity in the equation At/A0=e−ki·t where At is the activity at time t and A0 is the initial activity of the system. This parameter can be equivalently expressed as tau, the mean active lifetime of a given enzyme, by taking the inverse of ki, or as a half-life by multiplying tau by the natural logarithm of 2. Enzymes which are more stable have a smaller value of ki and corresponding larger values of tau and half-life. As defined herein, therefore, “improved stability” means a larger, higher or increased value of tau, expressed in units of time, such as hours.
For the purposes of the present invention, a modified TrCel3A exhibits improved stability (i.e, a larger value of tau) with respect to the corresponding parental Family 3 glycosyl hydrolase or parental TrCel3A in aqueous solution (a) with a low pH, (b with low pH and high aeration, (c) with low pH and high agitation or (d) with low pH and elevated temperature.
By “low pH”, it is meant any pH from about 2 to about 4.5, or any pH therebetween, for example any pH from about 2.5 to about 4.0, or any pH therebetween; for example pH 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.5 or any pH therebetween.
By “high aeration”, it is meant provision of a gas to the aqueous solution at a superficial gas velocity of from about 0.1 to about 100 cm/s, or any rate therebetween, for example any rate from about 0.1 to about 1 cm/s, or any rate therebetween. An alternative parameter to measure aeration rate that is known to one of skill in the art is vessel volumes per minute (vvm). In the context of the present invention, therefore, “high aeration” may also be defined as provision of a gas to the aqueous solution at a rate of from about 0.5 to about 5 vvm, or any rate therebetween. The gas may be a single gas, such as oxygen, nitrogen, and carbon dioxide, or a mixture of gases such as air.
By high agitation, it is meant mixing of the aqueous solution by shaking from about 300 to about 1000 rpm, or any rate therebetween, or by impeller stifling with a tip speed of from about 0.5 to about 10 m/s, or any rate therebetween, for example from about 0.5 to about 3 m/s. An alternative parameter to measure agitation that is known to one of skill in the art, particularly as it relates to agitation in bioreactors, is horse power (hp) per 100 gallons. In the context of the present invention, therefore, “high agitation” may also be defined as mixing of the aqueous solution at from about 0.2 hp/100 gallons to about 15 hp/100 gallons.
By elevated temperature, it is meant any temperature from about 30° C. to about 60° C., or any temperature therebetween, for example any temperature from about 40° C. to about 60° C., or any temperature therebetween, for example 30, 35, 40, 45, 50, 52, 54, 56, 58, 60° C., or any temperature therebetween.
The modified TrCel3A exhibits improved stabilityrelative to a parental TrCel3A from which it is derived when the tau of the modified TrCel3A is at least 2-fold, for example from about 2-fold to about 500-fold, higher than the tau of the parental TrCel3A under identical conditions of low pH, low pH and high aeration, low pH and high agitation, or low pH and elevated temperature. For example, the tau of the modified TrCel3A may be from about 2-fold to 250-fold higher than the tau of the corresponding parental TrCel3A, or any value in between, from about 3-fold to about 200-fold higher than the tau of the corresponding parental TrCel3A, or any value in between, or for example the tau may be from about 2-, 3-, 5- 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 120-, 140-, 160-, 180-, 200-, 220-, 240-, 250-, 300-, 350-, 400-, 450-, or 500-fold higher, or any value therebetween, than the tau of the corresponding parental TrCel3A under identical conditions. Example 8 details an assay for measuring the tau of native and modified TrCel3A beta-glucosidases.
The stability of several modified TrCel6A beta-glucosidases were compared by incubation of the enzymes at low pH (3.0) under conditions of severe agitation produced by swirling the enzyme solution in baffled flasks at 400 rpm and measuring the residual activity at several time points taken over a period of 30 minutes. The residual beta-glucosidase activity was determined via a chromogenic assay using para-nitrophenyl-beta-D-glucopyranoside as a substrate as described in Example 1
The effect of amino acid substitutions at positions 66, 72, 101, 235, 248, 369 and 386, was determined via a comparative study of the modified TrCel3A and the parental wild-type TrCel3A. The relative increase in tau over that of the parental TrCel3A (where the value of tau for the parental TrCel3A is set to 1.0) is shown in Table 2 below. Inactivation curves for these variants are shown in FIGS. 5, 7 and 8.







TABLE 2







Increased stability of Modified TrCel3A










Amino acid substitution
Relative Tau













None (TrCel3A)
1.0



S72N
4.3



V66I-S72N
13.9



V101M
2.9



T235S
2.9



N248K
3.8



N369K
8.2



A386T
5.1



S72N-F96L-V101M
19.4



S72N-F96L-V101M-N369K
145



S72N-F96L-V101M-N369K-A386T
134



S72N-V101M-N369K-A386T
205



V66I-S72N-V101M-N369K-A386T
97



V66I-S72N-F96L-V101M-N369K-A386T
59



S72E-F96L-V101M-N369K-A386T
194



S72N-F96L-V101M-N369P-A386T
330









Genetic Constructs Encoding Modified TrCel3A

The present invention also relates to a genetic construct comprising a nucleic acid sequence encoding the modified TrCel3A operably linked to regulatory nucleic acid sequences directing the expression and secretion of the modified TrCel3A from a host microbe. By “regulatory nucleic acid sequences” it is meant nucleic acid sequences directing the transcription and translation of the modified TrCel3A-encoding nucleic acid sequence and a nucleic acid sequence encoding a secretion signal peptide capable of directing the secretion of the modified TrCel3A from the host microbe. The regulatory nucleic acid sequences are preferably functional in a fungal host. The regulatory nucleic acid sequences may be derived from genes that are highly expressed and secreted in the host microbe under industrial fermentation conditions. For example, the regulatory nucleic acid sequences may be derived from any one or more of the Trichoderma reesei cellulase or hemicellulase genes.
The genetic construct may further comprise a selectable marker gene to enable isolation of a genetically modified microbe transformed with the construct as is commonly known to those of skill in the art. The selectable marker gene may confer resistance to an antibiotic or the ability to grow on medium lacking a specific nutrient to the host organism that otherwise could not grow under these conditions. The present invention is not limited by the choice of selectable marker gene, and one of skill in the art may readily determine an appropriate gene. For example, the selectable marker gene may confer resistance to hygromycin, phleomycin, kanamycin, geneticin, or G418, or may complement a deficiency of the host microbe in one of the trp, arg, leu, pyr4, pyr, ura3, ura5, his, or ade genes or may confer the ability to grow on acetamide as a sole nitrogen source.
The genetic construct may further comprise other nucleic acid sequences, for example, transcriptional terminators, nucleic acid sequences encoding peptide tags, synthetic sequences to link the various other nucleic acid sequences together, origins of replication, and the like. The practice of the present invention is not limited by the presence of any one or more of these other nucleic acid sequences.
Genetically Modified Microbes Producing Modified TrCel3A
The modified TrCel3A may be expressed and secreted from a genetically modified microbe produced by transformation of a host microbe with a genetic construct encoding the modified TrCel3A. The host microbe may be a yeast or a filamentous fungus, particularly those microbes that are members of the phylum Ascomycota. Genera of yeasts useful as host microbes for the expression of modified TrCel3A beta-glucosidase of the present invention include Saccharomyces, Pichia, Hansenula, Kluyveromyces, Yarrowia, and Arxula. Genera of fungi useful as microbes for the expression of modified TrCel3A beta-glucosidases of the present invention include Trichoderma, Hypocrea, Aspergillus, Fusarium, Humicola, Neurospora, and Penicillium. For example, the host microbe may be an industrial strain of Trichoderma reesei. Typically, the host microbe is one which does not express a parental TrCel3A.
The genetic construct may be introduced into the host microbe by any number of methods known by one skilled in the art of microbial transformation, including but not limited to, treatment of cells with CaCl2, electroporation, biolistic bombardment, PEG-mediated fusion of protoplasts (e.g. White et al., WO 2005/093072). After selecting the recombinant fungal strains expressing the modified TrCel3A, the selected recombinant strains may be cultured in submerged liquid fermentations under conditions that induce the expression of the modified TrCel3A.
Production of Modified TrCel3A
The modified TrCel3A of the present invention may be produced in a fermentation process using a genetically modified microbe comprising a genetic construct encoding the modified TrCel3A in submerged liquid culture fermentation.
Submerged liquid fermentations of microorganisms, including Trichoderma and related filamentous fungi, as one of skill in the art would know are typically conducted as a batch, fed-batch or continuous process. In a batch process, all the necessary materials, with the exception of oxygen for aerobic processes, are placed in a reactor at the start of the operation and the fermentation is allowed to proceed until completion, at which point the product is harvested. In a fed-batch process, the culture is fed continuously or sequentially with one or more media components without the removal of the culture fluid. In a continuous process, fresh medium is supplied and culture fluid is removed continuously at volumetrically equal rates to maintain the culture at a steady growth rate,
The process for producing the modified TrCel3A of the present invention may be performed as a batch, fed-batch, a repeated fed-batch, a continuous process or any combination thereof. For example, the process may be a fed-batch process.
One of skill in the art is aware that fermentation medium comprises a carbon source, a nitrogen source and other nutrients, vitamins and minerals can be added to the fermentation media to improve growth and enzyme production of the host cell. These other media components may be added prior to, simultaneously with or after inoculation of the culture with the host cell.
For the process for producing the modified TrCel3A of the present invention, the carbon source may comprise a carbohydrate that will induce the expression of the modified TrCel3A from a genetic construct in a genetically modified microbe. For example, if the genetically modified microbe is a strain of Trichoderma and the genetic construct comprises a cellulase or hemicellulase promoter operably linked of the modified TrCel3A nucleic acid sequence, the carbon source may comprise one or more of cellulose, cellobiose, sophorose, xylan, xylose, xylobiose and related oligo- or poly-saccharides known to induce expression of cellulases, hemicellulases and beta-glucosidase in Trichoderma. 
In the case of batch fermentation, the carbon source may be added to the fermentation medium prior to or simultaneously with inoculation. In the cases of fed-batch or continuous operations, the carbon source may also be supplied continuously or intermittently during the fermentation process.
The process for producing the modified TrCel3A of the present invention may be carried at a temperature from about 20° C. to about 40° C., or any temperature therebetween, or from 20, 22, 25, 26, 27, 28, 29, 30, 32, 35, 37, 40° C. or any temperature therebetween.
The process for producing the modified TrCel3A of the present invention may be carried out at a pH from about 3.0 to 6.5, or any pH therebetween, for example from about pH 3.0, 3.2, 3.4, 3.5, 3.7, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.2, 5.4, 5.5, 5.7, 5.8, 6.0, 6.2, 6.5 or any pH therebetween.
The process for producing the modified TrCel3A of the present invention may be carried out aerobically, with a superficial gas velocity of from about 0.1 to about 100 cm/s. For example, the superficial gas velocity may be from about 0.1 to about 1.0 cm/s. Alternatively, the superficial gas velocity used in the process for producing the modified TrCel3A of the present invention may be from about 0.5 to about 5 vvm, or any rate therebetween.
The process for producing the modified TrCel3A of the present invention may be carried out in a shakeflask which is shaken from about 300 to about 1000 rpm, or in a bioreactor which is agitiated by impeller with an impeller tip speed of from about 0.5 to about 10.0 m/s, or any speed therebetween for example at an impeller tip speed from about 0.5 to 3 m/s, or any speed therebetween. Alternatively, the bioreactor may be agitated at a power from about 0.2 hp/100 gallons to about 15 hp/100 gallons, or any power therebetween.
Following fermentation, the fermentation broth containing the cellulase enzyme may be used directly, or the cellulase enzyme may be separated from the fungal cells, for example by filtration or centrifiguation. Low molecular solutes such as unconsumed components of the fermentation medium may be removed by ultrafiltration. The cellulase enzyme maybe concentrated, for example, by evaporation, precipitation, sedimentation or filtration. Chemicals such as glycerol, sucrose, sorbitol and the like may be added to stabilize the cellulase enzyme. Other chemicals, such as sodium benzoate or potassium sorbate, may be added to the cellulase enzyme to prevent growth of microbial contamination.
The Use of Modified TrCel3A for the Hydrolysis of Cellulosic Substrates
The modified TrCel3A of the invention, may be combined with one or more cellulases to produce a cellulase mixture for use in the enzymatic hydrolysis of cellulose. For the purpose of the present invention, cellulases include all enzymes and proteins known to participate in the conversion of cellulose to soluble sugars, including but not limited to cellobiohydrolases (EC 3.2.1.91), endoglucanases (E.C 3.2.1.4), and other accessory enzymes that enhance the enzymatic conversion of cellulose to soluble sugars such as swollenins, expansins, and the like. In addition to the modified TrCel3A and cellulases, the cellulase mixture may comprise other enzymes such as other beta-glucosidases, hemicellulases, glucuronidases, galacturonases, esterases, galactosidases, amylases, and glucoamylases. It is understood that the enzymatic hydrolysis of cellulose by cellulase mixtures comprising the modified TrCel3A beta-glucosidases of the present invention is not limited by the composition of the cellulase mixture.
The cellulase mixture comprising the modified TrCel3A of the present invention may be used for enzymatic hydrolysis of cellulose present in “pretreated lignocellulosic feedstock.” A pretreated lignocellulosic feedstock is a material of plant origin that, prior to pretreatment, contains at least 20% cellulose (dry wt), more preferably greater than about 30% cellulose, even more preferably greater than 40% cellulose, for example 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90% or any % therebetween, and at least 10% lignin (dry wt), more typically at least 12% (dry wt) and that has been subjected to physical and/or chemical processes to make the fiber more accessible and/or receptive to the actions of cellulolytic enzymes.
After pretreatment, the lignocellulosic feedstock may contain higher levels of cellulose. For example, if acid pretreatment is employed, the hemicellulose component is hydrolyzed, which increases the relative level of cellulose. In this case, the pretreated feedstock may contain greater than about 20% cellulose and greater than about 12% lignin. Lignocellulosic feedstocks that may be used in the invention include, but are not limited to, agricultural residues such as corn stover, wheat straw, barley straw, rice straw, oat straw, canola straw, and soybean stover; fiber process residues such as corn fiber, sugar beet pulp, pulp mill fines and rejects or sugar cane bagasse; forestry residues such as aspen wood, other hardwoods, softwood, and sawdust; or grasses such as switch grass, miscanthus, cord grass, and reed canary grass. The lignocellulosic feedstock may be first subjected to size reduction by methods including, but not limited to, milling, grinding, agitation, shredding, compression/expansion, or other types of mechanical action. Size reduction by mechanical action can be performed by any type of equipment adapted for the purpose, for example, but not limited to, a hammer mill.
Non-limiting examples of pretreatment processes include chemical treatment of a lignocellulosic feedstock with sulfuric or sulfurous acid, or other acids; ammonia, lime, ammonium hydroxide, or other alkali; ethanol, butanol, or other organic solvents; or pressurized water (See U.S. Pat. Nos. 4,461,648, 5,916,780, 6,090,595, 6,043,392, and 4,600,590).
The pretreatment may be carried out to hydrolyze the hemicellulose, or a portion thereof, that is present in the lignocellulosic feedstock to monomeric sugars, for example xylose, arabinose, mannose, galactose, or a combination thereof. Preferably, the pretreatment is carried out so that nearly complete hydrolysis of the hemicellulose and a small amount of conversion of cellulose to glucose occurs. During the pretreatment, typically an acid concentration in the aqueous slurry from about 0.02% (w/w) to about 2% (w/w), or any amount therebetween, is used for the treatment of the lignocellulosic feedstock. The acid may be, but is not limited to, hydrochloric acid, nitric acid, or sulfuric acid. For example, the acid used during pretreatment is sulfuric acid.
One method of performing acid pretreatment of the feedstock is steam explosion using the process conditions set out in U.S. Pat. No. 4,461,648. Another method of pretreating the feedstock slurry involves continuous pretreatment, meaning that the lignocellulosic feedstock is pumped through a reactor continuously. Continuous acid pretreatment is familiar to those skilled in the art; see, for example, U.S. Pat. No. 5,536,325; WO 2006/128304; and U.S. Pat. No. 4,237,226. Additional techniques known in the art may be used as required such as the process disclosed in U.S. Pat. No. 4,556,430.
As noted above, the pretreatment may be conducted with alkali. In contrast to acid pretreatment, pretreatment with alkali does not hydrolyze the hemicellulose component of the feedstock, but rather the alkali reacts with acidic groups present on the hemicellulose to open up the surface of the substrate. The addition of alkali may also alter the crystal structure of the cellulose so that it is more amenable to hydrolysis. Examples of alkali that may be used in the pretreatment include ammonia, ammonium hydroxide, potassium hydroxide, and sodium hydroxide. The pretreatment is preferably not conducted with alkali that is insoluble in water, such as lime and magnesium hydroxide.
The pretreated lignocellulosic feedstock may be processed after pretreatment but prior to the enzymatic hydrolysis by any of several steps, such as dilution with water, washing with water, buffering, filtration, or centrifugation, or a combination of these processes, prior to enzymatic hydrolysis, as is familiar to those skilled in the art.
The pretreated lignocellulosic feedstock is next subjected to enzymatic hydrolysis. By the term “enzymatic hydrolysis”, it is meant a process by which cellulase enzymes act on cellulose to convert all or a portion thereof to soluble sugars. Soluble sugars are meant to include water-soluble hexose monomers and oligomers of up to six monomer units that are derived from the cellulose portion of the pretreated lignocellulosic feedstock. Examples of soluble sugars include, but are not limited to, glucose, cellobiose, cellodextrins, or mixtures thereof. The soluble sugars may be predominantly cellobiose and glucose. The soluble sugars may be predominantly glucose.
The enzymatic hydrolysis process preferably converts about 80% to about 100% of the cellulose to soluble sugars, or any range therebetween. More preferably, the enzymatic hydrolysis process converts about 90% to about 100% of the cellulose to soluble sugars, or any range therebetween. In the most preferred embodiment, the enzymatic hydrolysis process converts about 98% to about 100% of the cellulose to soluble sugars, or any range therebetween. The enzymatic hydrolysis process may be batch hydrolysis, continuous hydrolysis, or a combination thereof. The hydrolysis process may be agitated, unmixed, or a combination thereof.
The enzymatic hydrolysis of cellulase using a cellulase mixture comprising the modified TrCel3A may be batch hydrolysis, continuous hydrolysis, or a combination thereof. The hydrolysis may be agitated, unmixed, or a combination thereof.
The enzymatic hydrolysis may be carried out at a temperature of about 45° C. to about 75° C., or any temperature therebetween, for example a temperature of 45, 50, 55, 60, 65, 70, 75° C., or any temperature therebetween, and a pH of about 3.0 to about 7.5, or any pH therebetween, for example a pH of about 3.0 to about 5.5, or any pH therebetween, for example a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or pH therebetween. The initial concentration of cellulose in the hydrolysis reactor, prior to the start of hydrolysis, is preferably about 4% (w/w) to about 15% (w/w), or any amount therebetween, for example 4, 6, 8, 10, 12, 14, 15% or any amount therebetween. The dosage of the cellulase enzyme mixture comprising the modified TrCel3A may be about 1 to about 100 mg protein per gram cellulose, or any amount therebetween, for example 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg protein per gram cellulose or any amount therebetween. The hydrolysis may be carried out for a time period of about 12 hours to about 200 hours, or any time therebetween, for example, the hydrolysis may be carried out for a period of 15 hours to 100 hours, or any time therebetween, or it may be carried out for 12, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200 hours, or any time therebetween. It should be appreciated that the reaction conditions are not meant to limit the invention in any manner and may be adjusted as desired by those of skill in the art.
The enzymatic hydrolysis is typically carried out in a hydrolysis reactor. The enzyme mixture is added to the pretreated lignocellulosic feedstock (also referred to as the “substrate”) prior to, during, or after the addition of the substrate to the hydrolysis reactor.
All of the enzymes in the cellulase mixture may be secreted from one strain of an organism, referred to herein as a “complete blend” of secreted enzymes. By the term “complete blend”, it is meant all proteins secreted extracellularly into the growth medium by a specific microorganism. The enzyme mixture may include the complete blend of enzymes secreted by Trichoderma reesei. 
The individual enzymes of the cellulase mixture comprising the modified TrCel3A beta-glucosidase may be expressed individually or in sub-groups from different strains of different organisms and the enzymes combined to make the cellulase enzyme mixture. It is also contemplated that the individual enzymes of the cellulase mixture may be expressed individually or in sub-groups from different strains of a single organism, such as from different strains of Trichoderma reesei, and the enzymes combined to make the cellulase mixture. Preferably, all of the enzymes are expressed from a single strain of Trichoderma reesei. 
EXAMPLES
The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention in any manner.
Example 1 describes evaluation of the stability of parental TrCel3A at different pH conditions with low and high agitation/aeration. Example 2 describes the strains and vectors used in the following examples. Example 3 describes the cloning of the TrCel3A gene and transformation of yeast. Example 4 describes the making of error prone-PCR libraries. Examples 5 and 6 describe the expression of parental and modified TrCel3A beta-glucosidases from yeast microculture and the high-throughput screening to identify modified TrCel3As with improved stability. Examples 7 and 8 describe the larger-scale expression and characterization of modified and native TrCel3A beta-glucosidases. Example 9 describes evaluation of the activity of parental and modified TrCel3A beta-glucosidases. Examples 10 and 11 describe the generation of an aggregate modified TrCel3A with multiple amino acid substitution and the preparation and screening of site-saturation mutagenesis libraries of the aggregate modified TrCel3A. Examples 12 and 13 describe the production of a modified TrCel3A beta-glucosidase from Trichoderma reesei. Example 14 describes the assay of the relative specific activity of the parental and modified TrCel3A beta-glucosidases. Examples 15 and 16 describe stability assays for parental and modified TrCel3A beta-glucosidases.
Example 1
Inactivation of TrCel3A in a Trichoderma reesei Cellulase Mixture
This example demonstrates that parental TrCel3A is inactivated under conditions of low pH, low pH and high agitation, low pH and high aeration, or low pH and elevated temperature
Samples of a cellulase mixture with enhanced levels of parental TrCel3A produced by T. reesei strain P59G were adjusted to pH 3.0, 3.5, 4.0 and 5.0 (FIG. 3A) and mixed at 30° C. or 50° C. in unbaffled flasks by orbital shaking at 200 rpm for up to 80 hours. Samples of a cellulase mixture with enhanced levels of parental TrCel3A produced by T. reesei strain P59G were adjusted to pH 3.0 or pH 5.0 (FIG. 3B) and incubated at 30° C. in unbaffled flask with no shaking or stiffing, in unbaffled flasks with orbital shaking at 200 rpm (“Shaking”) or in baffled flasks with a magnetic stirrer (“Stirring”) for up to 400 hours (FIG. 3B). Samples of the cellulase mixture containing the parental TrCel3A were removed at various time points and assayed for residual beta-glucosidase activity using a para-nitrophenyl-beta-D-glucoside (pNPG) as substrate.
Release of para-nitrophenol from pNPG is readily detected by its absorbance at 340 nm. The pNPGase assay is carried out at 50° C. in a Cary300 spectrophotometer, the concentration of substrate is 0.4 mM in 3 mL, to which 2 μg of a cellulase mixture comprising the parental TrCel3A (P59G cellulase) is added. The total protein concentration was also measured at each time point using the method of Bradford et al. (Analytical Biochemistry, 72:248-254, (1976)). For the P59G cellulase, this represents the addition of roughly 0.4 μg of TrCel3A. The TrCel3A activity is taken as the slope of the initial increase in A340. The pNPG activity at each time point was divided by the activity at t=0h in order to calculate the relative specific pNPG activity at each time point.
The results in FIG. 3 show that the parental TrCel3A is sensitive to inactivation under conditions of low pH and high agitation. FIG. 3A shows that at 50° C., the beta-glucosidase activity is essentially stable at pH 5, inactivates slowly at pH 4 and inactivates rapidly at pH 3.5 and 3.0 under conditions of low agitation (shaking at 200 rpm in unbaffled flask). At 30° C., the enzyme is stable at both pH 3 and 5 under these shaking conditions. The results shown in FIG. 3B demonstrate that parental TrCel3A is sensitive to inactivation in aqueous solution at low pH with high agitation even at 30° C. In the aqueous solution at pH 3.0 with stifling in a baffled flask, inactivation of the TrCel3A was observed; however, the parental TrCel3A is stable in aqueous solutions at low pH with low agitation (pH 3.0 with 200 rpm shaking in an unbaffled flask) and in aqueous solutions at higher pH with high agitation (pH 5.0 with stifling in a baffled flask).
Example 2
Strains and Vectors
Saccharomyces cerevisiae strain BJ3505 (pep4::HIS3 prb-Δ1.6R HIS3 lys2-208 trp1-Δ101 ura3-52 gal2 can 1) was obtained from Sigma and was a part of the Amino-Terminal Yeast FLAG Expression Kit. Escherichia coli strain DH5α(F−φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk−, mk+) phoA supE44 thi-1 gyrA96 relA1λ−) was obtained from Invitrogen. The YEp352/PGK91-1 vector was obtained from the National Institute of Health. The pGEM T-easy vector was obtained from Promega.
Trichoderma reesei strain P59G is a genetically modified strain that produces and secretes high levels of the TrCel3A beta-glucosidase, encoded by T. reesei bgl1, as described in U.S. Pat. No. 6,015,703. BTR213 is a derivative of RutC30 (ATCC #56765; Montenecourt and Eveleigh, 1979) produced by random mutagenesis and first selected for ability to produce larger clearing zones on minimal media agar containing 1% acid swollen cellulose and 4 g/L 2-deoxyglucose, and then selected for the ability to grow on lactose media containing 0.2 μg/ml carbendazim. Strain P107B is a derivative of strain BTR213 generated by replacing the cel6a gene with the Neurospora crassa pyr4 gene. The BTR213aux and P107Baux strains, deficient in uridine production, were isolated by the ability to grow on 5-FOA (5-fluororotic acid) and inability to grow prototrophically in the absence of uridine.
Example 3
Cloning of the Parental TrCel3A Gene into YEp352/PGK91-1 and Transformation in Yeast
The TrCel3A gene contains two introns. One intron is located in the secretion signal at position 323 bp to 391 bp, while the other is located within the gene at position 2152 bp to 2215 bp. The TrCel3A gene contains a unique NheI site located at position 1203 bp. In order to facilitate expression from yeast and cloning using NheI and KpnI restriction enzymes, the unique NheI located within TrCel3A at position 1203 bp and the second intron were removed by a three step PCR. The TrCel3A gene was amplified in three segments from a plasmid containing TrCel3A, pc/xBG(Xbal)-TV (U.S. Pat. No. 6,015,703) using iPROOF DNA polymerase (BioRad). The first fragment (A) was amplified using primers which introduced an NheI site at the 5′ end of the gene downstream of the secretion signal (AT048) and which removed the internal NheI site (AT051). The second fragment (B) was amplified using primers which removed the internal NheI site (AT050) and the intron at position 2152 to 2215 bp (AT053). The third fragment (C) was amplified using primers which removed the intron at position 2152 to 2215 bp (AT052) and introduced a KpnI site at the 3′ end of the gene, downstream of the stop codon (AT049). Gene products B and C were joined together (to make gene product D) using PCR with primers AT050 and AT049. Gene product D was joined with gene product A using PCR with primers AT048 and AT049 to obtain TrCel3A without introns and with unique NheI and KpnI sites at the 5′ and 3′ ends, respectively. The final gene product was cloned into the pGEM T-easy vector (Promega) as per the manufacturer's instructions to make plasmid pGEM-TrCel3A. Primer sequences are shown below:







AT048:







(SEQ ID NO: 17)







5′ CGC CAG GCT AGC GTT GTA CCT CCT GC


 


AT049:







(SEQ ID NO: 18)







5′ CTG AGG GTA CCG CTA CGC TAC CGA C


 


AT050:







(SEQ ID NO: 19)







5′ CCC GCT AGT ATT GCC GTC GTT GGA TC


 


AT051:







(SEQ ID NO: 20)







5′ CCA ACG ACG GCA ATA CTA GCG GGC TTC


 


AT052:







(SEQ ID NO: 21)







5′ GTT CGG CTA TGG ACT GTC TTA CAC CAA GTT CAA CTA


C


 


AT053:







(SEQ ID NO: 22)







5′ GTT GAA CTT GGT GTA AGA CAG TCC ATA GCC GAA CTC






A DNA adapter containing NheI, KpnI, and EcoRI restriction sites was prepared by annealing primers AT046 and AT047 together. The adapter was inserted into a YEp based-plasmid containing the pgk1 promoter, alpha mating factor secretion signal, and pgk1 terminator sequences to make plasmid YEp352/PGK91-1/αssNKE. Specifically, the adapter was inserted as an NheI/EcoRI fragment into the NheI and EcoRI sites located downstream of the alpha mating factor secretion signal and upstream of the pgk1 terminator. Primer sequences are shown below:








AT046:



5′ CTA GCT GAT CAC TGA GGT ACC G
(SEQ ID NO: 23)


 


AT047:


5′ AAT TCG GTA CCT CAG TGA TCA G
(SEQ ID NO: 24)






Plasmid pGEM-TrCel3A was digested with NheI and EcoRI to release the 2235 bp TrCel3A gene. The fragment was purified and ligated into the NheI and EcoRI sites of YEp352/PGK91-1/αssNKE to obtain YEp352/PGK91-1/αssCel3A.
A DNA adapter containing SpeI, NheI, KpnI, and EcoRI restriction sites was prepared by annealing primers AT044 and AT045 together. The adapter contains sequences coding for six histidine residues downstream of the SpeI site and upstream of the NheI site. The adapter was inserted into a YEp based-plasmid containing the pgk1 promoter, alpha mating factor secretion signal, and pgk1 terminator sequences to make plasmid YEp352/PGK91-1/αss6HNKE. Specifically, the linker was inserted as an NheI/EcoRI fragment into the NheI and EcoRI sites located downstream of the alpha mating factor secretion signal and upstream of the pgk1 terminator. Primer sequences are shown below:







AT044:







(SEQ ID NO: 25)







5′ CTA GTC ATC ACC ATC ACC ATC ACG CTA GCT GAT CAC


TGA GGT ACC G


 


AT045:







(SEQ ID NO: 26)







5′ AAT TCG GTA CCT CAG TGA TCA GCT AGC GTG ATG GTG


ATG GTG ATG A






Plasmid pGEM-TrCel3A was digested with NheI and EcoRI to release the 2235 bp TrCel3A gene. The fragment was purified and ligated into the NheI and EcoRI sites of YEp352/PGK91-1/αss6HNKE to obtain YEp352/PGK91-1/αss6H-TrCe13A (FIG. 1). The vector YEp352/PGK91-1/αss6H-TrCel3A has been deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209. The deposit was made on Dec. 22, 2010, and was assigned ATCC Deposit No. PTA-11562.
A random mutagenesis library was generated using a Mutazyme® II DNA polymerase method. A series of four independent PCR were performed using 5, 10, 15, 20 ng of YEp352/PGK91-1/αss6H-TrCel3A vector and the Mutazyme® II DNA polymerase with primers YalphaN21 and 3′PGK-term. Annealing temperature was set to 50° C. The amplification was done for 20 cycles. The four PCR products were pooled and diluted to 16 ng/μL. The YEp352/PGK91-1/αss6H-TrCel3A vector was digested with NheI and KpnI and the empty vector fragment was isolated. This linear fragment and the final amplicon were transformed simultaneously and cloned by in vivo recombination into yeast strain BJ3505 (Butler, T. and Alcalde, M. 2003).








YalphaN21:



5′AGC ACA AAT AAC GGG TTA TTG
(SEQ ID NO: 27)


 


3′PGK-term:


5′GCA ACA CCT GGC AAT TCC TTA CC
(SEQ ID NO: 28)






Example 5
Expression of Parental and Modified TrCel3A Beta-glucosidases from Microplate Cultures
This example describes the selection and expression of TrCel3A from Saccharomyces cerevisiae for use in a high-throughput screening assay.
S. cerevisiae transformants were grown on plates containing synthetic complete medium (SC: 2% agar w/v, 0.17% yeast nitrogen base w/v, 0.078%-Ura drop-out supplement w/v, 2% glucose w/v, 2% casamino acids w/v, 0.5% ammonium sulfate w/v, pH 5.5) for 4-5 days at 30° C. Each growth plate was replicated by transferring a portion of each colonies, using sterilized velvet, to a screen-out plate containing SC medium plus 0.1% esculin hydrate and 0.03% FeCl3. Colonies which turned black after incubation for 3-4 days at 30° C. were identified as expressing active enzyme. Colonies were correlated back to their original growth plate and selected for liquid media expression cultures by toothpick inoculation of 1 mL SC media in 96-deepwell plates containing one glass bead (1.5-2.0 mm). Expression cultures were grown for 3 days at 30° C. and 250 rpm with humidity control. Glycerol stocks were prepared by transferring 0.050 mL of liquid culture to the corresponding wells of a microplate containing 0.050 mL of 40% glycerol and stored at −80° C. Expression culture plates were centrifuged at 1600×g for 5 minutes to pellet cells and supernatant was aspirated for screening assays.
Example 6
Screening of Gene Libraries for Modified TrCel3A Beta-glucosidases with Improved Stability at Low pH
This example describes the screening of modified Trichoderma reesei TrCel3As for improved stability at low pH by comparison to parent TrCel3A that had been cloned into Saccharomyces cerevisiae. 
Modified TrCel3As from yeast microcultures, as described in Example 5, were pre-incubated using two distinct 0.1 mL citrate buffered conditions in a 96-well PCR plate format. An aliquot of supernatant from each microculture was pre-incubated at both pH 5.0 and pH 3.5 (200 mM citrate buffer) for 30 minutes at 46° C. Residual beta-glucosidase activity of each modified TrCel3A was assessed by adding an aliquot of the pre-incubated mixture to 0.5 mM 4-nitrophenyl-β-D-gluco-pyranoside (pNPG) in 200 mM pH 5.0 citrate buffer and incubating at 50° C. for 20 minutes. The reaction was stopped by the addition of 400 mM Na2CO3 buffer. Absorbance was measured at 420 nm. Contained in each 96-well PCR plate were six parental TrCel3A controls used for comparison. A 3.5 pH/5.0 pH stability ratio was calculated for all modified TrCel3As and parental TrCel3A by dividing the activity after pre-incubation at pH 3.5 by the activity after pre-incubation at pH 5.0. The pH 3.5/pH 5.0 stability ratio for each modified TrCel3A was compared to the average ratio of the six parental TrCel3A controls on each plate and positives were selected at the 95% confidence level using a t-test. A sample of the data from one screening plate can be found in FIG. 4. All positive modified TrCel3A beta-glucosidases were produced again in microculture and re-screened to reduce the number of false positives.
Example 7
Expression of Parental and Modified TrCel3As from Large Scale Cultures
500 mL of sterile YPD media (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) was inoculated with 10 mL of overnight cultures of transformed Saccharomyces cerevisiae in the same media which had been inoculated with cells freshly picked from an agar plate. The culture was then incubated for 96 hours at 30° C. with shaking at 250 rpm.
After incubation, the 500 mL yeast culture was centrifuged for 10 minutes at 16,000×g and the yeast cell pellet was discarded. A standard antibody capture ELISA was followed (Harlow and Lane, 1988, p. 564-565). Yeast filtrates were diluted with carbonate buffer (pH 9.6) and boiled for 5 min prior to microplate (Costar #9018) coating. Primary His-tag antibody (Sigma #H1029) and peroxidase labeled secondary antibody (Sigma #A4416) were used at a dilution of 1:2000.
Example 8
Characterization of Modified TrCel3A Beta-glucosidases at Low pH and High Agitation
Yeast culture supernatants containing the parental and modified TrCel3As were collected as described in Example 7 and adjusted to pH 3.0 or pH 5.0 with 10 mL of 250 mM citrate and 500 mM phosphate buffers in the proportions 4:1 or 1:1, respectively. The samples were then incubated in baffled flasks at 30° C. with agitation at 400 rpm. The concentration of TrCel3A for the different samples in each assay was normalized to the sample with the lowest concentration. The difference in volume was made up with cell-free spent medium from fermentation of Saccharomyces containing the empty vector to a total volume of 50 mL. The range of TrCel3A concentration in the different assays was 4.8-10.8 μg/mL.
Aliquots were sampled over a period of 96 hours and measured for activity on 0.4 mM para-nitrophenyl β-D-glucopyranoside (pNPG) in 167 mM citrate pH 5.0 at 50° C. The concentration of enzyme was 1.9-4.3 μg/mL. The slope of the change in absorbance at 340 nm (A340) was used as a measure of enzyme activity. Data were plotted as a function of time and fit with a first-order decay model using the Solver function in Microsoft Excel. 95% confidence intervals of the fit for the ki values obtained were calculated using standard methods (Motulsky and Christopolous, 2003). For each modified TrCel3A the tau (in hours), which is the inverse of the inactivation constant ki, was compared to that of the parental TrCel3A using a type 2, two-tailed t-test.
The results in FIG. 5 and Table 2 show that the following amino acid substitutions increase the tau value, and hence improve the stability, of TrCel3A at low pH: V66I, S72N, V101M, T235S, N248K, N369K, A386T.
Example 9
Determining the Enzymatic Activity of Modified TrCel3A Beta-glucosidases
The concentration of parental or modified TrCel3A in yeast filtrates was determined by ELISA as described in Example 7.
The enzymatic activity of yeast culture filtrates containing parental and modified TrCel3A beta-glucosidases was measured in a pNPG assay (0.4 mM pNPG, 50 mM citrate pH 5.0, 50° C.) as described in Example 1. For each activity assay, sufficient yeast culture filtrate was added to the pNPG substrate solution to bring the concentration of parental or modified TrCel3A to a final concentration of 5 μg/mL (based on the concentrations determined by ELISA) and the change in absorbance at 340 nm was monitored. The initial slope of the pNP production curve was determined using Microsoft Excel and taken as a measure of the activity of the variant. Activities were measured in triplicate and the mean activity of each variant was compared to the mean activity of the parental TrCel3A using a t-test (type 2, two-tailed).
The activities of the modified and parental TrCel3A beta-glucosidases are plotted versus the tau value for each in FIG. 6. The activity of the modified TrCel3A beta-glucosidases are within 20% of the activity of the parental TrCel3A demonstrating that the amino acid substitutions that lead to improved stability are not detrimental to the activity of the enzyme.
Example 10
Construction of Aggregate Modified TrCel3A Beta-glucosidases with Multiple Amino Acid Substitutions
Using YEp352/PGK91-1/αss 6H-Cel3A-S72N as a template, additional mutations were introduced using a two-step PCR method involving megaprimer synthesis followed by megaprimer PCR using the High Fidelity iProof Taq Polymerase (BioRad). The internal primers were modified to introduce the desired amino acid substitutions into the TrCel3A construct. The external plasmid primers (YalphaN21 and 3′PGK-term) were used to amplify the final product. The megaprimers and final products were purified using the Wizard® SV Gel and PCR Clean-Up System.







TABLE 3







Generation of aggregate modified TrCel3A enzymes by PCR.



















Modified


PCR
Step
Template
Primer 1
Primer 2
Amplicon
TrCel3A





1
1
YEp352/PGK91-1-αss-
YalphaN21
DK006
PCR 1 Step 1
TrCel3A-S72N-




6H-TrCel3A-S72N



F96L-V101M



1
YEp352/PGK91-1-αss-
DK005
3′PGK-term
PCR 1 Step 1




6H-TrCel3A-S72N



2
Both PCR 1 Step 1
YalphaN21
3′PGK-term
YEp352/PGK91-1-




megaprimers


αss-6H-TrCel3A-







S72N-F96L-V101M


2
1
YEp352/PGK91-1-αss-
YalphaN21
DK010
PCR 2 Step 1
TrCel3A-S72N-




6H-TrCel3A-S72N-



F96L-V101M-




F96L-V101M



N369K-A386T



1
YEp352/PGK91-1-αss-
DK009
3′PGK-term
PCR 2 Step 1




6H-TrCel3A-S72N-




F96L-V101M



2
Both PCR 2 Step 1
YalphaN21
3′PGK-term
YEp352/PGK91-1-




megaprimers


αss-6H-TrCel3A-







S72N-F96L-V101M-







N369K-A386T


3
1
YEp352/PGK91-1-αss-
YalphaN21
DK186
PCR 3 Step 1
TrCel3A-S72N-




6H-TrCel3A-S72N-



F96L-V101M-




F96L-V101M-N369K-



N369K




A386T



1
YEp352/PGK91-1-αss-
DK185
3′PGK-term
PCR 3 Step 1




6H-TrCel3A-S72N-




F96L-V101M-N369K-




A386T



2
Both PCR 3 Step 1
YalphaN21
3′PGK-term
YEp352/PGK91-1-




megaprimers


αss-6H-TrCel3A-







S72N-F96L-V101M-







N369K


4
1
YEp352/PGK91-1-αss-
YalphaN21
DK068
PCR 4 Step 1
TrCel3A-S72N-




6H-TrCel3A-S72N-



V101M-N369K-




F96L-V101M-N369K-



A386T




A386T



1
YEp352/PGK91-1-αss-
DK067
3′PGK-term
PCR 4 Step 1




6H-TrCel3A-S72N-




F96L-V101M-N369K-




A386T



2
Both PCR 4 Step 1
YalphaN21
3′PGK-term
YEp352/PGK91-1-




megaprimers


αss-6H-TrCel3A-







S72N-V101M-







N369K-A386T


5
1
YEp352/PGK91-1-αss-
YalphaN21
DK066
PCR 5 Step 1
TrCel3A-V66I-




6H-TrCel3A-S72N-



S72N-V101M-




V101M-N369K-A386T



N369K-A386T



1
YEp352/PGK91-1-αss-
DK065
3′PGK-term
PCR 5 Step 1




6H-TrCel3A-S72N-




V101M-N369K-A386T



2
Both PCR 5 Step 1
YalphaN21
3′PGK-term
YEp352/PGK91-1-




megaprimers


αss-6H-TrCel3A-







V66I-S72N-V101M-







N369K-A386T


6
1
YEp352/PGK91-1-αss-
YalphaN21
DK066
PCR 6 Step 1
TrCel3A-V66I-




6H-TrCel3A-S72N-



S72N-F96L-




F96L-V101M-N369K-



V101M-N369K-




A386T



A386T



1
YEp352/PGK91-1-αss-
DK065
3′PGK-term
PCR 6 Step 1




6H-TrCel3A-S72N-




F96L-V101M-N369K-




A386T



2
Both PCR 6 Step 1
YalphaN21
3′PGK-term
YEp352/PGK91-1-




megaprimers


αss-6H-TrCel3A-







V66I-S72N-F96L-







V101M-N369K-







A386T









To facilitate cloning, the final product was digested with NheI+BamHI and ligated into vector YEp352/PGK91-1/αss 6H-Cel3A linearized with NheI+BamHI. The ligation mix was transformed into DH5α chemically-competent E. coli cells, plasmid extracted, and sequenced. Plasmids encoding the modified beta-glucosidases were transformed into yeast strain BJ3505.







5′YalphaN21







(SEQ ID NO: 27)







5′-AGCACAAATAACGGGTTATTG-3′


 


3′PGK-term







(SEQ ID NO: 28)







5′-GCAACACCTGGCCCTTACC-3′


 


5′DK005







(SEQ ID NO: 37)







5′-CGCGAACGTGGACAGCTGATCGGTGAGGAGATGAAGGCCTC-3′


 


3′DK006







(SEQ ID NO: 38)







5′-GAGGCCTTCATCTCCTCACCGATCAGCTGTCCACGTTCGCG-3′


 


5′DK009







(SEQ ID NO: 39)







5′-CGACGGGGCCTTGGGCATGGGTTGGGGTTCCGGCACCGTCAACT


A-3′


 


3′DK010







(SEQ ID NO: 40)







5′-CCATGCCCAAGGCCCCGTCGTCGCAGCCTTTGTCCTTGCACGAG


G-3′


 


5′DK065







(SEQ ID NO: 41)







5′-GACGGACCCCTCGGTATCCGATACTCGACAGGC-3′


 


3′DK066







(SEQ ID NO: 42)







5′-GCCTGTCGAGTATCGGATACCGAGGGGTCCGTC-3′


 


5′DK067







(SEQ ID NO: 43)







5′-CGCGAACGTGGACAGTTCATCGGTGAGGAGATG-3′


 


3′DK068







(SEQ ID NO: 44)







5′-CATCTCCTCACCGATGAACTGTCCACGTTCGCG-3′


 


5′DK185







(SEQ ID NO: 45)







5′-GGGTTCCGGCGCCGTCAACTATC-3′


 


3′DK186







(SEQ ID NO: 46)







5′-GATAGTTGACGGCGCCGGAACCC-3′






The aggregate modified TrCel3A beta-glucosidases produced by the PCR reactions in Table 3 were expressed from the yeast transformants using the methods described in Example 7. The stability of the aggregate modified TrCel3A beta-glucosidases were characterized using the methods described in Examples 8. As shown in Table 2 and FIG. 7, aggregates comprising a combination of two, three, four or five of the amino acid substitutions selected from V66I, S72N, V101M, N369K, A386T, exhibit tau values that are from 19-fold to over 200-fold higher than the tau value for the parental TrCel3A.
The vector encoding the aggregate modified TrCel3A comprising amino acid substituions S72N, F96L, V101M, N369K, and A386T (YEp352/PGK91-1/αss6H-Cel3A-AT003) was used as a template for site-saturation mutagenesis in Example 11. The aggregate modified TrCel3A encoded by this vector (TrCel3A-AT003) was expressed in Trichoderma reesei as described in Example 12 and the yeast vector encoding this aggregate modified TrCel3A.
Example 11
Construction of Site-Saturation Mutagenesis Libraries
Four amino acid positions in TrCel3A (S72, V101, N369 and A386) were chosen for site-saturation mutagenesis in order to find an amino acid which further improves stability at low pH. Site-saturation mutagenesis was performed by PCR (one-step PCR reaction and ligation of both fragments) using NNS primers (listed below). The YEp352/PGK91-1/αss6H-Cel3A-AT003 (S72N, F96L, V101M, N369K and A386T) vector was used as template, PCR was performed with iProof High-Fidelity DNA Polymerase (Biorad) and PCR fragments were ligated with T4 DNA ligase (Fermentas). One SSM library was generated for each position, keeping the other positions unchanged in the template. The PCR for fragment 1 was done using the NNS primer and the complementary external primer 3′PGK-term. The PCR for the second fragment was done with the second primer which did not contain NNS and the complementary external primer YalphaN21. No purification step was performed and both amplified PCR fragments were ligated since primers were phosphorylated. The ligated amplicons were cloned in YEp352/PGK91-1/αss 6HNKE using the gap repair method in yeast.









5′N72X-F:







(SEQ ID NO: 29)









5′P-GA TAC TCG ACA GGC NNS ACA GCC TTT ACG



 



5′ N72X-R:







(SEQ ID NO: 30)









5′P-GAA CAC CGA GGG GTC CGT CTT G



 



5′M101X-F:







(SEQ ID NO: 31)









5′P-C GGT GAG GAG NNS AAG GCC TCG G



 



5′M101-R:







(SEQ ID NO: 32)









5′P-ATG AAC TGT CCA CGT TCG CGG



 



5′K369X-F:







(SEQ ID NO: 33)









5′P-G CCC TCG TGC NNS GAC AAA GGC TG



 



5′K369X-R:







(SEQ ID NO: 34)









5′P-GAG TTT CTG GCG TGG TTA CC



 



5′T386X-F:







(SEQ ID NO: 35)









5′P-G GGT TCC GGC NNS GTC AAC TAT CC



 



5′T386X-R:







(SEQ ID NO: 36)









5′ P-CAA CCC ATG CCC AAG GCC






To perform a gap repair the vector YEp352/PGK91-1/αss6HNKE was digested with Nhe I and Kpn I and purified on gel. Saccharomyces cerevisiae strain BJ3505 was used as the host. The digested YEp352/PGK91-1/αss6HNKE vector and the ligated amplicons were transformed in the yeast strain BJ3505 using the procedure described by Gietz, R. D. and Woods, R. A. (Gietz, R. D. and Woods, R. A. 2002). The resulting site-saturation libraries were screened for modified TrCel3A beta-glucosidases with improved stability at low pH using the methods described in Examples 5 and 6. As shown in FIG. 3 and Table 2, two modified TrCel3A beta-glucosidases (TrCel3A-S72E-F96L-V101M-N369K-A386T and TrCel3A-S72N-F96L-V101M-N369P-A386T) were identified with significantly improved stability at low pH over the parental TrCel3A-S72N-F96L-V101M-N369K-A386T), indicating that the S72E and N369P substitutions are superior to the S72N and N369K substitutions for improving the stability of TrCel3A at low pH.
Example 12
Expression of an Aggregate Modified TrCel3A Beta-glucosidase in Trichoderma reesei 
12.1. Construction of T. reesei Transformation Vector
The backbone for the T. reesei vector expressing the aggregate modified TrCel3A containing amino acid substitutions S72N, F96L, V101M, N369K, and A386T (TrCel3A-AT003) was constructed as described bellow. The Spacer DNA required to introduce additional unique restriction sites, MluI and NotI, was amplified from pCAMBIA1301 (see URL: cambia.org/daisy/cambia/materials/vectors/585.html#dsy585_Description and GenBank Accession No. AF234297) using primers AC168 and AC169 and cloned into the SacI/BamHI sites of pUC19 to form pUC19-SP. The expression cassette, c/x-Cel6A-cbh2, containing cel7a promoter, cel6a coding gene and cel6a terminator was isolated from vector pC/X-S413P-TV (U.S. Publication No. US2008-0076152A1). The vector was digested with NdeI restriction enzyme, blunt-ended and digested with XbaI. This fragment was then cloned into the EcoRI/XbaI sites for pUC19-SP to form the vector pUC19-SP-c/xCel6A. To construct the TrCel3A-AT003 expression cassette, the cel7-xyn2 promoter and xyn2 secretion signal were amplified using primers AC230 and AC231 and the pC/X-S413P-TV vector (U.S. Publication No. 2008-0076152A1) as a template. The TrCel3A-AT003 coding sequence was amplified using AC232 and AC233 and YEp352/PGK91-1/αss6H-Cel3A-AT003 vector (Example 10) as a template. The generated fragments had short overlapping identical sequences at the 3′ and 5′ ends, respectively. Thus, both fragments were used as primers and templates in a ten-cycle PCR reaction, annealed to each other and filled ends to generate the c/x-Cel3A-AT003 fragment. The generated fragment was amplified using outside primers, AC231 and AC232. The amplified fragment was cloned into pJET (see URL: fermentas.com/catalog/kits/clonejetperclon.htm) to generate pJET-c/xCel3A-AT003, which was verified by sequencing. The c/x-Cel3A-AT003 fragment was then isolated from pJET-c/xCel3A-AT003 vector as a MluI/KpnI fragment and ligated into the same sites of pUC19-SP-c/xCel6A to generate pUC19-SP-c/xCel3A-AT003. A selectable marker cassette containing the Neurospora crassa pyr4 gene was amplified from pNCBgl-NSNB(r) (U.S. Publication No. 2008-0076152A1) using primers AC323 and AC343 digested with PacI/NotI restriction enzymes and cloned into PvuI/NotI sites of pUC19-SP-c/xCel3A-AT003 vector generating final T. reesei transformation vector, pc/xCel3A-AT003-pyr4 (FIG. 2).







AC168







(SEQ ID NO: 47)







5′-GCAGAGCTCGCGGCCGCGAACCGACGACTCGTCCGTC-3′


 


AC169







(SEQ ID NO: 48)







5′-CTGGGATCCGATATCACGCGTGTGACATCGGCTTCAAATGGC-3′


 


AC230







(SEQ ID NO: 49)







5′-TTTACGCGTGATTATGGCGTACTAGAGAGCGG-3′


 


AC231







(SEQ ID NO: 50)







5′-CTGCAGGAGGTACAACCTGGCGCTTCTCCACAGCCACGG-3′


 


AC232







(SEQ ID NO: 51)







5′-GTGGAGAAGCGCCAGGTTGTACCTCCTGCAGGGACTCCATG-3′


 


AC233







(SEQ ID NO: 52)







5′-TTTGGTACCCTACGCTACCGACAGAGTGCTCG-3′


 


AC323







(SEQ ID NO: 53)







5′-TTTGCGGCCGCCATCATTCGTCGCTTTCGG-3′


 


AC343







(SEQ ID NO: 54)







5′-TTCGATCGACTATACCACCACCCACCG-3′






12.2. Transformation of Trichoderma reesei 

Trichoderma strains BTR213aux and P107Baux (Example 2) were transformed with the pc/xCel3A-AT003-pyr4 vector by biolistic gold particle bombardment using PDS-1000/He system (BioRad; E.I. DuPont de Nemours and Company). Gold particles (median diameter of 0.6 um, BioRad Cat. No. 1652262) were used as microcarriers. The following parameters were used in the optimization of the transformation: a rupture pressure of 1100 psi, a helium pressure of 29 mm Hg, a gap distance of 0.95 cm, a macrocarrier travel distance of 16 mm, and a target distance of 9 cm. The spore suspension was prepared by washing T. reesei spores from the PDA plates incubated for 4-5 days at 30° C. with sterile water. Approximately 1×106 spores was plated on 60 mm diameter plates containing minimal medium agar (MM). After particle delivery, all transformation plates were incubated at 30° C. for 5-10 days. Transformants arising on the transformation plates were transferred to MM media and incubated at 30° C. Isolated stable transformants were used for subsequent analysis.
Minimal medium (MM) agar:














Amount for



Component
1 L of medium



















KH2PO4
10
g



(NH4)2SO4
6
g



Na3Citrate-2H2O
3
g



FeSO4—7H2O
5
mg



MnSO4—H2O
1.6
mg



ZnSO4—7H2O
1.4
mg



CaCl2—2H2O
2
mg



Agar
20
g



20% Glucose f.s.
50
ml



1 M MgSO4—7H2O f.s.
4
mL











pH 5.5









12.3. Production of Modified TrCel36A-AT003 in Microcultures

To identify the transformants expressing the aggregate modified TrCel3A protein, all isolated stable transformants were grown in microculture. Approximately 5000 T. reesei spores were inoculated in each well of 24-well culture dish (COSTAR) containing 1 mL of Trichoderma microculture media. Plates were incubated for 5-7 days at 30° C. with shaking at 250 rpm.
Trichoderma Microculture Media













Component
Concentration g/L


















Cellulase-inducing cocktail
35



Ammonium sulphate
12.7



KH2PO4
8.0



MgSO4—7H2O
4.0



CaCl2—2H2O
1.0



FeSO4—7H2O
0.1



MnSO4—7H2O
0.032



ZnSO47H2O
0.028



CaCO3
20



Corn Steep Liquor (powder)
5




pH 4.24





**cellulase-inducing cocktail comprising, as a function of total carbohydrate, 56% gentiobiose, 14% sophorose, 6% cellobiose, 10% trehalose, 6% maltotriose, 4% glucose and 14% other carbohydrates






Cultures were transferred to microfuge tubes, cells were pelleted by microcentrifugation at 12,000 rpm, and the culture supernatants transferred to clean microfuge tubes. The total protein concentration of each supernatant was measured by Bradford protein assay as described in Example 1. The relative concentration of the aggregate modified TrCel3A produced by transformants was determined by ELISA as follows. Culture supernatents and purified component standards were diluted 0.01-10 μg/mL (based on total protein) in phosphate-buffered saline, pH 7.2 (PBS) and incubated overnight at 4° C. in microtitre plates (Costar EIA #9018). These plates were washed with PBS containing 0.1% Tween-20 (PBS/Tween) and then incubated in PBS containing 1% bovine serum albumin (PBS/BSA) for 1 h at room temperature. Blocked microtitre wells were washed with PBS/Tween. Rabbit polyclonal antisera specific for TrCel3A was diluted (1:16,000) in PBS/BSA, added to separate microtitre plates and incubated for 2 h at room temperature. Plates were washed and incubated with a goat anti-rabbit antibody coupled to horseradish peroxidase (Sigma #A6154), diluted 1/2000 in PBS/BSA, for 1 hr at room temperature. After washing, tetramethylbenzidine was added to each plate and incubated for 30 min at room temperature. The absorbance at 360 nm was measured in each well and converted into protein concentration using the TrCel3A standard curve. The relative concentration of TrCel3A protein was calculated by dividing TrCel3A concentration by the total amount of protein produced and the transformants possessing a relative TrCel3A abundance at about 20-30% of total protein were selected for analysis in 14 L fermentation (Table 3).







TABLE 3







Relative Cel3A expression levels produced by T. reesei transformants


grown in microcultures with cellulase inducible carbohydrates and


selected for further analysis in 14L fermentation











Relative amount



Strain name
of Cel3A, % of total protein













P59G (control)
42.7



BTR213aux (host)
6.0



BTRc/x-penta 54
29.1



BTRc/x-penta 46
54.3



BTRc/x-penta 69S
51.8



P107Baux (host)
6.4



P107Bc/x-penta 15S
36.1



P107Bc/x-penta 22
42.9









Example 13
Production of a Cellulase Mixture Comprising Modified TrCel3A
Spores of the selected T. reesei transformants were inoculated onto standard 85 mm Petri plates containing potato dextrose agar (PDA). These plates were incubated at 30° C. for 5 days to achieve a confluent growth of fresh green spores. To prepare the inoculum for fermentation testing, spores from a single PDA plate were transferred to 2 L, baffled Erlenmeyer flask containing 750 mL of liquid Berkley media (pH 5.5). Flasks were incubated at 28° C. for 3 days using an orbital agitator (Model G-52 New Brunswick Scientific Co.) running at 100 rpm.
Berkley Media for Flasks













Component
Concentration, g/L



















(NH4)2SO4
1.4




KH2PO4
2.0




MgSO4•7H2O
0.31




CaCl2•2H2O
0.53




Dry Corn Steep Liquor
5.1




Glucose
10




Trace elements*
1
mL/L





*Trace elements solution contains 5 g/L FeSO4•7H20; 1.6 g/L MnSO4•H20; 1.4 g/L ZnSO4•7H20.






The contents of each inoculum flask were transferred to a 14 L pilot scale fermentation vessel (Model MF114 New Brunswick Scientific Co.) set up with 10 L of Initial Media for Feb-Batch fermentation (pH 5.5). The vessel was run in batch mode until the glucose in the media was depleted. At this point, a cellulase-inducing cocktail comprising, as a function of total carbohydrate, 56% gentiobiose, 14% sophorose, 6% cellobiose, 10% trehalose, 6% maltotriose, 4% glucose and 14% other carbohydrates, was feed on a continuous basis from a stock that was 35% w/v of solids dissolved in water. Peristaltic pumps were used to deliver the carbon source at a feed at a rate of 0.4 grams of carbon per liter culture per hour. Operational parameters during both the batch and fed-batch portions of the run were: mixing by impeller agitation at 500 rpm, air sparging at 8 standard liters per minute, and a temperature of 28° C. Culture pH was maintained at 4.0-4.5 during batch growth and pH 3.0 or 5.0 during cellulase production using an automated controller connected to an online pH probe and a pump enabling the addition of a 10% ammonium hydroxide solution. Periodically, 100 mL samples of broth were drawn for biomass and protein analysis. After 165 hours of fermentation time 1 L of fermentation media was collected and filtered for further protein analysis.
Initial Media for Fed-Batch Fermentations













Component
Concentration, g/L



















(NH4)2SO4
2.20




KH2PO4
1.39




MgSO4•7H2O
0.70




CaCl2•2H2O
0.185




Dry Corn Steep Liquor
6.00




Glucose
13.00




Trace elements*
0.38
mL/L





*Trace elements solution contains 5 g/L FeSO4•7H20; 1.6 g/L MnSO4•H20; 1.4 g/L ZnSO4•7H20.






The total protein concentration of the final fermentation filtrates was measured by Bradford assay as described in Example 1. The concentration of TrCel3A-type enzymes in the final fermentation filtrates was measured by ELISA as described in Example 12.3.
Example 14
Assay of Specific Cellobiase Activity of Parental and Modified TrCel3A Beta-glucosidases Produced in pH 3.0 and pH 5.0 Fermentations
This example demonstrates that the relative specific activity, in this instance the specific activity of the beta-glucosidase produced in Trichoderma reesei fermentations conducted at pH 3.0 divided by the specific activity of the beta-glucosidase produced in Trichoderma reesei fermentations conducted at pH 5.0, is higher for the aggregate modified TrCel3A-AT003 (TrCel3A with amino acid substitutions S72N, F96L, V101M, N369K, and A386T) than for the parental TrCel3A.
Initial rate assays were used to measure the specific activity of the parental TrCel3A and the aggregate modified TrCel3A-AT003 beta-glucosidase in cellulase mixtures produced from Trichoderma reesei fermentations conducted at pH 5.0 or 3.0, on cellobiose. The cellulase enzyme mixtures comprising the beta-glucosidases were incubated with 30 mM cellobiose in 50 mM citrate buffer at pH 5.0. Six dilutions of the cellulase mixture, ranging from 1000- to 6000-fold were used. Samples were incubated at 50° C. for 30 min in deep well plates and then placed in a boiling water bath for 10 min to stop the reaction. The concentration of glucose produced at each dilution of cellulase mixture was measured using a glucose oxidase/horseradish peroxidise coupled system (Trinder P., 1969). The specific activity, in IU/mg, was determined by dividing the number of μmoles of glucose produced by the length of the assay, 30 min, and then by the number of milligrams of TrCel3A-type enzyme present in the reaction. The concentration of TrCel3A-type enzyme (parental or aggregate modified) in each experiment was determined using the protein concentration of the crude enzyme, as determined by the Bradford method as described in Example 1, and the fractional TrCel3A-type enzyme content of each culture filtrate, as determined by the ELISA as described in Example 13. Specific activity determinations were performed in triplicate.
As shown in FIG. 9, the specific cellobiase activity of the TrCel3A-type beta-glucosidase enzyme produced at pH 3.0 divided by the specific cellobioase activity of the TrCel3A-type beta-glucosidase enzyme produced at pH 5.0 is significantly higher for the aggregate TrCel3A-AT003 produced by the transformants of the BTR213aux or P107Baux host strains than for the parental TrCel3A produced by the P59G control strain. Therefore, the TrCel3A-AT003 beta-glucosidase is more stable under the low pH, highly aerated and highly agitated conditions of the Trichoderma reesei fermentations than is the parental TrCel3A.
Example 15
Stability of Modified and Parental TrCel3A under Conditions Mimicking Those of a Cellulose Hydrolysis Reaction
This example demonstrates that the stability of the aggregate modified TrCel3A is improved at reduced pH at a standard hydrolysis temperature, improved at a higher temperature at a standard pH, and improved under conditions of both increased temperature and reduced pH.
Trichoderma strains expressing cellulase mixtures comprising the parental and aggregate modified TrCel3A variants were fermented at pH 5.0 as described in Example 13. 10 mL samples of pH-adjusted cellulase mixtures were prepared in 35 mL screw-top glass centrifuge tubes through the addition of 9 mL cellulase mixture to 1 mL of 1.0 M citrate buffer, pH 5.0, 4.0, 3.5 and 3.0. These samples were incubated at 50° C. or 60° C. in air-heated incubators with 250 rpm orbital shaking. Samples were taken at 0, 0.5, 1, 2, 4, 6, 11.5, 24.5, 74 and 98 h and assayed for beta-glucosidase activity using the pNPG method described in Example 1.
As shown in FIG. 10 and in Table 4, the aggregate modified TrCel3A-AT003 beta-glucosidase is significantly more stable than the parental TrCel3A at 50° C. and pH 3.5 or 3.0 and at 60° C. at all pH's tested. These results suggest that the amino acid substitutions in TrCel3A-AT003 (S72N, F96L, V101M, N369K, and A386T) confer improved stability at low pH and at elevated temperature.







TABLE 4







Stability of parental TrCel3A and aggregate modified TrCel3A-AT003 at


low pH and elevated temperature.










Tau (h)














pH
TrCel3A
TrCel3A-AT003















50° C.
5.0
1000
1000




4.0
761
800




3.5
22.4
322




3.0
1.13
17.0



60° C.
5.0
130
639




4.0
12.6
176




3.5
2.20
11.9




3.0
0.224
1.38









The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.
REFERENCES


    Bhatia, Y., Mishra, S., and Bisaria, V. S. (2002) Microbial beta-Glucosidases: Cloning, Properties and Applications. Crit. Rev. Biotech. 22:375-407.
    Bommarius, A. S, and Karau, A. (2005) Deactivation of Formate Dehydrogenase (FDH) in Solution and at Gas-Liquid Interfaces. Biotechnol. Prog. 21:1663-72.
    Butler, T. and Alcalde, M. 2003. In Methods in Molecular Biology, vol. 231: (F. H. Arnold and G. Georgiou, editors), Humana Press Inc. Totowa (New Jersey), pages 17-22
    Cummings, C. and Fowler, T. (1996) Secretion of Trichoderma reesei beta-glucosidase by Saccharomyces cerevisiae. Curr. Genet. 29:227-33.
    Eijsink V G, Gaseidnes C., Borchert T V, van den Burg B. 2005. Directed Evolution of Enzyme Stability. Biomol. Eng. 22:21-30
    Elias, C. B. and Joshi, J. B. (1998) Role of Hydrodynamic Shear on Activity and Structure of Proteins. Adv. Biochem. Eng. 59:47-71.
    Fersht, A. (1998) Structure and Mechanism in Protein Science. W.H. Freeman and Co. USA
    Gietz, R. D. and Woods, R. A. 2002. Meth. Enzym. 350: 87-96
    Gunjikar, T. P., Sudhir S. B., and Joshi, J. B. (2001) Shear Deactivation of Cellulase, Exoglucanase, Endoglucanase and beta-Glucosidase in a Mechanically Agitated Reactor. Biotechnol. Prog. 17:1166-8.
    Harlow E. and Lane D. (1988) Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory press (Cold Spring Harbor, N.Y.), pages 564-565.
    Henrissat, B. (1991) A classification of glycosyl-dydrolases based on amino acid sequence similarities. Biochemical Journal, 293:781-788;
    Henrissat, B. and Bairoch, A. (1996) Updating the sequence-based classification of glycosyl hydrolases. Biochemical Journal, 316:695-696
    Jones, E. O., and Lee, J. M. (1988) Kinetic analysis of bioconversion of cellulose in attrition bioreactor. Biotechnol. Bioeng. 31:35-40.
    Montenecourt, B. and Eveleigh, D. 1979. Adv. Chem. Ser. 181: 289-301
    Motulsky, H. J. and Christopolous, A. (2003) Fitting models to biological data using linear and nonlinear regression. A pratical guide to curve fitting. GraphPad Software Inc., San Diego, Calif.
    Reese, E. T. (1980) Inactivation of Cellulase by Shaking and its Prevention by Surfactants. J. Appl. Biochem. 2:36-9.
    Sachse, H., Kude, J., Kerns, G., and Berger, R. (1990) Production of cellulase in a rotating disc fermenter using immobilized Trichoderma reesei cells. Acta Biotechnol. 10:523-29.
    Sinnott, M. L. (1990) Catalytic mechanisms of enzymic glycosyl transfer. Chem. Rev. 90:1171-202.
    Trinder, P. (1969) Determination of glucose in blood using glucose oxidase with an alternative oxygen accepter. Annals of Clinical Biochemistry, 6:24-27.
    Varghese, J. N., Hrmova, M., Fincher, G. B. (1999) Three-dimensional structure of a barley beta-D-glucan exohydrolase, a family 3 glycosyl hydrolase. Structure Fold. Des. 7: 179-190
    Weijers, S. R. and Van't Riet, K. (1992) Enzyme Stability in Downstream Processing 2: Quantification of Inactivation. Biotech. Adv. 10:251-73.
    Woodward, J. and Arnold, S. L. (1981) The Inhibition of beta-Glucosidase Activity in Trichoderma reesei C30 Cellulase by Derivatives and Isomers of Glucose. Biotech. Bioeng. 23:1553-62.

## Claims

1. An isolated Trichoderma reesei TrCel3A beta-glucosidase comprising one or more amino acid substitutions selected from the group consisting of V66I, S72E, S72N, F96L, V101M, T235S, N248K, N369K, N369P and A386T, wherein the amino acid sequence of the isolated TrCel3A beta-glucosidase comprises a sequence which is from about 90% to about 99.9% identical to wild-type Trichoderma reesei TrCel3A beta-glucosidase encoded by SEQ ID NO:100 and exhibits at least a 2-fold improvement in stability, relative to said wild-type Trichoderma reesei TrCel3A beta-glucosidase when incubated in an aqueous solution at a pH from about 2.0 to about pH 4.5.

2. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 1, comprising an amino acid sequence which from about 95% to about 99.9% identical to SEQ ID NO: 100.

3. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the isolated Trichoderma reesei TrCel3A beta-glucosidase exhibits from about a 2-fold to about a 500-fold improvement in stability, relative to said wild-type Trichoderma reesei TrCel3A beta-glucosidase when incubated in an aqueous solution at a pH from about 2.0 to about pH 4.5.

4. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 1, further comprising one or more amino acid substitutions selected from the group consisting of V43X, F96X, F260X, and 1543X.

5. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 4, further comprising one or more amino acid substitutions selected from the group consisting of V43I, V43C, F96L, V101A, V101G, F260I, F260V, F260Q, F260D, I543N, I543W, I543A, I543S, I543G, and I543L.

6. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the aqueous solution is subject to aeration at a superficial gas velocity of from about 0.1 to about 100 cm/s, to agitation by shaking at from about 300 to about 1000 rpm, or to agitation by stirring with impeller at a tip speed of from about 0.5 to about 10 m/s.

7. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 1, wherein the aqueous solution is subject to aeration at a superficial gas velocity of from about 0.5 to about 5 vvm, or to agitation in a bioreactor at from about 0.2 to about 15 hp/100 gallons.

8. The isolated Trichoderma reesei TrCel3A beta-glucosidase of claim 1, which exhibits at least a 2-fold improvement in stability, relative to said wild-type Trichoderma reesei TrCel3A beta-glucosidase when incubated in an aqueous solution at a pH from about 2.0 to about pH 4.5 and a temperature between about 30° C. and 60° C.

9. An isolated genetic construct comprising a nucleic acid sequence encoding the Trichoderma reesei TrCel3A beta-glucosidase according to claim 1.

10. The isolated genetic construct of claim 9, further comprising regulatory nucleic acid sequences that direct the expression and secretion of the Trichoderma reesei TrCel3A beta-glucosidase.

11. An isolated genetically modified microbe comprising the genetic construct of claim 9.

12. The isolated genetically modified microbe of claim 11, wherein said microbe is a species of yeast or filamentous fungus.

13. The isolated genetically modified microbe of claim 12, wherein said microbe is Saccharomyces cerevisiae or Trichoderma reesei.

14. A process for producing a Trichoderma reesei TrCel3A beta-glucosidase, comprising the steps of culturing the isolated genetically modified microbe of claim 11 under conditions that induce the expression and secretion of the Trichoderma reesei Tr Cel3A beta-glucosidase and recovering the Trichoderma reesei TrCel3A beta-glucosidase from the culture medium.

15. The process of claim 14, wherein the step of culturing is conducted at a pH from about 2.0 to about 4.5, a superficial gas velocity of about 0.1 to about 100 cm/s and an impeller tip speed of from about 0.5 to 10 m/s.

16. The process of claim 14, wherein the step of culturing is conducted at a pH from about 3.0 to about 4.5, a superficial gas velocity of about 0.5 to 5 vvm and is agitated at from about 0.2 to about 15 hp/100 gallons.

17. A process for the hydrolyzing a cellulose substrate comprising contacting said substrate with a cellulase mixture comprising the Trichoderma reesei TrCel3A beta-glucosidase of claim 1.

18. The process of claim 17, wherein the cellulose substrate is a pretreated lignocellulosic feedstock.

19. The process of claim 18, wherein the pretreated lignocellulosic feedstock is contacted with the cellulase mixture for about 12 hours to 200 hours at a pH from about 3.0 to 7.5 and a temperature from about 45° C. and 75° C.

20. A process for producing a Trichoderma reesei TrCel3A beta-glucosidase, comprising the steps of (i) transforming fungal host cells with the isolated genetic construct as defined in claim 9 to produce recombinant fungal strains; (ii) selecting the recombinant fungal strains expressing the Trichoderma reesei TrCel3A beta-glucosidase; and (iii) culturing selected recombinant strains in submerged liquid fermentations under conditions that induce the expression of the Trichoderma reesei TrCel3A beta-glucosidase.

21. An isolated Trichoderma reesei TrCel3A beta-glucosidase comprising the amino acid sequence of:
SEQ ID NO: 111 (TrCel3A-S72N-F96L-V101M-N369K full).