# Patent

## Title
Beta-glucosidase variants having improved activity, and uses thereof

## Abstract

The present invention relates to the expression and optimization of enzymes involved in lignocellulosic biomass decomposition. The present invention relates more particularly to beta-glucosidase variants comprising at least one modification among the amino acids located at positions 225, 238, 240 and 241, according to the numbering in SEQ ID No. 2 of Trichoderma reesei beta-glucosidase, and also relates to the use of said variants having improved effectiveness in methods for cellulose decomposition and biofuel production.

## Background
The present invention relates to the expression and optimization of enzymes involved in lignocellulosic biomass decomposition. The present invention relates more particularly to beta-glucosidase variants comprising at least one modification among the amino acids located at positions 225, 238, 240 and 241, according to the numbering in SEQ ID No. 2, of the beta-glucosidase of Trichoderma reesei, and also to the use of these variants with improved effectiveness in methods for cellulose decomposition and methods for producing biofuel (for example: ethanol, butanol, isopropanol).
The possibility of producing ethanol from cellulose has received a great deal of attention owing to the availability of large amounts of starting material and also to the cleanliness of ethanol as a fuel.
The natural cellulosic starting materials for such a process are denoted using the term “biomass”. Many types of biomass, including wood, agricultural residues, herbaceous crops and solid municipal waste, have been considered as starting materials for biofuel production. These materials are constituted mainly of cellulose, hemicellulose and lignin.
Cellulose is a polymer constituted of glucose molecules linked by beta 1-4 linkages, which is very resistant to decomposition or to depolymerization using acids, enzymes or microorganisms. Once the cellulose has been converted to glucose, said glucose is easily fermented to biofuel, for example ethanol, using a yeast.
The oldest methods studied for converting cellulose to glucose are based on acid hydrolysis. This process can be carried out in the presence of acid concentrates or dilute acids. However, several drawbacks, such as poor recovery of the acid when acid concentrates are used and low glucose production in the case of the use of dilute acids, prevent the acid hydrolysis process from reaching commercialization.
In order to overcome the drawbacks of the acid hydrolysis process, cellulose conversion processes have more recently involved enzymatic hydrolysis, using cellulose-type enzymes. This enzymatic hydrolysis of the lignocellulosic biomass (for example, cellulose) has, however, the drawback of being an expensive industrial process. As a result, it is necessary to use strains of microorganisms that secrete cellulases which are increasingly effective.
In this respect, many microorganisms contain enzymes which hydrolyze cellulose, such as the fungi Trichoderma, Aspergillus, Humicola or Fusarium and also bacteria such as Thermomonospora, Bacillus, Cellulomonas and Streptomyces. The enzymes present in these microorganisms have three types of activities that are of use in the conversion of cellulose to glucose and can be divided up into three groups: endoglucanases, which attack cellulose fibers randomly internally, exoglucanases, which will attack the ends of the fibers, releasing cellobiose therefrom, and beta-glucosidases, which will hydrolyze this cellobiose to glucose. These beta-glucosidases constitute the limiting step of the method for cellulose conversion. This is because the primary difficulty of the method lies in the conversion of the cellobiose to glucose, since any cellobiose not hydrolyzed at the end of the method represents a loss of yield during the production of biofuel.
This accumulation of cellobiose is a major problem in enzymatic hydrolysis, given that several microorganisms which produce cellulases, including Trichoderma, produce very little beta-glucosidase. Specifically, less than 1% of the total proteins produced by industrial Trichoderma strains are of beta-glucosidase type. This low amount of beta-glucosidase therefore results in a low capacity for hydrolyzing cellobiose to glucose, hence the accumulation of said cellobiose in the system. Moreover, a high concentration of cellobiose inhibits the activity of the other cellulases, and in particular the exoglucanases, for which cellobiose is the final reaction product.
Several approaches have been proposed for increasing the beta-glucosidase activity in the microorganisms and, consequently, the conversion of the cellobiose to glucose.
A first approach consists in adding exogenously produced beta-glucosidase to the mixtures secreted by the microorganisms, in order to improve the hydrolysis. However, this method is not commercially viable since it is much too expensive.
A second approach, as described in WO 92/010581, is to use genetic engineering to insert new copies of the beta-glucosidase gene into the genome of the microorganisms, in such a way that said microorganisms produce a larger amount of enzyme.
A third approach, described in WO 99/46362, consists in genetically modifying the microorganisms using a genetic construct which comprises a promoter, the mature beta-glucosidase gene and the xylanase secretion signal sequence. The presence of the xylanase secretion signal sequence makes it possible to significantly increase the amount of beta-glucosidase produced by the microorganisms.
However, in order for a hydrolysis of the lignocellulosic biomass to be effective and economically profitable, the enzymatic mixture must be produced by a one and only microbial strain, and must comprise balanced proportions of various enzymatic activities (inter alia, but not exclusively, exoglucanases, endoglucanases, xylanases and beta-glucosidases). By way of example, in the natural mixtures of Trichoderma reesei, the presence of 70-80% of exoglucanases, 15-20% of endoglucanases, a few percent of hemicellulases and approximately 0.5% of beta-glucosidases is generally noted. This mixture is perfectly suitable for hydrolyzing the majority of pretreated substrates (for example, such as wheat straw which has been steam-exploded under acid conditions) with acceptable yields. In summary, if the increase in beta-glucosidase activity is carried out by enrichment of the amount of enzyme, this must not be done to the detriment of the other enzymatic activities.
Consequently, the possibility of obtaining high beta-glucosidase activities without notably modifying the proportion of all the enzymes of the mixture would be a significant gain for the method for converting lignocellulosic biomass to biofuel.
With this perspective, the applicant companies have, to their great credit, found, after a great deal of research, an isolated or purified polypeptide having an improved beta-glucosidase activity compared with the beta-glucosidase activity of the wild-type BGL1 protein (SEQ ID No. 2), comprising an amino acid sequence in which at least one amino acid is modified compared with the amino acid sequence according to SEQ ID No. 2, said modified amino acid being chosen from positions 225, 238, 240 and 241 of the amino acid sequence SEQ ID No. 2, and said amino acid sequence having at least 75% sequence identity with SEQ ID No. 2 and preferably at least 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity with SEQ ID No. 2.
Furthermore, the polypeptides according to the invention have the advantage of being less sensitive to inhibition by glucose and, as a result, maintain a better beta-glucosidase activity in the presence of a high glucose concentration.
In one embodiment, the polypeptide as described above is characterized in that it has a beta-glucosidase activity determined in the presence of glucose which is improved compared with the beta-glucosidase activity of the wild-type BGL1 protein (SEQ ID No. 2) determined in the absence of glucose.
Those skilled in the art may, for example, determine the increase or, in other words, the improvement in the enzymatic activity of a polypeptide according to the invention by means of an enzymatic activity test using the substrate pnp-glucopyranoside. The amount of para-nitrophenol obtained after action of the beta-glucosidase may, for example, be determined by reading the optical density at 414 nm.
An example of a protocol, that those skilled in the art may use to determine whether a polypeptide according to the invention has an improved enzymatic activity compared with that of the wild-type BGL1 protein, is the following:

    
    
        formation of a stock culture of E. coli expressing a polypeptide according to the invention, overnight at 37° C.;
        inoculation of an LB culture medium with 1% of stock culture for 24 h at 20° C.;
        centrifugation for 2 minutes at 13 000 rpm;
        resuspension of the cell pellets with 100 mM succinate buffer at pH 5 (final OD600 of 100);
        incubation of 50 μl of cells with 100 μl of 100 mM succinate buffer, at pH 5, containing 15 mM of pnp-glucopyranoside for 1 h 30 at 50° C., followed by 5 minutes on ice;
        addition of 150 μl of 0.2 M Na2CO3;
        centrifugation for 2 minutes at 13 000 rpm;
        reading of the optical density at 414 nm on 150 μl of supernatant.
    
    


In addition, those skilled in the art may use the protocol described above, incubating the 50 μl of cells with 100 μl of 100 mM succinate buffer, at pH 5, containing 15 mM of pnp-glucopyranoside and 60 g/l of glucose for 1 h 30 at 50° C., in order to determine whether a polypeptide according to the invention is less sensitive to inhibition by glucose than the wild-type BGL1 protein.
In the context of the invention, a “modified” amino acid means a “substituted”, “inserted” or “deleted” amino acid.
According to one embodiment, the “modified” amino acid is “substituted” compared with the amino acid sequence according to SEQ ID No. 2.
According to one embodiment, the “modified” amino acid is “inserted” compared with the amino acid sequence according to SEQ ID No. 2.
According to one embodiment, the “modified” amino acid is “deleted” compared with the amino acid sequence according to SEQ ID No. 2.
According to one embodiment, the polypeptide as described above is characterized in that at least two amino acids of the amino acid sequence are modified compared with the amino acid sequence SEQ ID No. 2, said modified amino acids being chosen from positions 225, 238, 240 and 241 of the sequence SEQ ID No. 2.
According to one embodiment, the polypeptide as described above is characterized in that at least three amino acids of the amino acid sequence are modified compared with the amino acid sequence SEQ ID No. 2, said modified amino acids being chosen from positions 225, 238, 240 and 241 of the amino acid sequence SEQ ID No. 2.
According to one embodiment, the polypeptide as described above is characterized in that at least four amino acids of the amino acid sequence are modified compared with the amino acid sequence SEQ ID No. 2, said modified amino acids being those of positions 225, 238, 240 and 241 of the amino acid sequence SEQ ID No. 2.
According to one embodiment, the polypeptide as described above is characterized in that at least one, at least two, at least three or at least four amino acids of the amino acid sequence are modified compared with the amino acid sequence SEQ ID No. 2, said modifications being chosen from Q225H, V238I, T240G and T241S.
According to one embodiment, the polypeptide as described above is characterized in that one amino acid is modified compared with the sequence SEQ ID No. 2, said modification being Q225H.
According to one embodiment, the polypeptide as described above is characterized in that three amino acids are modified compared with the sequence SEQ ID No. 2, said modifications being V238I, T240G and T241S.
According to one embodiment, the polypeptide as described above is characterized in that four amino acids are modified compared with the sequence SEQ ID No. 2, said modifications being Q225H, V238I, T240G and T241S.
According to one embodiment, the polypeptide as described above also comprises at least one additional modified amino acid chosen from positions 97, 99, 100, 118, 119, 121, 123, 126, 127, 128, 130, 132, 134, 135, 140, 147, 151, 153, 163, 168, 173, 174, 177, 179, 182, 187, 193, 206, 207, 212, 217 and 621 of the amino acid sequence SEQ ID No. 2.
According to one embodiment, the polypeptide as described above is characterized in that said additional modified amino acid comprises one or more modifications selected from the group consisting of V97I, Y99F, S100G, V118T, N119E, I121M, E123Q, Q126E, F127Y, I128L, E130A, V132A, A134G, S135C, S135V, 1140L, P147A, T151I, Q153H, V163T, T168A, G173A, G173S, Q174E, N177E, I179L, V182A, V182N, I187C, L193V, N206D, P207V, L212M, T217L, L621F and L621T.
According to one embodiment, the polypeptide as described above is selected from the group consisting of:

    
    
        an amino acid sequence chosen from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14; or
        an amino acid sequence SEQ ID No. X having:
        
            i) a percentage of identical residues relative to the length of SEQ ID No. 4, 6, 8, 10, 12 or 14 of at least 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98% or 99%;
            ii) a percentage of identical residues relative to the length of SEQ ID No. X of at least 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98% or 99%.
        
        
    
    


Variants of the amino acid sequences of the present invention can be prepared by various conventional methods, such as random mutagenesis, site-directed mutagenesis, gene synthesis or shuffling, using all or a part of the nucleotide or peptide sequences presented in the present text. Such variants comprise, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the enzyme. The present invention relates to any variant obtained from the sequences presented in this text, provided that the variants of said amino acid sequences retain an improved beta-glucosidase function (as defined above) compared with Bgl-1.
In one embodiment, the invention relates to an amino acid sequence, hereinafter referred to, for practical reasons, as SEQ ID No. X, which, when aligned with SEQ ID No. 4, 6, 8, 10, 12 or 14, comprises:
a) a percentage of identical residues relative to the length of SEQ ID No. 4, 6, 8, 10, 12 or 14 of at least 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98% or 99%; and
b) a percentage of identical residues relative to the length of SEQ ID No. X of at least 70% identity, preferably 75%, 80%, 85%, 90%, 95%, 98% or 99%.
According to the invention, the percentage of identical residues relative to the length of SEQ ID No. 4, 6, 8, 10, 12 or 14 corresponds to the number of residues that are identical between SEQ ID No. X and SEQ ID No. 4, 6, 8, 10, 12 or 14, divided by the number of residues in SEQ ID No. 4, 6, 8, 10, 12 or 14. When using the GenomeQuest database, said percentages of identical residues relative to the length of SEQ ID No. 4, 6, 8, 10, or 14 correspond to Query percentage identities (% id Query), where Query corresponds to the sequence SEQ ID No. 4, 6, 8, 10, 12 or 14.
According to the invention, the percentage of identical residues relative to the length of SEQ ID No. X corresponds to the number of residues that are identical between SEQ ID No. X and SEQ ID No. 4, 6, 8, 10, 12 or 14, divided by the number of residues in SEQ ID No. X. When using the GenomeQuest database, said percentages of identical residues relative to the length of SEQ ID No. X correspond to Subject percentage identities (% id Subject), where Subject corresponds to SEQ ID No. X.
The subject of the invention is also a purified or isolated nucleic acid encoding at least one polypeptide as described above. Table 1 below comprises the identifications of the nucleic and peptide sequences for the Bgl-1 genes and the genes A and C, and also for the polypeptides of the invention.











TABLE 1







Clones
Nucleic acid
Polypeptide









BGL1 (wild-type)
SEQ ID No. 1
SEQ ID No. 2



 



10H7
SEQ ID No. 3
SEQ ID No. 4



 



59B8
SEQ ID No. 5
SEQ ID No. 6



 



164A2
SEQ ID No. 7
SEQ ID No. 8



 



100B11
SEQ ID No. 9
SEQ ID No. 10



 



115E1
SEQ ID No. 11
SEQ ID No. 12



 



149G7
SEQ ID No. 13
SEQ ID No. 14



 



Gene A
SEQ ID No. 15
SEQ ID No. 16



 



Gene C
SEQ ID No. 17
SEQ ID No. 18










The invention also relates to a vector comprising a nucleic acid as described above.
According to the invention, the term “vector” is intended to mean any DNA sequence into which it is possible to insert fragments of foreign nucleic acid, the vectors making it possible to introduce foreign DNA into a host cell. Examples of vectors are plasmids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) and P1 bacteriophage-derived artificial chromosomes (PACs), and virus-derived vectors.
According to the invention, the nucleic acid as described above may be functionally linked to a promoter, a terminator or any other sequence necessary for the expression of said nucleic acid in the host cell.
The vector according to the invention may also carry a selectable marker. The term “selectable marker” is intended to mean a gene, the expression of which confers, on the cells which contain it, a characteristic which makes it possible to select said cells. It is, for example, an antibiotic resistance gene.
The subject of the invention is also an isolated host cell comprising either at least one of the polypeptides as described above, or at least one of the nucleic acids as described above, or at least one of the vectors as described above.
Those skilled in the art may introduce at least one of the polypeptides, at least one of the nucleic acids or at least one of the vectors as described above into the host cell by well-known conventional means. For example, mention may be made of calcium chloride treatment, electroporation, or the use of a particle gun.
According to one embodiment, those skilled in the art may introduce into the host cell, and by conventional means, several copies of a nucleic acid encoding a polypeptide having an improved beta-glucosidase activity according to the invention.
According to one embodiment, the isolated host cell as described above is chosen from Trichoderma, Aspergillus, Neurospora, Humicola, Penicillium, Fusarium, Thermomonospora, Bacillus, Pseudomonas, Escherichia, Clostridium, Cellulomonas, Streptomyces, Yarrowia, Pichia and Saccharomyces. 
According to one preferred embodiment, the isolated host cell as described above is chosen from Trichoderma reesei, Trichoderma viridae, Trichoderma koningii, Aspergillus niger, Aspergillus nidulans, Aspergillus wentii, Aspergillus oryzae, Aspergillus phoenicis, Neurospora crassa, Humicola grisae, Penicillium pinophilum, Penicillium oxalicum, Escherichia coli, Clostridium acetobutylicum, Clostridium saccharolyticum, Clostridium benjerinckii, Clostridium butylicum, Pichia pastoris, Yarrowia lipolityca and mixtures thereof.
According to one preferred embodiment, the isolated host cell as described above is Trichoderma reesei. 
The invention also relates to the use of any one of the polypeptides described above, for the hydrolysis of beta-oligosaccharides.
The invention also relates to the use of any one of the polypeptides described above, for the hydrolysis of cellobiose to glucose.
The subject of the invention is also the use of any one of the polypeptides described above, for the production of biofuel.
According to the invention, the term “biofuel” can be defined as any product which results from biomass conversion and which can be used for energy purposes. Furthermore, and without wanting to be limited, mention may be made, by way of example, of biogases, products that can be incorporated (optionally after subsequent conversion) into a fuel or that can be a fuel in their own right, such as alcohols (ethanol, butanol and/or isopropanol according to the type of fermentative organism used), solvents (acetone), acids (butyric acid), lipids and derivatives thereof (short-chain or long-chain fatty acids, fatty acid esters), and also hydrogen.
Preferably, the biofuel according to the invention is an alcohol, for example ethanol, butanol and/or isopropanol. More preferably, the biofuel according to the invention is ethanol.
In another embodiment, the biofuel is biogas.
In addition to the production of biofuel, the polypeptides having an improved beta-glucosidase activity according to the invention can also be used in other types of applications by catalyzing the hydrolysis of various substrates, thus allowing the release of a variety of flavors. By way of example, they can be used in order to release fruit flavors by catalyzing several glucosides present inside these fruits or, alternatively, they can hydrolyze the monoterphenyl beta-glucosidases of grapes, thus representing an important source of flavors for wine. Consequently, the polypeptides having an improved beta-glucosidase activity according to the invention can be used in several fields, in particular in perfumery, in the food industry, in enology, etc.
The strains of filamentous fungi, preferably Trichoderma, more preferably T. reesei, capable of expressing at least one polypeptide according to the invention are cultured in fermenters, in the presence of a carbon-based substrate, such as lactose or glucose, chosen for the growth of the microorganism. In one embodiment, this carbon-based substrate, depending on its nature, is introduced into the fermenter before sterilization or is sterilized separately and introduced into the fermenter after sterilization of the latter, so as to obtain an initial concentration of 20 to 35 g/l.
An aqueous solution containing the substrate chosen for the production of the enzymes is then added. An enzymatic composition which acts on lignocellulosic biomass, produced by the fungi, is finally recovered by filtration of the culture medium. This composition contains in particular endoglucanase, exoglucanase and the beta-glucosidase according to the invention. In one embodiment, the aqueous solution containing the substrate chosen for the production of the enzymes is prepared at the concentration of 200-250 g/l; this solution should contain the inducing substrate such as lactose. This aqueous solution is injected after exhaustion of the initial carbon-based substrate so as to provide an optimized amount, of between 35 and 45 mg/g of cells (“fed batch”). During this “fed batch” phase, the residual sugar concentration in the culture medium is less than 1 g/l and the enzymes which act on lignocellulosic biomass are secreted by the fungus. Said enzymes can be recovered by filtration of the culture medium.
The subject of the invention is an enzymatic composition which acts on lignocellulosic biomass, said enzymatic composition being produced by filamentous fungi and comprising at least one polypeptide having an improved beta-glucosidase activity compared with the beta-glucosidase activity of the wild-type BGL1 protein.
Finally, the subject of the invention is a method for producing biofuel from biomass, comprising the following steps:

    
    
        suspending in an aqueous phase the material to be hydrolyzed;
        adding an enzymatic composition which acts on lignocellulosic biomass, as described above;
        assaying the sugars released;
        separating the sugar solution from the nonhydrolyzed solid fraction;
        fermenting the sugar solution;
        separating the biofuel from the fermentation must.
    
    


In one embodiment, the material to be hydrolyzed is suspended in an aqueous phase in a proportion of from 6% to 40% of dry matter, preferably 20% to 30%. The pH is adjusted to between 4 and 5.5, preferably between 4.8 and 5.2, and the temperature is adjusted to between 40 and 60° C., preferably between 45 and 50° C. The hydrolysis reaction is initiated by adding the enzymatic composition which acts on lignocellulosic biomass; the amount normally used is from 10 to 30 mg of excreted proteins per gram of pretreated substrate or less. The reaction generally lasts from 15 to 48 hours. The reaction is followed by assaying the sugars released, in particular the glucose. The sugar solution is separated from the nonhydrolyzed solid fraction, essentially constituted of lignin, by filtration or centrifugation; it is used for the fermentation.
In one embodiment, the biofuel may be separated from the fermentation must by distillation.
Another subject of the invention is a method for producing biofuel from biomass, characterized in that it comprises the following steps:

    
    
        suspending in an aqueous phase the biomass to be hydrolyzed;
        simultaneously adding an enzymatic composition which acts on lignocellulosic biomass, as defined above, and a fermentative organism;
        separating the biofuel from the fermentation must.
    
    


According to this embodiment, the cellulose present in the biomass is converted to glucose, and at the same time, in the same reactor, the fermentative organism (for example a yeast) converts the glucose to final product according to an SSF (simultaneous saccharification and fermentation) process known to those skilled in the art. Depending on the metabolic and hydrolytic capacities of the fermentative organism, the addition of a more or less large amount of exogenous cellulolytic mixture may be required in order for the operation to run smoothly.
In another embodiment, one and the same fermentative organism may be capable of converting the biomass to glucose and then the glucose to final product.
The use of the polypeptide having a better beta-glucosidase activity according to the present invention thus provides the advantage of obtaining a better glucose production yield. The present invention thus makes it possible to use less enzyme than previously, which provides an economic advantage, the production cost of the biofuel, for example, being lower.



Other aspects, subjects, advantages and characteristics of the invention will be presented on reading the nonrestrictive description which follows and which describes preferred embodiments of the invention given by means of examples and of FIG. 1.
FIG. 1 is a graph representing the improvement in the beta-glucosidase activity for the variants 149G7, 100B11 and 115E1 in comparison with the parent BGL1 gene, when in the presence of glucose.
FIG. 2 is a graph representing the specific beta-glucosidase activity of the enzymatic mixtures produced in flasks by the transformed clones (100B11, 164A2 and 115E1) and the starting strain CL847. The CL847-bg11+ strain is indicated by way of comparison.
FIG. 3 is a graph representing the results of the hydrolyses (glucose released) for the enzymes produced by the reference strain (CL847) and the variant (100B11).



EXAMPLES
Example 1
1st Round of Shuffling
The sequence of the Trichoderma reesei beta-glucosidase gene (parental BGL1 gene, SEQ ID No. 1) was subjected to a first round of shuffling according to the patented method described in EP 1104457B1 with the putative glucosidase gene of Chaetomium globosum (gene A) (SEQ ID No. 15 and SEQ ID No. 16 (protein sequence)) having 70% identity with the parental BGL1 gene.
1—High-throughput Screening
A high-throughput screening test made it possible to select the best clones resulting from the shuffling of these two sequences, i.e. those having an improvement factor greater than 2 in terms of the beta-glucosidase activity when compared with the parental BGL1 gene of T. reesei. 
The tests for screening of the library of the first round of shuffling was carried out according to the following steps:

    
    
        isolation on agar of the various colonies of E. coli expressing the shuffling variants for the recombinant enzyme according to the invention and preculturing of said colonies in LB medium overnight at 37° C.;
        inoculation of an LB medium at 3% with the preculture, then incubation for 4 h at 37° C.;
        induction of the expression of the variants by addition of 100 μM isopropyl-beta-thiogalactoside (IPTG), then incubation at 20° C. overnight;
        centrifugation for 2 minutes at 13 000 rpm;
        resuspension of the cell pellets in 100 μL of 0.1M succinate buffer containing 2.2 mM of para-nitrophenyl-D-glucoside-6-phosphate (pNPGlc);
        incubation for 3 h at room temperature;
        reading of the optical density at 414 nm after alkalinization.
    
    


Under these screening conditions, an improvement in the beta-glucosidase activity compared with the BGL1 reference enzyme was found in several clones, including in particular the clones 10H7 (SEQ ID Nos. 3 and 4), 59B8 (SEQ ID Nos. 5 and 6) and 164A2 (SEQ ID Nos. 7 and 8).
2—Determination of the Improvement in the β-glucosidase Activity
2-1/On the Substrate pNPGlc
In order to determine the relative kcat of the variants selected in the first round of shuffling, the following procedure is carried out:

    
    
        formation of a stock culture of E. coli expressing a recombinant enzyme according to the invention, overnight at 37° C.;
        inoculation of an LB culture medium with 1% of stock culture for 24 h at 20° C. with induction using IPTG (250 μM);
        centrifugation for 2 minutes at 13 000 rpm;
        resuspension of the cell pellets with 100 mM succinate buffer at pH 5 (final OD600 of 100);
        incubation of 50 μl of cells with 100 μl of 100 mM succinate buffer at pH 5 containing 15 mM of pnp-glucopyranoside for 1 h 30 at 50° C., followed by 5 minutes on ice;
        addition of 150 μl of 0.2M Na2CO3;
        centrifugation for 2 minutes at 13 000 rpm;
        reading of the optical density at 414 nm on 150 μl of supernatant.
    
    


Table 2 gives the values of the kcats and also the improvement factors obtained for the clones 10H7, 59B8 and 164A2 under these experimental conditions.







TABLE 2







Improvement in beta-glucosidase activity


(results of the induced cultures)













Improvement



Clones
Kcat (min−1)
factor
















1st round clones
10H7
590.0
8




59B8
518.6
7




164A2
1437.3
20



reference protein
BGLI
71.0
1










The results show very large improvements in enzymatic activities compared with the reference enzyme (BGL1) for the 3 clones 10H7, 59B8 and 164A2.
2-2/On Cellobiose
The improvement in activity of the clones 10H7, 59B8 and 164A2 was then confirmed on a second substrate: cellobiose.
This test was carried out on cultures of E. coli expressing a recombinant enzyme according to the invention. The steps of the test are as follows:

    
    
        Inoculation of an LB culture medium with 1% of stock culture induced with IPTG, then incubation overnight at 37° C.
        Culturing of said cells at 37° C. until an optical density at 600 nm of 0.4 is obtained.
        Induction of said cells with 250 μM IPTG 250 at 20° C. for 20 hours.
        Washing of the cell pellets three times in a 100 mM succinate buffer, pH 5, in order to remove the glucose of the culture medium.
        Incubation of 10 μl of said cells with 190 μl of 263.2 mM cellobiose (250 mM final concentration) for 12 hours at 50° C. in a microplate.
    
    


Developing:

    
    
        Mixing and incubation, for 1 h at room temperature, of:
        
            10 μl of above reaction
            90 μl of 100 mM succinate buffer at pH 5
            5 μl of glucose oxidase, 44 U/ml,
        
        
        Mixing and incubation, for 30 min at room temperature, of:
        
            10 μl of glucose oxidase reaction
            2 μl of horse radish peroxidase at 10 U/ml
            5 μl of 100 mM ABTS
            83 μl of 50 mM phosphate buffer, pH 7.4,
        
        
    
    


Reading of the optical densities at 420 nm.







TABLE 3







Improvement in beta-glucosidase activity


(results of the induced cultures)













Improvement



Clones
kcat (min−1)
factor
















1st round clones
10H7
69.1
13




59B8
37.7
7




164A2
213.2
41



reference protein
BGLI
5.2
1










Similarly, the results show very large improvements in enzymatic activities compared with the starting enzyme (BGL1) for the clones 10H7, 59B8 and 164A2 where cellobiose is used as substrate.
Example 2
2nd Round of Shuffling
The sequences of the improved genes obtained in the first round of shuffling were subsequently subjected to a second round of shuffling (still according to the patented method described in EP1104457B1). In order to increase the genetic diversity, at least one gene encoding a beta-glucosidase having 70% identity was added. In this specific example, the gene of the putative glucosidase of Neurospora crassa (gene C) (SEQ ID No. 17 and SEQ ID No. 18 (protein sequence)) was used.
1—High-throughput Screening
A high-throughput screening test as described previously (with the exception of the IPTG induction step, since the improvement provided in the first round of shuffling allows detection of the beta-glucosidase activity based only on the leakage of the promoter) was carried out on the clones obtained following this second round of shuffling, in order to select the best clones, i.e. those having an improvement factor greater than 2 in terms of the beta-glucosidase activity when compared with the clone 164A2.
Under these screening conditions, an improvement in the beta-glucosidase activity compared with the reference enzyme (164A2) was found in several clones, including in particular the clones 100B11 (SEQ ID Nos. 9 and 10), and 115E1 (SEQ ID Nos. 11 and 12).
2—Determination of the Improvement in the β-glucosidase Activity
2-1/On pNPGlc
In order to determine the relative kcat, the activities of the clones 100B11 and 115E1 were measured using the activity test as described previously.
Table 4 gives the values of the kcats and also the improvement factors obtained for the clones 100B11 and 115E1 under these experimental conditions.







TABLE 4







Improvement in beta-glucosidase activity


(results of the induced cultures)













Improvement



Clones
Kcat (min−1)
factor
















2nd round clones
100B11
4342.8
3.0




115E1
3989.2
2.8



reference protein
164A2
1437.3
1










The results show very large improvements in enzymatic activities compared with the reference enzyme (164A2) and with (BGL1) (×60) for the clones 100B11 and 115E1.
2-2/On Cellobiose
The improvement in activity of the clones 100B11 and 115E1 was then confirmed on a second substrate: cellobiose.
In order to determine the relative kcat, the activities of the clones 100B11 and 115E1 were measured by means of the activity test as described previously, using cellobiose as substrate.







TABLE 5







Improvement in beta-glucosidase activity


(results of the induced cultures)













Improvement



Clones
Kcat (min−1)
factor
















2nd round clones
100B11
387.2
1.8




115E1
406.4
1.9



reference protein
164A2
213.2
1










Similarly, the results show large improvements in enzymatic activities compared with the reference enzyme (164A2) for the clones 100B11 and 115E1 when cellobiose is used as substrate.
Example 3
3rd Round of Shuffling
The sequences of the improved genes obtained in the second round of shuffling were subsequently subjected to a third round of shuffling (still according to the patented method described in EP1104457B1). In order to increase the genetic diversity, at least one gene encoding a beta-glucosidase having 70% identity was added. In this specific example, the gene of the putative glucosidase of Neurospora crassa (gene C) (SEQ ID No. 17 and SEQ ID No. 18) and the gene of the putative glucosidase of Chaetomium globosum (gene A) (SEQ ID No. 15 and SEQ ID No. 16) were used.
1—High-throughput Screening
A high-throughput screening test as described previously (with the exception of the IPTG induction step, since the improvement provided in the first round of shuffling enables detection of the beta-glucosidase activity based only on the leakage of the promoter) was carried out on the clones obtained following this third round of shuffling, in order to select the best clones, i.e. those having an improvement factor greater than 2 in terms of the beta-glucosidase activity when compared with the clone 115E1.
Under these screening conditions, an improvement in the beta-glucosidase activity compared with the reference enzyme (115E1) was found in particular for the clone 149G7 (SEQ ID Nos. 13 and 14).
2—Determination of the Improvement in the β-glucosidase Activity
2-1/On pNPGlc
In order to determine the relative kcat, the activity of the clone 149G7 was measured by means of the activity test as described previously.
The results show a 2.4-fold improvement in the enzymatic activity of the clone 149G7 compared with the clone 115E1 and a more than 100-fold improvement compared with BGL1.
Example 4
Improved β-glucosidase Activity in the Presence of Glucose
In order to compare the activity of the clones 149G7, 100B11 and 115E1 with BGL1, the activity of these clones was measured by means of the activity test as described previously on pNPGlc in the presence of 60 g/l of glucose in the reaction medium (reaction product).
FIG. 1 shows that the clone 149G7 retains 61% of its activity in the presence of 60 g/l of glucose, whereas the BGL1 reference parental protein retains only 27% of its activity.
Although the present invention has been described above by means of examples of the preferred embodiments thereof, it is understood that it can be modified without departing from the spirit and nature of the invention as defined in the appended claims.
Example 5
Transformation of T. Reesei with the Improved Beta-glucosidase Variants
Each gene corresponding to the variants 115E1, 100B11 and 164A2 was cloned into a vector allowing expression in Trichoderma reesei with selection with hygromycin. The gene was placed under the control of a strong promoter, cbh1, inducible at the same time as the other cellulases of T. reesei. 
The transformation of Trichoderma reesei was carried out according to the conventional methods known to those skilled in the art (protoplast transformation by calcium shock and selection with 50 μg/ml of hygromycin). The transformants were purified by sporulation and then subcultured twice in selective medium in order to eliminate the unstable clones. The integration of the DNA of interest was then verified by PCR according to the method described by Yu et al., Fungal Genet. Biol. (2004); 41(11):973-981.
The PCR-positive clones were then evaluated in terms of cellulase production in flasks. A few spores of each clone were used to inoculate 50 ml of PD broth medium (Difco). The flasks were incubated for 72 h at 30° C. with shaking at 150 rpm. After 72 h, this preculture was used to inoculate, at 30%, a cellulase production medium having the following composition: lactose (20 g/l), Solka floc cellulose (20 g/l), peptone (5 g/l), KH2PO4 (15 g/l), (NH4)2SO4 (5 g/l), CaCl2 (0.6 g/l), MgSO4 (0.6 g/l), FeSO4 (0.005 g/l), MnSO4 (0.0014 g/l), ZnSO4 (0.0014 g/l), CoCl2 (0.0037 g/l), maleic acid (11.6 g/l), Tris (12.1 g/l) and NaOH (2.08 g/l).
The cultures were incubated at 30° C. with shaking at 150 rpm. After 5 days, the cultures were centrifuged and the protein concentration of the supernatant was measured by the Bradford method. The beta-glucosidase activity of the supernatants was measured by hydrolysis of the chromophoric substrate p-nitrophenyl-beta-D-glucoside (pNPG) under the following conditions:

    
    
        50 mM of citrate buffer at pH 4.8
        5 mM of pNPG
        10 μl of sample
        incubation at 50° C. for 30 min.
    
    


The reaction was stopped by adding 100 μl of 2% sodium carbonate. The amount of para-nitrophenol released by hydrolysis of the pNPG was measured by measuring the absorbance at 410 nm and compared with a para-nitrophenol range. The reaction was linear from 25 to 400 μM of para-nitrophenol.
FIG. 2 shows the results obtained for each variant (one example per variant), in comparison with the activities measured on a nontransformed strain (CL847) and with a strain transformed with the native beta-glucosidase of T. reesei (CL847-bg11+).
Table 6 gives the improvement factors relative to the initial strains CL847 (Durand et al., Enzyme Microb. Technol., 1988; 10:341-346) and CL847-bg11+, which overexpresses the native beta-glucosidase bg11 of T. reesei.







TABLE 6







Specific beta-glucosidase activity increase


factors relative to the reference


(data resulting from FIG. 2)










Clones
Increase factor







CL847
—



CL847-bgl1+
2.9



100B11
26.2



164A2
22.3



115E1
12.1










Example 6
Enzymatic Activities of a Composition of Cellulases Produced in a Fermenter by a T. Reesei Transformant Expressing an Improved Beta-glucosidase
The variant 100B11 of example 5 was used to carry out a production of cellulases in a 2 l fermenter.
The production of cellulases is carried out in a mechanically stirred fermenter. The medium has the following composition: KOH (1.66 g/l), 85% H3PO4 (2 ml/l), (NH4)2SO4 (2.8 g/l), MgSO4.7H2O (0.6 g/l), CaCl2 (0.6 g/l), MnSO4 (3.2 mg/l), ZnSO4.7H2O (2.8 mg/l), CoCl2 (4.0 mg/l), FeSO4.7H2O (10 mg/l), Corn Steep (1.2 g/l), antifoam (0.5 ml/l).
The fermenter containing 1.75 l of mineral medium and 70 g of lactose is sterilized at 120° C. and then inoculated with 0.25 l of a liquid preculture of the Trichoderma reesei strain CL847. The medium of the preculture, supplemented with potassium phthalate at 5 g/l in order to control the variations in pH, is identical to that of the fermenter. The fungus is grown in preculture on lactose, at the concentration of 30 g/l. The growth of the inoculum lasts from 2 to 3 days and is carried out between 27 and 30° C. on a shaker table.
After 46 hours of growth, the initial substrate of the fermenter is exhausted and the solution of lactose at 250 g/l is injected continuously at the flow rate of 4.5 ml/h up to 142 hours.
The temperature is regulated at 27° C. during the biomass growth phase, and then at 25° C. until the end of the culture. The pH is regulated at 5 during the growth phase, and then at 4 until the end of the culture by adding an aqueous ammonia solution which provides the nitrogen necessary for the synthesis of the excreted proteins. The dissolved oxygen content is maintained above 15 to 20% by action on the aeration and the shaking.
The production of enzymes is monitored by assaying the extracellular proteins by the Folin method (Lowry, Biol. Chem. 1951; 193:265-275), after separation of the mycelium by filtration or centrifugation. The beta-glucosidase activity was measured according to the method described above (see example 5) with the substrate pNPG. The FPase filter paper activity was measured according to the method recommended by Mandels et al., Biotechnology for Biofuels, 2009; 2:21. The results are given in table 7.









TABLE 7









Strains used for the production











CL847
CL847-bgl1+
100B11














Specific beta-glucosidase
10055
45176
119677


activity (IU/mg) of the


cellulolytic mixture produced


Specific FPase activity
0.5
0.4
0.65


(IU/mg) of the cellulolytic


mixture produced


Beta-glucosidase activity
—
4.5
11.9


increase factor relative to the


strain CL847









Example 7
Hydrolytic Efficiency on Pretreated Lignocellulosic Substrate of the Cellulolytic Mixture Produced by a T. reesei Transformant Expressing an Improved Beta-glucosidase
The cellulolytic mixtures produced by the reference strain CL847 and by the transformant expressing the improved beta-glucosidase, 100B11, resulting from example 6 were used to hydrolyze wheat straw pretreated by vapor explosion under acid conditions. The hydrolyses are carried out in a jacketed Bio-Laffite bioreactor stirred with two stirrers of “ship's anchor” type, under the following experimental conditions:

    
    
        lignocellulosic substrate diluted to 10% of dry matter
        reaction volume 2 l
        1M acetate buffer, pH 4.8 (pH tested daily)
        temperature 48° C.
    
    


The whole is impregnated for 12 h at 300 rpm before the addition of 20 mg/g of dry matter of enzyme and is switched to 500 rpm. Samples are taken at 0 h, 5 h, 24 h, 48 h and 72 h after the addition of the enzymes. The enzymes are inactivated by placing the sample in a boiling water bath for 10 minutes. The sample is then centrifuged and the supernatant is filtered before assaying the glucose by HPLC.
The results are given in FIG. 3. As early as 24 h, the enzymatic mixture containing the improved beta-glucosidase produced by the transformant 100B11 released twice as much glucose as the enzymatic mixture produced by the reference strain (CL847). The maximum yield is reached as early as 24 h for the mixture resulting from the strain 100B11. At 72 h, this yield has still not been reached for the reference enzymatic mixture (CL847).
The enzymatic mixture produced by the transformant 100B11 therefore has a much greater efficiency than that of the reference enzymatic mixture (CL847) for the same dose of enzyme. This property results in a greater yield and a greater productivity, enabling a more complete hydrolysis of the substrate. Alternatively, it would make it possible to reduce the dose of enzyme to be used in order to obtain an equivalent hydrolysis result. Since the cost of the cellulolytic enzymes represents a large part of the cost price of lignocellulosic bioethanol, any significant reduction in the amount of enzymes to be used can be considered to be a considerable improvement in the method.

## Claims

1. An isolated or purified modified polypeptide comprising an amino acid sequence in which at least one amino acid is modified compared with the amino acid sequence SEQ ID No. 2, said modified amino acid being chosen from positions 225, 238, 240 and 241 of the amino acid sequence SEQ ID No. 2, wherein said modified polypeptide has the amino acid sequence SEQ ID No. 10; or at least 85% identity with said amino acid sequences SEQ ID No. 2 or SEQ ID NO. 10, and wherein said modified polypeptide has an improved beta-glucosidase activity compared with the beta-glucosidase activity of the wild-type protein having the sequence set forth in SEQ ID No. 2.

2. The modified polypeptide as claimed in claim 1, wherein at least one amino acid of the amino acid sequence is modified compared with the amino acid sequence SEQ ID No. 2, said modification being chosen from Q225H, V238I, T240G and T241S.

3. The modified polypeptide as claimed in claim 1, further comprising at least one additional modified amino acid chosen from positions 97, 99, 100, 118, 119, 121, 123, 126, 127, 128, 130, 132, 134, 135, 140, 147, 151, 153, 163, 168, 173, 174, 177, 179, 182, 187, 193, 206, 207, 212, 217 and 621 of the amino acid sequence SEQ ID No. 2.

4. The modified polypeptide as claimed in claim 1 further comprising at least one additional modified amino acid, said modification being selected from the group consisting of V97I, Y99F, S100G, V118T, N119E, I121M, E123Q, Q126E, F127Y, I128L, E130A, V132A, A134G, S35C, S135V, I140L, P147A, T151I, Q153H, V163T, T168A, G173A, G173S, Q174E, N177E, I179L, V182A, V182N, T187C, L193V, N206D, P207V, L212M, T217L, L621F and L621T.

5. The isolated or purified modified polypeptide as claimed in claim 1, wherein said modified polypeptide has the amino acid sequence SEQ ID No. 10.

6. An enzymatic composition which acts on lignocellulosic biomass, said enzymatic composition being produced by filamentous fungi and comprising at least one modified polypeptide according to claim 1.

7. A method for hydrolyzing a beta-oligosaccharide, comprising contacting a beta-oligosaccharide with a modified polypeptide as claimed in claim 1.

8. A method for hydrolyzing cellobiose to glucose comprising contacting cellobiose with a modified polypeptide as claimed in claim 1.

9. A method for producing biofuel comprising contacting a source of cellulose with a modified polypeptide as claimed in claim 1 to produce glucose and fermenting the glucose to produce a biofuel.

10. A method for producing biofuel from biomass, comprising:
suspending in an aqueous phase the biomass to be hydrolyzed;
adding an enzymatic composition which acts on lignocellulosic biomass, as claimed in claim 6, in order to begin the hydrolysis;
assaying the sugars released;
separating the sugar solution from the nonhydrolyzed solid fraction;
fermenting the sugar solution; and
separating the biofuel from the fermentation must.

11. A method for producing biofuel from biomass, comprising:
suspending in an aqueous phase the biomass to be hydrolyzed;
simultaneously adding an enzymatic composition which acts on lignocellulosic biomass, as claimed in claim 6, and a fermentative organism; and
separating the biofuel from the fermentation must.