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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2445—Beta-glucosidase (3.2.1.21)
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/02—Monosaccharides
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  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/16—Butanols
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  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01021—Beta-glucosidase (3.2.1.21)
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  • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/30—Fuel from waste, e.g. synthetic alcohol or diesel
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

• biomass Natural cellulosic raw materials for such a process are referred to as "biomass”. Many types of biomass, such as wood, agricultural residues, herbaceous crops and municipal solid waste, have been considered as potential raw materials for biofuel production. These materials consist mainly of cellulose, hemicellulose and lignin.
• Cellulose is a polymer consisting of glucose molecules linked by beta 1-4 bonds, which are highly resistant to degradation or depolymerization. Once the cellulose is converted to glucose, it is easily fermented to biofuel, for example ethanol, using yeast.
• the cellulose conversion processes have more recently focused on enzymatic hydrolysis, using cellulase enzymes.
• this enzymatic hydrolysis of lignocellulosic biomass has the disadvantage of being an expensive industrial process. Therefore, it is necessary to use strains of microorganisms secreting cellulase increasingly efficient.
• many microorganisms include enzymes that hydrolyze cellulose, such as Trichoderma, Aspergillus, Humicola, Fusarium fungi as well as bacteria such as Thermomonospora, Bacillus, Cellulomonas and Streptomyces.
• the enzymes secreted by these microorganisms have three types of activities useful in the conversion of cellulose to glucose and are divided into three groups: the endoglucanases, which attack the celluloses fibers randomly internally, the exoglucanases that go attack the ends of the fibers by releasing cellobiose, and beta-glucosidases that will hydrolyze this cellobiose into glucose. These are the limiting step of the cellulose conversion process. Indeed, the first difficulty of the process lies in the conversion of cellobiose to glucose, because any unhydrolyzed cellobiose at the end of the process represents a loss of yield during the production of biofuel.
• the inventors have developed in their patent application WO2010 / 029259 beta-glucosidase genes for obtaining enzymes with increased specific activity, which significantly improves the process of converting lignocellulosic biomass into biofuel.
• the hydrolysis and fermentation can be carried out according to different schemes.
• SSF-Simultaneous Saccharification and Fermentation Another type of process (SSF-Simultaneous Saccharification and Fermentation) can be envisaged.
• SSF the two stages (hydrolysis and fermentation of hexoses) take place simultaneously preventing the accumulation of sugars at inhibitory concentrations for the enzymes.
• Investment costs are also reduced following the use of a single reactor.
• the rate Hydrolysis is higher due to the absence of inhibition because the sugars released are used immediately for fermentation in ethanol.
• the reactor temperature necessarily constitutes a compromise between the optimum hydrolysis and fermentation temperatures, typically between about 30 ° C and about 35 ° C.
• the activity of cellulolytic enzymes, including beta-glucosidase is reduced by about 30%.
• the inventors have developed a polypeptide having an improved beta-glucosidase activity at a temperature of between about 30 ° C. and about 35 ° C., especially with respect to the beta-glucosidase activity of the wild-type BGL1 protein of sequence SEQ ID No. 3 .
• BGL1 corresponds to the beta-glucosidase of Trichoderma reesei.
• the inventors have previously identified several clones having a specific activity beta-glucosidase improved compared to the beta-glucosidase activity of the wild-type protein BGL1. Such results are presented in their patent application WO 2010/029259. More specifically, they have demonstrated a particular clone encoding a polypeptide of sequence SEQ ID No. 5 (designated 100B11) whose expression in Trichoderma reesei under the control of a strong promoter causes a factor of increase in activity. beta-glucosidase of 26.2 (Table 6 of the patent application WO 2010/029259) of the enzymatic cocktail produced relative to that produced by a strain not expressing this enzyme.
• the invention therefore relates to a polypeptide having a beta-glucosidase activity of amino acid sequence SEQ ID No. 1.
• amino acid sequence of the polypeptide of the invention is as follows:
• said amino acid sequence polypeptide SEQ ID No. 1 has a beta-glucosidase activity at a temperature of between about 30 ° C. and about 35 ° C. improved, in particular with respect to the beta-glucosidase activity of the protein.
• wild BGL1 sequence SEQ ID No. 3 at these same temperatures.
• the BGL1 protein is encoded by the nucleic acid sequence SEQ ID No. 4.
• said amino acid sequence polypeptide SEQ ID NO is a beta-glucosidase activity at a temperature of between about 30 ° C and about 35 ° C improved over the beta-glucosidase activity of the sequence polypeptide 100B11. of amino acids SEQ ID No. 5 at these same temperatures.
• the 100B11 polypeptide is encoded by the nucleic acid sequence SEQ ID NO: 6.
• the polypeptide according to the invention has the advantage of being less sensitive to inhibition by glucose and thus retains a better beta-glucosidase activity in the presence of a high glucose concentration.
• the polypeptide as described above has a beta-glucosidase activity determined in the presence of glucose which is improved with respect to the beta-glucosidase activity of the wild-type BGL1 protein (SEQ ID No. 3) determined in the absence of glucose.
• the polypeptide of the invention has an improved beta-glucosidase activity of at least 10%, preferably at least 20%, preferably at least 30%, even more preferably at least 40% at a temperature between about 30 ° C and about 35 ° C relative to the beta-glucosidase activity of the 100B 11 polypeptide of amino acid sequence SEQ ID NO: 5.
• Those skilled in the art may, for example, determine the increase or, in other words, the improvement of the enzymatic activity of a polypeptide according to the invention by an enzymatic activity test using the para-nitrophenyl beta-D substrate. -glucopyranoside (pNPG).
• the amount of para-nitrophenol obtained after the action of beta-glucosidase may, for example, be determined by reading the optical density at 414 nm.
• the invention also relates to a nucleic acid encoding the amino acid sequence polypeptide SEQ ID No. 1.
• said nucleic acid comprises the nucleic acid sequence SEQ ID NO: 2.
• the invention also relates to a vector comprising a nucleic acid as described above.
• vector is intended to mean any DNA sequence in which it is possible to insert foreign nucleic acid fragments, the vectors making it possible to introduce foreign DNA into a host cell.
• vectors are plasmids, cosmids, artificial yeast chromosomes (YAC), artificial chromosomes of bacteria (BAC) and artificial chromosomes derived from bacteriophage PI (PAC), vectors derived from viruses.
• the nucleic acid as described above may be operably linked to a promoter, a terminator or any other sequence necessary for its expression in the host cell.
• the vector according to the invention may also carry a selection marker.
• selection marker is meant a gene whose expression confers on cells containing it a characteristic allowing them to be selected. This is for example an antibiotic resistance gene.
• the invention also relates to an isolated host cell capable of producing the polypeptide of the invention as described above, or comprising a nucleic acid encoding said polypeptide of the invention.
• Those skilled in the art can introduce at least the polypeptide, the nucleic acid or the vector as described previously in the host cell by conventional methods well known. For example, mention may be made of calcium chloride treatment, electroporation and the use of a particle gun.
• one skilled in the art can introduce into the host cell and by conventional methods, several copies of a nucleic acid encoding a polypeptide having improved beta-glucosidase activity according to the invention.
• the isolated host cell as described above is chosen from among Trichoderma, Aspergillus, Neurospora, Humicola, Myceliophthora, Chrysosporium, Penicillium, Fusarium, Thermomonospora, Bacillus, Pseudomonas, Escherichia, Clostridium, Cellulomonas, Streptomyces, Yarrowia, Pichia and Saccharomyces.
• the isolated host cell as described above is chosen from Trichoderma reesei, Trichoderma viridae, Trichoderma koningii, Aspergillus niger, Aspergillus nidulans, Myceliophthora thermopila, Chrysosporium lucknowense, 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, Saccharomyces cerevisiae, and mixtures thereof. According to a preferred embodiment, the isolated host cell as described above is chosen from Trichoderma reesei,
• the invention also relates to the use of the polypeptide as described above or any of the cells previously described for the hydrolysis of beta-oligosaccharides.
• the invention also relates to the use of the polypeptide as described above or any of the cells previously described for the hydrolysis of cellobiose to glucose.
• the subject of the invention is also the use of the polypeptide as described above or any of the cells previously described for the production of biofuel.
• the term biofuel can be defined as any product resulting from the transformation of biomass and can be used for energy purposes.
• biogas products which can be incorporated (possibly after further processing) into a fuel or be a fuel in their own right, such as alcohols (the ethanol, butanol and / or isopropanol depending on the type of fermentative organism used), solvents (acetone), (butyric) acids, lipids and their derivatives (short or long chain fatty acids, esters of fat), as well as hydrogen.
• 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.
• the biofuel is biogas.
• the product is a molecule of interest to the chemical industry such as, for example, other alcohols such as 1,2-propane diol, 1,3-propane diol, 1,4-butanediol. 2,3-butanediol, organic acids such as acetic acid, propionic acid, acrylic acid, butyric acid, succinic acid, malic acid, fumaric acid, citric, itaconic, or hydroxy acids such as glycolic, hydroxypropionic, or lactic acid.
• other alcohols such as 1,2-propane diol, 1,3-propane diol, 1,4-butanediol. 2,3-butanediol, organic acids such as acetic acid, propionic acid, acrylic acid, butyric acid, succinic acid, malic acid, fumaric acid, citric, itaconic, or hydroxy acids such as glycolic, hydroxypropionic, or lactic acid.
• the polypeptide having enhanced beta-glucosidase activity at a temperature between 30 ° C and 35 ° C can also be used in other types of applications by catalyzing the hydrolysis of various substrates, thereby allowing the release of a variety of flavors.
• it can be used to release fruit flavors by hydrolyzing several glucosides present in these fruits or it can hydrolyze monoterphenyl betaglucosides of grapes thus representing an important source of flavors. for the wine. Therefore, the polypeptide as described above can be used in several fields, including perfumery, food industry, oenology, etc.
• Strains of filamentous fungi preferably Trichoderma, more preferably T. reesei, capable of expressing the polypeptide according to the invention are cultured in fermentors, in the presence of a carbon substrate, such as lactose or glucose, chosen for growth of the microorganism.
• a carbon substrate such as lactose or glucose
• this carbon substrate according to its nature, is introduced into the fermentor before sterilization or is sterilized separately and introduced into the fermentor after sterilization of the latter to obtain an initial concentration of 20 to 35 g / l.
• aqueous solution containing the substrate selected for the production of the enzymes is then added.
• An enzymatic composition acting on the lignocellulosic biomass produced by the fungi is finally recovered by filtration of the culture medium.
• this composition there are in particular endoglucanases, exoglucanases and beta-glucosidase according to the invention.
• the aqueous solution containing the substrate selected for the production of the enzymes is prepared at a concentration of 200-250 g / L; this solution must contain an inducing substrate such as lactose.
• This aqueous solution is injected after depletion of the initial carbonaceous substrate so as to provide an optimized amount of between 35 and 45 mg / g of cells ("fed batch").
• the residual concentration of sugar in the culture medium is less than 1 g / L and the enzymes acting on the lignocellulosic biomass are secreted by the fungus. These can be recovered by filtration of the culture medium.
• the subject of the invention is an enzymatic composition acting on lignocellulosic biomass, said enzymatic composition being produced by filamentous fungi or yeasts and comprising the polypeptide as described above.
• the subject of the invention is a process for producing biofuel from biomass comprising the following steps:
• hydrolysis in the presence of an enzymatic composition of the lignocellulosic biomass as described above so as to produce a hydrolyzate containing glucose;
• Another subject of the invention is a process for producing biofuel from biomass, characterized in that it comprises the following steps:
• Another subject of the invention is a process for producing biofuel from biomass, characterized in that it comprises the following successive stages:
• the cellulose present in the biomass is converted into glucose, and at the same time, in the same reactor, the fermentative organism (for example a yeast) converts the glucose into the final product according to a method of SSF (Simultaneous Saccharification and Fermentation) known to those skilled in the art.
• the smooth progress of the operation may require the addition of a greater or lesser amount of exogenous cellulolytic mixture.
• the fermentative organism produces the polypeptide which is the subject of the invention, by secretion or at the surface of its cell, optionally together with other enzymes acting on lignocellulosic biomass, thus limiting or eliminating the need for enzymes. produced by the filamentous fungus.
• the use of the polypeptide having a better beta-glucosidase activity at a temperature of between about 30 ° C. and about 35 ° C. according to the present invention thus has the advantage of obtaining a better production yield of glucose.
• the present invention allows to use less enzyme than before, which has an economic advantage, the cost of production of biofuel, for example, being less.
• Trichoderma reesei beta-glucosidase gene (parental gene BGL1, SEQ ID No. 4) 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: 7, encoded by the nucleic acid sequence SEQ ID No. 8) having 70% identity with the parental gene BGL1.
• a high throughput screening test was used to select the best shuffling clones from these two sequences, ie those with a factor greater than 2 at the level of beta-glucosidase activity when compared with the parental gene BGLI of T. reesei.
• the bank screening test of the first round of shuffling was conducted according to the following steps:
• Table 2 presents the kcat values as well as the improvement factors obtained for three previously identified clones (designated 10H7, 59B8 and 164A2) under these experimental conditions.
• the improved gene sequences obtained in the first round of shuffling were subsequently subjected to a second round of shuffling (still according to the patented process described in EP1104457B1).
• at least one gene coding for a beta-glucosidase having 70% identity with the wild-type enzyme BGL1 has been added.
• the putative glucosidase gene of Neurospora crassa (gene C) (SEQ ID No. 9 encoded by the nucleic acid sequence SEQ ID No. 10) was used.
• a high throughput screening assay as described above (with the exception of the IPTG induction step, since the improvement in the first round of shuffling allows detection of beta-glucosidase activity based only on on the escape of the promoter) was carried out on the clones obtained following this second round of shuffling, in order to select the best clones, that is to say those having an improvement factor greater than 2 at the level of beta-glucosidase activity when compared with clone 164A2.
• beta-glucosidase activity was found in several clones, in particular the 100B11 clones (SEQ ID NO: 5 encoded by the nucleic acids SEQ ID NO: 6), 115E1 (SEQ ID NO: 11 encoded by the nucleic acid sequence SEQ ID NO: 12).
• Table 4 presents the kcat values as well as the improvement factors obtained for the clones 100B11 and 115E1 under these experimental conditions. TABLE 4: Improvement of beta-glucosidase activity (induced culture results)
• the activities of the 10161 and 115E1 clones were measured by the activity test at 50 ° C as described above using cellobiose as substrate as described in point 2-2 of Example 1 .
• results show significant enzyme activity improvements over the reference enzyme (164A2) for clones 10101 and 115E1 when cellobiose is used as a substrate.
• the improved 14 gene sequences (138E12, 134G2, 10161, 115E1, 99G11, 127B12, 91F6, 135F9, 116D9, 212D11, 210A6, 124F5, 129D2 and 141F7) obtained in the second round of shuffling were subsequently subjected to a third round of shuffling (still according to the patented process described in EP1104457B1).
• at least one gene encoding a beta-glucosidase having 70% identity with these genes has been added.
• the putative betaglucosidase gene of Neurospora crassa (gene C) (SEQ ID No.
• a high throughput screening assay as described above (with the exception of the IPTG induction step, since the improvement in the first round of shuffling allows detection of beta-glucosidase activity based only on on the escape of the promoter) was performed on the clones obtained following this third round of shuffling. The activity of these clones was measured at 30 ° C and 50 ° C.
• the clone 17E5 (amino acid sequence SEQ ID No. 1, encoded by the nucleic acid sequence SEQ ID No. 2) was selected because it has a 30 ° C activity ratio. / 50 ° C interesting.
• Table 6 shows the relative activities obtained at 50 ° C. and at 30 ° C. for clone 17E5 and for clone 100B11 (reference clone resulting from the second round of shuffling).
• the activity of clone 17E5 was measured at 30 ° C and 50 ° C by the activity assay as previously described.
• Table 7 shows the value of the kcat as well as the improvement factor obtained for clone 17E5 under these experimental conditions.
• the results show an improvement in the enzymatic activity of clone 17E5 by a factor of 2 relative to the reference clone and at both temperatures.
• Trichoderma reesei The 17E5 gene was cloned into a vector allowing expression in a strain Trichoderma reesei derived from RUT C30 (ATCC 56765), CL847 (Durand et al., Enzyme Microb Technol, 1988; 10: 341-346). with selection by hygromycin (Hph gene of Streptomyces hygroscopicus).
• the 17E5 gene was placed under control of a strong cbh1 promoter inducible at the same time as the other T. reesei cellulases.
• Trichoderma reesei The transformation of Trichoderma reesei was carried out according to the standard methods known to those skilled in the art (transformation of protoplasts by calcium shock and selection with hygromycin 50 ⁇ g / ml). The transformants were purified by sporulation and then subcultured twice in selective medium to eliminate unstable clones. Thirty clones were then evaluated for cellulase production in 24-well plates.
• a few spores of each clone were seeded with 2 ml of medium having the following composition: lactose 20g / L, cellulose Solka floc 20g / L, Peptone 5g / L, KH 2 PO 4 15g / L, (NH 4 ) 2 S0 4 5g / L, CaCl 2 0.6g / L, MgSO 4 0.6g, FeS0 4 0.005g / L, MnSO 4 0.0014g / L, ZnSO 4 0.0014g / L, CoCl 2 0.0037g / L, 11.6 g / L maleic acid, 12.1 g / L Tris and 2.08 g / L NaOH.
• the flasks were incubated at 30 ° C with shaking at 150 rpm.
• the beta-glucosidase activity of the supernatants was measured by hydrolysis of the para-nitrophenyl beta-D-glucopyranoside chromophore substrate (pNPG) under the following conditions:
• the reaction was stopped by adding 100 ⁇ ⁇ sodium carbonate 2%.
• the amount of para-nitrophenol liberated by hydrolysis of pNPG was measured by measuring the absorbance at 410 nm and compared to a range of para-nitrophenol.
• the reaction was linear from 25 to 400 ⁇ of para-nitrophenol.
• the samples were optionally diluted so that the measured absorbance remains in the linearity of the range.
• the beta-glucosidase activity was also measured at 50 ° C., under the same conditions as above, for comparison. Clones exhibiting the highest beta-glucosidase activity (at least 5-fold higher than the original strain) were selected.
• Table 8 shows the beta-glucosidase pNPase 35 ° C / 50 ° C activities measured in ⁇ / min / mg enzyme for supernatants derived respectively from a wild type CL847 strain, a strain expressing the 100B11 variant and from one of the clones expressing the 17E5 variant obtained according to the method described above.
• Table 8 Beta-glucosidase activities of wild-type CL847, 10OBll polypeptide and 17E5 polypeptide
• the wild-type beta-glucosidase gene of Trichoderma reesei (BGL1) as well as those of variants 10161 and 17E5 were cloned without signal peptide in the vector pESC-Leu (Agilent Technologies).
• This construction allows the expression of the protein in the cytoplasm of the strain Saccharomyces cerevisiae EBYIOO, auxotrophic for leucine and tryptophan (Boder ET and Wittrup KD, Biotechnol Prog, 1998, 14: 55-62).
• This plasmid makes it possible to place the expression of genes under the control of the galactose-inducible promoter GAL1, and has the auxotrophic selection marker gene (Leu2) which allows the selection of transformants.
• the protein produced is also fused to the N-terminal c-myc tag, allowing the detection and purification of the enzyme produced by affinity chromato graphy.
• the transformation of S. cerevisiae EBYIOO was carried out according to the standard methods known to those skilled in the art (yeast transformation by thermal shock and lithium acetate). The transformants were selected on YNB-Glc-Trp medium containing 0.67% Yeast Nitrogen Base (YNB), 2% glucose and 0.01% tryptophan.
• the total protein concentration in the cytoplasmic extract was estimated on average by Bradford assay (Bradford MM., Anal Biochem, 1976, 72: 248-54) at 1.7 mg / mL.
• beta-glucosidase activity cytoplasmic extracts was measured by hydrolysis of p-nitrophenyl beta-D-glucopyranoside substrate (pNPG) in a volume of 600 ⁇ ⁇ under the following conditions:
• the reaction was stopped by adding 100 ⁇ ⁇ of 1M sodium carbonate to 100 ⁇ ⁇ of hydrolysis reaction.
• the para-nitrophenol (pNP) concentration released by hydrolysis of pNPG was determined by measuring the absorbance at 415 nm and compared to a standard range of para-nitrophenol (linear from 0.36 ⁇ to 360 ⁇ ).
• the cytoplasmic extracts were optionally diluted to be in the initial reaction rate condition.
• Table 9 shows the activity ratios beta-glucosidase 30 ° C / 50 ° C measured in ⁇ 1. ⁇ "1 . ⁇ ” 1 of total proteins for cytoplasmic extracts from a strain respectively expressing the wild-type enzyme (Sc -BGL1), a strain expressing 10101 (Sc-100B11) and a strain expressing 17E5 (Sc-17E5).
• the cytoplasmic extracts of Sc-BGL1 and Sc-100B 11 and Sc-17E5 variants of Example 5 were used to purify the corresponding enzymes, BGL1, 10OB11 and 17E5 according to the following protocol:
• the concentration of the purified enzymes is obtained by measuring the absorbance at 280 nm nanodrop, using a molar extinction coefficient equal to 120 125 M "1. Cm" 1 for native BglI and 120 250 M "1. Cm” 1 for 100B11 and 17E5. It is on average equal to 0. 19 mg / ml. The purity of each enzyme was verified by 10% polyacrylamide gel electrophoresis in the presence of SDS with staining of Coomassie Blue proteins.
• BGL1 and purified variants 100B11 and 17E5 were measured at 30 ° C and 50 ° C as previously described.
• Table 10 shows the specific activities of each enzyme (in
• results show an improvement at 30 ° C of the specific activity of the variant 17E5 by a factor of 2 relative to wild-type BGL1 and of 1.4 relative to the 100B11 variant.

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