Variants of exoglucanases having improved activity and uses thereof

Abstract

The present invention relates to the expression and optimisation of enzymes involved in the breakdown of lignocellulosic biomass. The present invention relates to variants of the exoglucanase 2 of Trichoderma reesei, as well as to the use of said variants with improved efficiency in methods for breaking down cellulose and for producing biofuel.

Claims

1. An isolated or purified polypeptide having an improved exoglucanase activity of at least 10% at a temperature of 35 C. compared with the exoglucanase activity of the exoglucanase 2 reference protein of SEQ ID NO: 2, said polypeptide being selected from the group consisting of: i. the amino acid sequence selected from SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 28; and ii. an amino acid sequence having a percentage identity of at least 98% compared with the sequence SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 28.

2. An enzymatic composition capable of acting on lignocellulosic biomass, said enzymatic composition being produced by filamentous fungi and comprising at least one polypeptide as claimed in claim 1.

3. An isolated or purified polypeptide according to claim 1, said polypeptide being selected from the group consisting of: an amino acid sequence having a percentage identity of at least 99% compared with the sequence SEQ ID NO: 22, SEQ ID NO: 24, and SEQ ID NO: 28.

4. A process for producing biofuel from lignocellulosic biomass, comprising the following successive steps: the biomass to be hydrolyzed is suspended in an aqueous phase; the lignocellulosic biomass is hydrolyzed in the presence of an enzymatic composition comprising at least one polypeptide as claimed in claim 1 the biomass so as to produce a hydrolysate containing glucose; the glucose of the hydrolysate is fermented in the presence of a fermentative organism so as to produce a fermentation must; the biofuel is separated from the fermentation must.

5. A process for producing biofuel from biomass, comprising the following successive steps: the biomass to be hydrolyzed is suspended in an aqueous phase; an enzymatic composition comprising at least one polypeptide as claimed in claim 1 and a fermentative organism are added simultaneously to the suspension and the mixture is fermented so as to produce a fermentation must; the biofuel is separated from the fermentation must.

6. A process for hydrolyzing cellulosis comprising the use of a polypeptide according to claim 1.

7. A process for producing biofuel comprising the use of a polypeptide according to claim 1.

Description

(1) Other aspects, subjects, advantages and features of the invention will be presented on reading the non-restrictive description which follows and which describes preferred embodiments of the invention, given by means of examples and of the FIGURES.

(2) FIG. 1 is a MALDI-TOF mass spectrum representing the DP3 to DP11 cellodextrins used for the screening.

EXAMPLES

Example 1: Preparation Reduced Cellodextrins (DP 3-11)

(3) 1Cellulose Hydrolysis

(4) (Adapted from Y-H. Percival Zhang, L. R. Lynd Analytical Biochemistry 322 (2003), 225-232.)

(5) ##STR00001##

(6) 20 g of cellulose (Avicel, CAS Number 9004-34-6, Sigma-Aldrich Saint-Quentin Fallavier) are added portionwise and with vigorous stirring to 160 ml of a hydrochloric acid solution cooled to 0 C. Precooled sulfuric acid is added to the solution in several steps (410 ml). The reaction is kept stirring for four hours at 24 C. before being poured into 1.8 l of acetone cooled to 20 C. After two hours of stirring, the precipitate is filtered off, taken up in 400 ml of cooled acetone and then again filtered. The solid is then taken up in 600 ml of water, and then stirred overnight in order to dissolve the cellodextrins (CDs). After the solid has been filtered off, the soluble fraction containing the cellodextrins is neutralized with 300 g of Amberlite IRA 400 OH.sup. resin and then lyophilized. The lyophilisate is then resuspended in 500 ml of methanol in the presence of ultrasound for 30 minutes in order to dissolve the low-molecular-weight sugars, before being filtered and then lyophilized again so as to give 6.8 g of DP 3-11 cellodextrins.

(7) For the screening, it was chosen to work with substrates of the highest possible molecular weight in order to mimic as closely as possible the structure of cellulose. However, high-molecular-weight cellodextrins are not soluble, which prevents good reproducibility of the tests.

(8) A range of cellodextrins of DP 5-7 was therefore chosen, which represents a good compromise between the high molecular weight required and the solubility of the cellodextrins.

(9) FIG. 1 presents a MALDI-TOF mass spectrum typically obtained according to the process described above.

(10) FIG. 1 shows that the isolated oligosaccharides are predominantly of DP 5-7.

(11) 2Cellodextrin Reduction

(12) 400 mg of sodium borohydride are added to 2 g of DP 3-11 cellodextrins diluted in 120 ml of water. After three hours with stirring at ambient temperature, the solution is neutralized by adding Amberlite H.sup.+ IR 120 resin, filtered, and then lyophilized, so as to give 2 g of quantitatively reduced cellodextrins (C. Schou, G. Rasmussen, M-B. Kaltoft, B. Henrissat, M. Schulein Eur. J. Biochem. 217, 947-953 (1993)).

(13) Assaying of the isolated cellodextrins with BCA (bicinchoninic acid) makes it possible to verify the total reduction of the ends (Y.-H. Percival Zhang, L. R. Lynd Biomacromolecules 2005, 6, 1510-1515).

Example 2: Evolution by L-Shuffling

(14) The sequence of the cellobiohydrolase 2 gene from Trichoderma reesei (SEQ ID NO: 1) was subjected to a round of L-shuffling according to the patented process described in patent EP 1 104 457 with the genes of a putative exoglucanase from Giberella zeae PH-1 (SEQ ID NO: 29) and of a hypothetical protein NECHADRAFT 73991 from Nectria haematococca mpVI (SEQ ID NO: 31) having respectively 63% and 69% homology with the parental gene CBH2 (SEQ ID NO: 1). The nucleic sequence encoding the signal peptide (SEQ ID NO: 33) was deleted during the cloning, and replaced with that of yeast, of sequence SEQ ID NO: 34 (sequence of the corresponding signal peptide: SEQ ID NO: 35).

(15) 1High-Throughput Screening

(16) A high-throughput screening test was developed in order to select the best clones resulting from the L-shuffling, i.e. those exhibiting at least 20% improvement in the cellobiohydrolase activity compared with the CBH2 reference enzyme (SEQ ID NO: 2).

(17) The high-throughput screening test was carried out according to the following steps: isolation on agar of the clones of Y. lipolytica expressing the L-shuffling variants of the enzyme according to the invention and preculturing in YNB casa medium (yeast nitrogen base 1.7 g/l, NH.sub.4Cl 10 g/l, glucose 10 g/l, casamino acids 2 g/l, pH 7) of said colonies for 36 hours at 28 C.; inoculation of a YTD medium (yeast extract 10 g/l, tryptone 20 g/l, glucose 2.5 g/l, pH 6.8) supplemented with tetracycline at 12.5 g/ml at 5% with the preculture and then incubation for 20 hours at 28 C.; inoculation of the expression medium containing the inducer (oleic acid) in an amount of 20 g/l at 10% with the previous culture and then incubation for 96 hours at 28 C.; centrifugation for five minutes at 1500 rpm; removal of 100 l of supernatant; addition of 100 l of reduced CDs at 1 g/l in 0.1 M citrate phosphate buffer at pH 6; incubation for 24 hours at 35 C.; centrifugation for five minutes at 2500 rpm; removal of 80 l of supernatant; addition of 80 l of DNS reagent; incubation for 12 minutes at 105 C. and then five minutes on ice; reading of the optical density (OD) at 540 nm on 120 l.

(18) Under these screening conditions, an improvement in the cellobiohydrolase activity (increase in the OD at 540 nm) compared with the CBH2 reference enzyme (SEQ ID NO: 2) was found in several clones. Among these clones, mention may be made of the 35B7, 95B7, 100F11, 139F12, 157B11, 161A1, 161C12, 189H8, 196D9, 198E11, 251B4, 251C4 and 382A2, encoding respectively the enzymes SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28.

(19) 2Determination of the Improvement in the Cellobiohydrolase Activity

(20) 2-1/ On the Reduced-Cellodextrin Substrate

(21) In order to estimate the relative kcat of the variants selected in the first round of L-shuffling with respect to the CBH2 reference enzyme (SEQ ID NO: 2), the following process is carried out: preparation of a stock culture of Y. lipolytica expressing a recombinant enzyme according to the invention, overnight at 28 C.; inoculation of an expression medium with a volume of stock culture making it possible to have an optical density at 600 nm equal to 0.2 at the beginning of the culture; culture of said cells at 28 C. for 96 hours; centrifugation at 8000 rpm for five minutes; incubation of 100 l of supernatant with 100 l of 0.1 M citrate phosphate buffer, pH 6, containing 1% of reduced CDs, for 4 hours at 35 C. and 50 C.; removal of 100 l of reaction; addition of 100 l of DNS reagent; incubation for five minutes at 100 C.; incubation for three minutes on ice; centrifugation for 10 minutes at 3000 rpm; reading of the optical density at 540 nm on 150 l.

(22) According to the invention, the calculation of the kcats is carried out in the following way: plotting the curve of the ODs at 540 nm as a function of the volume of culture supernatant in the test; subtracting the value of the negative control; dividing by the coefficient of the glucose standard rate (various amounts of glucose are revealed with the DNS); dividing by the reaction time (240 minutes).

(23) Table 2 gives the value of the kcats and also the improvement factor obtained for the 35B7, 95B7, 100F11, 139F12, 157B11, 161A1, 161C12, 189H8, 196D9, 198E11, 251B4, 251C4 and 382A2 clones encoding respectively the enzymes SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28 compared with the CBH2 reference protein (SEQ ID NO: 2) under these experimental conditions.

(24) TABLE-US-00002 TABLE 2 improvement in the cellobiohydrolase activity on reduced CDs 35 C. 50 C. Kcat Improvement Kcat Improvement Clone (min.sup.1) factor (min.sup.1) factor First-round 35B7 0.166 3.8 0.2345 1.6 clones 95B7 0.287 6.6 0.715 4.8 100F11 0.0508 1.2 0.1375 0.9 139F12 0.1719 3.9 0.2328 1.6 157B11 0.113 2.6 0.2061 1.4 161A1 0.0577 1.3 0.1175 0.8 161C12 0.1086 2.5 0.2162 1.4 189H8 0.0872 2.0 0.1792 1.2 196D9 0.1055 2.4 0.1969 1.3 198E11 0.1218 2.8 0.1757 1.2 251B4 0.0495 1.1 0.0865 0.6 251C4 0.0623 1.4 0.1315 0.9 382A2 0.315 7.2 0.552 3.7 Reference cbh2 0.0436 1 0.1501 1 protein

(25) The results show an improvement in enzymatic activity compared with the CBH2 reference enzyme (SEQ ID NO: 2) for the 35B7, 95B7, 100F11, 139F12, 157B11, 161A1, 161C12, 189H8, 196D9, 198E11, 251B4, 251C4 and 382A2 clones encoding respectively the enzymes SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28, whether at 35 C. or at 50 C.

(26) 2-2/ On the Avicel Substrate

(27) The improvement in activity of the 35B7, 95B7, 100F11, 139F12, 157B11, 161A1, 161C12, 189H8, 196D9, 198E11, 251B4, 251C4 and 382A2 clones encoding respectively the enzymes SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28 was then measured with a second substrate: Avicel.

(28) The determination of the improvement in the activity on this substrate is carried out by end-point measurement according to the following protocol: preparation of a stock culture of Y. lipolytica expressing a recombinant enzyme according to the invention, overnight at 28 C.; inoculation of an expression medium with a volume of stock culture making it possible to have an optical density at 600 nm equal to 0.2 at the beginning of the culture; culture of said cells at 28 C. for 96 hours; centrifugation at 8000 rpm for 5 minutes; incubation of 100 l of supernatant with 100 l of 0.1 M citrate phosphate buffer, pH 6, containing 1% of Avicel, for 18 hours at 35 and 50 C.; removal of 100 l of reaction; addition of 100 l of DNS reagent; incubation for 5 minutes at 100 C.; incubation for 3 minutes on ice; centrifugation for 10 minutes at 3000 rpm; reading the optical density at 540 nm on 150 l.

(29) Table 3 presents the value of the ODs at 540 nm (after subtraction of the value of the negative control) and also the improvement factor obtained for the 35B7, 95B7, 100F11, 139F12, 157B11, 161A1, 161C12, 189H8, 196D9, 198E11, 251B4, 251C4 and 382A2 clones under these experimental conditions.

(30) TABLE-US-00003 TABLE 3 improvement in the cellobiohydrolase activity on Avicel 35 C. 50 C. Delta OD Improvement Delta OD Improvement Clone 540 nm factor 540 nm factor First- 35B7 0.0617 1.0 0.0948 0.8 round 95B7 0.0396 0.6 0.0555 0.4 clones 100F11 0.038 0.6 0.0159 0.1 139F12 0.06 0.9 0.0365 0.3 157B11 0.0456 0.7 0.0319 0.3 161A1 0.0508 0.8 0.0237 0.2 161C12 0.0564 0.9 0.0595 0.5 189H8 0.0676 1.0 0.0573 0.5 196D9 0.0565 0.9 0.0874 0.7 198E11 0.0867 1.3 0.0546 0.4 251B4 0.0765 1.2 0.0622 0.5 251C4 0.063 1.0 0.0889 0.7 382A2 0.2476 3.8 0.2256 1.8 Reference cbh2 0.0644 1 0.1252 1 protein

(31) The results from table 3 show an improvement in the enzymatic activity, compared with the CBH2 reference enzyme (SEQ ID NO: 2) at 35 C. for the 198E11 and 251B4 clones (respectively SEQ ID Nos: 22 and 24) and also an improvement in the enzymatic activity compared with the CBH2 reference enzyme (SEQ ID NO: 2) at 35 C. and at 50 C. for the 382A2 clone (SEQ ID NO: 28).