Method for producing an enzymatic cocktail from fungal must

Abstract

The invention relates to a process for the production of an enzymatic cocktail from a cellulolytic microorganism producing cellulases and/or hemi-cellulases, comprising: a step for the production of enzymes with a medium being obtained containing enzymes and a microorganism must, said must is separated, or not separated, from the liquid containing said enzymes, a step of cooling said must to a temperature below the temperature of the enzyme production step and for a time such that the beta-glucosidase concentration of the liquid originating from the cooling step is greater than that of the liquid originating from the enzyme production step, and/or the solid volume/total volume ratio is less than said ratio for the enzyme production step, and an enzymatic cocktail is obtained at the end of the cooling step.

Claims

1. A process for the production of an enzymatic cocktail from a cellulolytic microorganism producing cellulases and/or hemi-cellulases, where the microorganism is a filamentous fungus comprising: aproducing enzymes to obtain a medium containing enzymes and a microorganism must, and separating said must from the liquid containing said enzymes, bcooling said must to a temperature between 4 and 20 C., which is a temperature below the temperature of the enzyme production step, for a time of 12 to 90 hours such that: the beta-glucosidase or -xylosidase concentration of liquid originating from cooling is greater than that of the liquid originating from the enzyme production (a), and/or the solid volume/total volume ratio is less than said ratio for the enzyme production and cobtaining an enzymatic cocktail at the end of cooling (b).

2. The process according to claim 1 in which said separated must is diluted with water.

3. The process according to claim 1 in which, after cooling (b), the enzymatic cocktail originating from the must is separated and a residual must is obtained.

4. The process according to claim 1 in which the separation takes place by at least one centrifugation or filtration/pressing or microfiltration, optionally preceded by settling.

5. The process according to claim 3 in which the enzymes of the separated liquid are concentrated.

6. The process according to claim 1 in which cooling (b) is carried out at a pH between 3.5 and 5.5.

7. A process for the production of an enzymatic cocktail from a cellulolytic microorganism producing cellulases and/or hemi-cellulases, where the microorganism is a filamentous fungus comprising: aproducing enzymes to obtain a medium containing enzymes and a microorganism must, optionally separating said must from the liquid containing said enzymes, bcooling said must to a temperature between 4 and 20 C., which is a temperature below the temperature of the enzyme production step, for a time such that: the beta-glucosidase or -xylosidase concentration of the liquid originating from cooling is greater than that of the liquid originating from the enzyme production (a), and/or the solid volume/total volume ratio is less than said ratio for the enzyme production, and cobtaining an enzymatic cocktail is obtained at the end of cooling (b), in which cooling (b) is carried out in an atmosphere depleted of oxygen.

8. A process for the production of an enzymatic cocktail from a cellulolytic microorganism producing cellulases and/or hemi-cellulases, where the microorganism is a filamentous fungus comprising: aproducing enzymes to obtain a medium containing enzymes and a microorganism must, optionally separating said must from the liquid containing said enzymes, bcooling said must to a temperature between 4 and 20 C., which is a temperature below the temperature of the enzyme production step, for a time such that: the beta-glucosidase or -xylosidase concentration of the liquid originating from cooling is greater than that of the liquid originating from the enzyme production (a), and/or the solid volume/total volume ratio is less than said ratio for the enzyme production, and cobtaining an enzymatic cocktail is obtained at the end of cooling (b), in which in cooling (b), a neutral gas is injected.

9. The process according to claim 1 in which the microorganism belongs to the genus Trichoderma.

10. The process according to claim 3 in which said residual must is subjected to cooling and an enzymatic cocktail, optionally separated from the residual must, is obtained at the end of the cooling.

11. The process according to claim 10 in which enzymatic cocktails originating from production of separate must are mixed.

12. The process according to claim 1 in which at least one enzymatic cocktail is mixed with separated liquid containing enzymes and originating from the enzyme production (a).

13. A process for enzymatic hydrolysis of a feed, comprising subjecting said feed to at least one enzymatic cocktail obtained according to claim 1.

14. The process according to claim 9, wherein the microorganism belongs to Trichoderma reesei.

15. The process according to claim 14, wherein the microorganism belongs to the strain CL 847.

16. The process according to claim 7, wherein cooling is carried out in an anaerobic atmosphere.

17. The process according to claim 8, wherein the cooling gas is carbon dioxide or nitrogen.

18. A process for the production of an enzymatic cocktail from a cellulolytic microorganism producing cellulases and/or hemi-cellulases, where the microorganism is a filamentous fungus comprising: aproducing enzymes to obtain a medium containing enzymes and a microorganism must, and separating said must from the liquid containing said enzymes, bcooling said must to a temperature between 4 and 20 C., which is a temperature below the temperature of the enzyme production step, for a time of 12 to 90 hours such that: the beta-glucosidase or -xylosidase concentration of liquid originating from cooling is greater than that of the liquid originating from the enzyme production (a), and/or the solid volume/total volume ratio is less than said ratio for the enzyme production and cobtaining an enzymatic cocktail at the end of cooling (b), by mixing the liquid containing said enzymes from (a) and the liquid originating from cooling in (b).

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The invention will be better understood from FIGS. 1 to 6. FIGS. 7 to 13 relate to the examples.

(2) FIG. 1 describes the preferred embodiment with a separation step between the enzyme production step and the cooling step. FIG. 2 is a representation without separation of the must. In FIGS. 3 and 4, an additional cooling step is added to FIGS. 1 and 2 respectively.

(3) FIG. 5 shows an embodiment combining the presence or absence of separation.

(4) FIG. 6 shows a preferred embodiment of the must separation step before and/or after the cooling step.

(5) FIG. 7 is a photo of the Trichoderma reesei CL847 must after separation and before the cooling step.

(6) FIG. 8 shows the change in the concentration of proteins in the culture supernatant.

(7) FIG. 9 shows the -glucosidase activity measured at the end of the production step and after cooling the fungus at 4 C. for 72 hours.

(8) FIG. 10 shows the -glucosidase activity measured at the end of the culture and after autolysis of the fungus at 4 and 20 C. for 72 hours.

(9) FIG. 11 shows the specific -glucosidase activity (IU/mg) obtained after the two separation series.

(10) FIG. 12 shows the specific FPase activity (IU/mg) obtained after the two separation series.

(11) FIG. 13 corresponds to the identification of the enzymes in the separation step.

(12) According to FIG. 1, a carbon source and an inductive source are introduced (pipe 1) into an enzyme production step (enzyme production zone 2), as well as a cellulolytic microorganism producing cellulases and/or hemi-cellulases (pipe 3) and the required nutrients (pipe 4).

(13) The product obtained is separated (separation zone 5) into a liquid containing the enzymes (pipe 6) and a must is recovered (pipe 7) containing the fungus and enzymes held within.

(14) The must is subjected to the cooling step (cooling zone 8).

(15) In an embodiment, the liquid is drawn off from the enzyme production zone (reactor) and the must remains in said zone where it is cooled down.

(16) In another preferred embodiment, the culture medium is drawn off from the enzyme production zone and separated in a separation means (filtration/pressing, centrifugation etc.), preferably after settling and drawing-off of the liquid, in order to obtain the must. This must is introduced into the cooling zone which can be the reactor of the enzyme production zone (in the case of a discontinuous process) or another reactor (in the case of a continuous process).

(17) The cooled must (pipe 9) is separated (zone 10) into a liquid containing the enzymatic cocktail (pipe 11) and a solid which is the residual must (pipe 12).

(18) The enzymes in the flows of pipes 6 and 11 are mixed and are sent (pipe 13) into the enzymatic hydrolysis zone (14).

(19) FIG. 3 repeats this diagram, adding a cooling step.

(20) The residual must (must 12) is subjected to the additional cooling step (cooling zone 15).

(21) In the same way as previously, the must sent into the additional cooling step (zone 15) may not have been separated in the zone 10 after the first cooling step (zone 8). The cooled must (pipe 16) is separated (zone 17) into a liquid containing the enzymatic cocktail (pipe 19) and a solid which is the residual must (pipe 18).

(22) The enzymes in the flow of the pipe 19 are mixed with those of the flows originating from the enzyme production step (pipe 6), from the first cooling step (pipe 11) and are sent (pipe 20) into the enzymatic hydrolysis zone (14).

(23) The reference numbers of FIG. 1 will be recognized in FIG. 2. The separation step (zone 5) after the enzyme production step is omitted.

(24) The same applies to FIG. 4 which is based on FIG. 2 and which includes the additional cooling step, the reference numbers relating to this addition are taken from FIG. 3.

(25) It is also possible to combine in one diagram the presence of one separation and the absence of the other separation. This is for example illustrated in FIG. 5. This shows the presence of the separation at the end of the enzyme production step (zone 5), the absence of separation at the end of the first cooling step (zone 8) and before the additional cooling step, and the presence of separation after the additional cooling step (zone 17).

(26) FIG. 6 shows a preferred embodiment of the must separation step before and/or after the cooling step.

(27) With reference to FIG. 1, this is an embodiment of zone 5 and/or of zone 10.

(28) At the end of the enzyme production zone 2, the culture medium is separated in a settling step (zone 30), the fungus is located in the settled sludge (pipe 31) and the cloudy liquor obtained (pipe 32) passes into a centrifugation step (centrifugation zone 33). This results in a cream (pipe 34) containing the fungus and a clear liquor (pipe 35). In order to achieve the separation, the clear liquor passes into a microfiltration step (zone 36), the retentate (pipe 37) contains the fungus, and the permeate (pipe 38) contains the enzymes.

(29) The different flows containing the fungus (sludge, cream, retentate) can be mixed and constitute the must which will be sent to the cooling step or constitute the residual must which will be subjected, or not subjected, to a new cooling step.

(30) In order to concentrate the enzymes, the permeate is passed into an ultrafiltration step. A concentrated retentate of enzymes (pipe 40) is then obtained, as well as a permeate (pipe 41) which can be reused in the process.

(31) It will be noted that all of the settling and centrifugation steps can be replaced by one filtration/pressing (filter/press).

EXAMPLES

Example 1: With FIGS. 7 to 9

(32) FIG. 7 is a photo of the Trichoderma reesei CL847 must after separation and before the cooling step.

(33) FIG. 8 shows the change in the concentration of proteins in the culture supernatant.

(34) FIG. 9 shows the -glucosidase activity measured at the end of the production step and after cooling the fungus at 4 C. for 72 hours.

(35) The production of cellulases is carried out in a 20 L bioreactor (of which 12 L are useful) stirred mechanically. The mineral medium has the following composition: KOH 1.66 g/L, H3PO4 85% 2 mL/L, (NH4)2SO4 2.8 g/L, MgSO4.7 H2O 0.6 g/L, CaCL2 0.6 g/L, MnSO4 3.2 mg/L, ZnSO4.7 H2O 2.8 mg/L, CoCl2 10 4.0 mg/L, FeSO4.7 H2O 10 mg/L, Corn Steep 1.2 g/L, antifoaming agent 0.5 mL/L.

(36) The bioreactor containing the mineral medium is sterilized at 120 C. for 20 minutes, the glucose carbon-containing source is sterilized separately at 120 C. for 20 minutes then added sterilely into the bioreactor so as to have a final concentration of 30 g/L. The bioreactor is seeded at 10% (v/v) with a liquid pre-culture of the strain of Trichoderma reesei CL847. The mineral medium of the pre-culture is identical to that of the bioreactor apart from the addition of potassium phthalate at 5 g/L in order to buffer the pH. The growth of the fungus in the preculture is carried out using glucose as carbon-containing substrate, at a concentration of 30 g/L. The growth of the inoculum lasts from 2 to 3 days and is carried out at 28 C. in a stirred incubator. The transfer to the bioreactor is carried out if the residual glucose concentration is less than 15 g/L.

(37) The production experiment carried out in the bioreactor comprises two phases: a growth phase on a glucose carbon-containing substrate (initial concentration=30 g/L) at a temperature of 27 C. and a pH of 4.8 (regulated with 5.5 M ammonia). Aeration is at 0.5 vvm and stirring is increased between 200 and 800 rpm depending on the pO2 (dissolved oxygen pressure), which is maintained higher than 30%. an enzyme production phase. When the original substrate of the fermenter is exhausted, a solution of lactose at 250 g/L is continuously injected at a flow rate of 35 to 45 mg per g of cells and per hour up to 164 hours. The temperature is reduced to 25 C. and the pH to 4 until the end of the culture. The pH is adjusted by the addition a 5.5 N solution of ammonia which supplies the nitrogen required for the synthesis of the excreted proteins. The dissolved oxygen content is maintained above 15 to 20% by the aeration and stirring action.

(38) The production of enzymes is followed by assay of the extracellular proteins by the Lowry method and BSA standard, after separation of the mycelium by filtration or of cells formed). The final concentration of proteins obtained is equal to 45 g/L.

(39) This production step was followed by separation of the fungal must from the culture supernatant. The must was pressed so as to extract a maximum amount of supernatant (FIG. 7) and weighed. The latter was placed in a closed container at 4 C. for 72 hours.

(40) Samples of the supernatant released following the autolysis of the fungus were taken every 24 hours up to 72 hours

(41) The weight of the liquid was determined after 72 hours. It is equivalent to 30% of the weight of the must. Assays of the protein concentrations are shown in FIG. 8. It can be seen that the concentration is multiplied by 3 relative to the end of the enzyme production as well as the -glucosidase activity (FIG. 9)

Example 2: With FIGS. 10 to 13

(42) FIG. 10 shows the -glucosidase activity measured at the end of the culture and after autolysis of the fungus at 4 and 20 C. for 72 hours.

(43) FIG. 11 shows the specific -glucosidase activity (IU/mg) obtained after the two separation series.

(44) FIG. 12 shows the specific FPase activity (IU/mg) obtained after the two separation series.

(45) FIG. 13 corresponds to the identification of the enzymes in the separation step.

(46) The process is implemented in a 6 m.sup.3 pilot unit. The fungus Trichoderma reesei CL847 is cultured under the same conditions described in Example 1. At the end of the production phase the enzymes are separated in 4 steps: a settling step, a centrifugation step, a microfiltration step and an ultrafiltration step.

(47) These four steps are shown diagrammatically in FIG. 6. The first three steps serve to eliminate the fungus and the last step (ultrafiltration) serves to concentrate the enzymes produced.

(48) All the fungal fractions recovered (settler sludge, centrifuge cream and microfiltration retentate), are placed in the bioreactor, cooled at 8 C. for 72 hours and diluted with water to a final volume equal to 4 m.sup.3. The must undergoes a second separation series: centrifuge, microfiltration and ultrafiltration.

(49) The quantity of enzymes recovered at the end of the second separation series is similar to that obtained at the end of the first, i.e. approximately 70 kg of proteins after the first separation series and 64 kg after the second separation series.

(50) By contrast, it is interesting to note that the -glucosidase activity (FIG. 11) and the FPase activity (FIG. 12) specific to the enzymatic cocktail obtained are 20% greater after the cooling step (autolysis of the fungus). The enzymatic cocktail recovered is consequently much more effective.

(51) Two-dimensional electrophoreses were carried out in order to see if there was a significant modification of the composition of the enzymatic cocktail during the separation steps. Starting from samples desalinated beforehand using FPLC [fast protein liquid chromatography], with 200 g deposits on the 2D gel, and Coomassie blue staining, the proteins are separated according to their molecular mass and their isoelectric point.

(52) The scanned gels are shown in FIG. 13; FIG. 13a corresponds to the cream and FIG. 13b to the clear liquor.

(53) The main enzymes were identified in the centrifugation step in the clear liquor and the cream. A significant difference was noted in the profiles of the gels. The centrifugation creams show -xylosidases. This activity was measured on pnp-xylose for confirmation and a specific activity was found which was approximately 3 times greater in the creams than in the enzymatic cocktail obtained at the end of production (1.2 IU/mg in the creams and 0.4 IU/mg in the final sample from the fermenter).