Resumen
The present invention provides a method of producing a lipolytic enzyme comprising the steps of: (i) providing a Trichoderma reesei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions; and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme.
Reivindicaciones
CLAIMS 1 A method of producing a lipolytic enzyme comprising the steps of. (i) providing a transformed or transfected Thchoderma reβsei cell comprising a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ !D NO. 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions, and (ii) culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme 2. A method of producing a lipolytic enzyme comprising the steps of: (i) transfecting or transforming a Tήchoderma rβesei cell with a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO; 1 or SEQ ID NO: 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO. 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions; (H) optionally repeating step (i) on the cell to sequentially transfect or transform the cell with at least one additional heterologous nucleotide sequence as defined In (i)(a), (i)(b) or (I)(C); and (iii) cuSturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme. 3. A method according to claim 1 or claim 2 wherein the heterologous nucleotide sequence further comprises a promoter sequence, which promoter sequence is a cellobiohydrolase promoter sequence 4. A method according to any one of claims 1 to 3 wherein the lipolytic enzyme is produced in an amount of at least 20g/litre of culture supernatant 5 A method according to any one of the preceding claims, wherein said method comprises the additional step of concentrating and/or isolating and/or recovering the lipolytic enzyme, 6. A method according to any one of the preceding claims, wherein the Trichoderma reesei cell is prepared by transforming or transfecting a Trichoderma reesei cell with the nucleotide sequence. 7 A method according to any one of the preceding claims, wherein the Trichoderma reesei celi is provided by transforming it with or is transformed with the nucleotide sequence using bioiistic transformation 8. A lipolytic enzyme obtainable by the method of any one of the preceding claims 9. A foodstuff comprising said lipolytic enzyme according to claim 8 10 A Trichoderma reesei cell comprising: a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 40 % sequence identity to SEQ ID NO: 1 or 2; and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO: 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises an the nucleotide sequence which hybridizes to SEQ ID NO. 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions 11. A Trichoderma reesei cell according to claim 10, wherein the cell comprises at least two (such as at least three, such as at least four, such as at least five, or such as at least six) heterologous nucleotide sequences encoding said lipolytic enzyme 12, An expression vector comprising i) at least one nucleotide sequence which nucleotide sequence: a) encodes a lipolytic enzyme protein having at least 40% sequence identity to SEQ ID NO: 1 or 2, and/or b) encodes a lipolytic enzyme and comprises the nucleotide sequence shown as SEQ ID NO. 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) encodes a lipolytic enzyme wherein the nucleotide sequence comprises an the nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO. 3 or SEQ ID NO. 4 or the complement of any thereof under stringent conditions, and ii) at least one ceSlobiohydrolase promoter, wherein said at least one nucleotide sequence is under the control of said at least one cellobiohydrolase promoter 13. An expression vector according to claim 12 wherein said vector further comprises a nucleotide sequence encoding a signal peptide, which nucleotide sequence encoding said signal peptide is operabiy linked to said nucleotide sequence encoding said lipolytic enzyme. 14. Use of a Trichoderma reesei cell in the expression of a) at least one heterologous nucleotide sequence encoding a lipolytic enzyme comprising an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO. 2 or an amino acid sequence which has at least 40% sequence identity to SEQ ID NO. 1 or 2, and/or b) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence shown as SEQ ID NO. 3 or SEQ ID NO: 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; and/or c) at least one heterologous nucleotide sequence encoding a lipolytic enzyme wherein the nucleotide sequence comprises the nucleotide sequence which hybridizes to SEQ ID NO. 3 or SEQ ID NO. 4 or a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions, for improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity or yield 15 The invention according to any one of claims 1 to 14 wherein the Trichoderma reesei eel! comprises one or more suppressed gene(s) encoding non-lipolytic enzyme(s), preferably two or more suppressed gene(s) encoding non-lipolytic enzyme(s), preferably three or more suppressed gene(s) encoding non-lipolytic enzyme(s), preferably four suppressed gene(s) encoding non-iipolytic enzyme(s). 16- The invention according to claim 15 wherein at least one or each of the suppressed gene(ε) encoding non-lipolytic enzyme(s) is a suppressed gene(s) encoding a cellulase 17. The invention according to any one of claims 1 to 16 wherein after expression of the nucleotide sequence the Trichoderma reesei ceil is removed from the medium into which the enzyme has been secreted 18. The invention according to any one of claims 1 to 17 wherein after expression of the nucleotide sequence the Trichoderma reesei cell is removed from the medium into which the enzyme has been secreted; and then the cell free medium is concentrated, 19. The invention according to any one of claims 1 to 16 wherein the pH of the medium into which the enzyme is secreted is raised after a period of time to yield sufficient levels of the secreted enzyme and prior to the isolation and/or purification and/or concentration of the enzyme 20 The invention according to any one of claims 1 to 19 wherein the following steps are carried out to the medium into which the enzyme of the present invention has been secreted following cuituring of the cell: adjusting the pH of the medium, diluting the medium with water, separating the celi(s) from the medium; concentrating the medium wherein said medium is cell-free, and optionally granulating said medium wherein said medium is cell-free 21 The invention according to any one of claims 1 to 20 wherein the enzyme is used in a method to prepare a food or foodstuff intended for human consumption said method comprising admixing said enzyme with a suitable food or foodstuff ingredient 22 The invention according to claim 21 wherein said enzyme is in the medium into which the enzyme of the present invention has been secreted following culturing of the cell. 23. The invention according to claim 21 or claim 22 wherein said medium is ceil-free 24 The invention according to any one of claims 21 to 23 wherein said medium is concentrated. 25 The invention according to any one of claims 21 to 24 wherein said medium is granulated, 26. A method as generally described herein with reference to the examples and figures. 27 A Trichoderma reesei cell as generally described herein with reference to the examples and figures 28 An expression vector as generally described herein with reference to the examples and figures 29 A use as generally described herein with reference to the examples and figures 30 A method of producing a lipolytic enzyme comprising the steps of: (i) providing a transformed or transfected Trichoderma reesei ceil comprising at least one heterologous nucleotide sequence encoding a lipolytic enzyme; (ii) cuituring the ceil at a pH 4 to pH 5 5 under conditions to allow for expression of said heterologous nucleotide sequeπce(s) encoding said lipolytic enzyme, (iii) isolating, purifying or concentrating the enzyme in a medium at pH 5 5 to pH 6 5 31 A method according to claim 30 wherein the lipolytic enzyme: a) comprises an amino acid sequence shown as SEQ ID NO. 1 or SEQ ID NO: 2 or comprises an amino acid sequence which has at least 40% sequence identity to SEQ ID NO. 1 or 2, and/or b) is encoded by a nucleotide comprising the sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4 or comprising a nucleotide sequence which has at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO. 4, and/or c) is encoded by a nucleotide sequence comprising a nucleotide sequence which hybridizes to SEQ ID NO: 3 or SEQ ID NO: 4 or comprises a nucleotide sequence which is at least 40% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under stringent conditions. 32. A method substantially as described herein and with reference to the accompanying figures.
