Method of producing a lipolytic enzyme

1. A method of producing a lipolytic enzyme comprising the steps of:

providing a transformed or transfected Trichoderma reesei cell having a promoter region of a Trichoderma reesei cellobiohydrolase 1, the Trichoderma reesei cell comprising one or more of:

(i) 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 95% sequence identity to SEQ ID NO: 1 or 2,

(ii) 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 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4, or

(iii) 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 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under high stringency conditions of 0.1 SSC (0.15 M NaCl, pH 7.0) at 65° C.;

culturing the cell under conditions to allow for expression of said heterologous nucleotide sequence(s) encoding said lipolytic enzyme; and

raising the pH at the end of fermentation that occurs during the culturing of the cell to a pH above the pH of the culture conditions.

2. The method of claim 1 , wherein the lipolytic enzyme is produced in an amount of at least 20 g/liter of culture supernatant.

3. The method of claim 1 , wherein said method comprises one or more of concentrating, isolating, or recovering the lipolytic enzyme.

4. The method of claim 1 , wherein the Trichoderma reesei cell is prepared by transforming or transfecting a Trichoderma reesei cell with the nucleotide sequence.

5. The method of claim 1 , wherein the Trichoderma reesei cell is provided by transforming it with or is transformed with the nucleotide sequence using biolistic transformation.

6. A Trichoderma reesei cell comprising:

a promoter region of a Trichoderma reesei cellobiohydrolase 1; and

a sequence comprising one or more of:

(i) at least one heterologous nucleotide sequence encoding a lipolytic enzyme protein having at least 95% sequence identity to SEQ ID NO: 1 or 2;

(ii) 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 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; or

(iii) 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 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under high stringency conditions of 0.1 SSC (0.15 M NaCl, pH 7.0) at 65° C.

7. A Trichoderma reesei cell according to claim 6 , 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.

8. A method of producing an active lipolytic enzyme by a Trichoderma reesei cell to use in foodstuff, the method comprising:

transforming or transfecting the Trichoderma reesei cell having a promoter region of a Trichoderma reesei cellobiohydrolase 1, the Trichoderma reesei cell comprising one or more of:

(i) 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 95% sequence identity to SEQ ID NO: 1 or 2;

(ii) 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 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4; or

(iii) 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 95% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4 or the complement of any thereof under high stringency conditions of 0.1 SSC (0.15 M NaCl, pH 7.0) at 65° C.; for improving one or more of the following: expression of the lipolytic enzyme, glycosylation of the lipolytic enzyme, enzyme activity, or yield.

9. The method of claim 8 , wherein after expression of the nucleotide sequence the Trichoderma reesei cell is removed from the medium into which the enzyme has been secreted.

10. The method of claim 8 , 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.

11. The method of claim 8 , 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.

12. The method of claim 8 , wherein the following steps are carried out to the medium into which the enzyme of the present invention has been secreted following culturing of the cell: adjusting the pH of the medium, diluting the medium with water; separating the cell(s) from the medium; concentrating the medium wherein said medium is cell-free; and optionally granulating said medium wherein said medium is cell-free.

13. The method of claim 8 , 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.

14. The method of claim 13 , wherein said enzyme is in the medium into which the enzyme of the present invention has been secreted following culturing of the cell.

15. The method of claim 14 , wherein said medium is cell-free.

16. The method of claim 15 , wherein said medium is concentrated.

17. The method of claim 16 , wherein said medium is granulated.