Compositions comprising digestive enzymes

• US 10,927,360 B1
• Filed: 06/03/2020
• Issued: 02/23/2021
• Est. Priority Date: 08/07/2019
• Status: Active Grant

• Current Assignee: Clara Foods, Co.
• Original Assignee: Clara Foods, Co.
• Inventors: Redfearn, Halle, Kshirsagar, Harshal, Mahadevan, Kritika, Chapeaux, Alexandre, Rutherford-Jenkins, Wesley
• Primary Examiner(s): Desai, Anand U
• Application Number:
  US16/891,835
• Publication Number:
  US 20210040466A1
• Time in Patent Office: 265 Days
• Field of Search: None
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12N 9/6481 Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Y 304/23001 Pepsin A (3.4.23.1)

Abstract

Provided herein are compositions with enhanced protein specific activity, protein combinations and methods for the preparation thereof.

Claims

1. A composition comprising a recombinant pepsin polypeptide, wherein the composition is (a) free from animal-derived proteins, (b) the pepsin polypeptide is in a proteolytically inactive form which does not self-digest, (c) the composition has a pH greater than 5.4, and (d) the composition has a specific activity of at least 30,000 FCC units/mg total protein;

wherein the composition is in powdered form.

2. The composition of claim 1, wherein the composition comprising the proteolytically inactive pepsin polypeptide form which does not self-digest and is stable for at least 6 months at room temperature or for at least 6 months at 4°

C.

3. The composition of claim 1, wherein the composition has a moisture content of less than 10%.

4. The composition of claim 1, wherein the composition has a specific activity of at least 40,000 FCC units/mg total protein.

5. The composition of claim 1, wherein the composition has a pH of at least 6.0.

6. The composition of claim 1, wherein the powdered form can be solubilized in an aqueous medium to create a liquid composition having a concentration of the recombinant pepsin polypeptide that is at least 20 g per liter.

7. The composition of claim 6, wherein the proteolytically inactive pepsin polypeptide form which does not self-digest is stable in the liquid composition for at least 30 days at a temperature of 4°

C.

8. The composition of claim 1, wherein the recombinant pepsin polypeptide comprises an amino acid sequence of a sheep, pig, cow, human, zebu, yak, Central European red deer, or goat pepsin.

9. The composition of claim 1, wherein the recombinant pepsin polypeptide comprises SEQ ID NO:

10, or an amino acid sequence with at least 90% identity thereto.

10. The composition of claim 1, wherein the recombinant pepsin polypeptide is produced in a Pichia species, a yeast, a filamentous fungi, a Saccharomyces species, a bacteria, a Trichoderma species or an Aspergillus species.

11. The powdered composition of claim 1, produced by a method comprising:

a. providing a microorganism expressing a recombinant pepsinogen, wherein the expressed pepsinogen is secreted by the microorganism into a growth media;

b. harvesting the growth media and removing the cells therefrom to obtain a liquid starting material;

c. lowering the pH of the liquid starting material to less than pH 4.0 to obtain an activated pepsin composition

d. raising the activated pepsin composition to a pH of greater than 5.4 to obtain the composition comprising the recombinant pepsin polypeptide; and

e. isolating the stable pepsin polypeptide from protein and small molecules in the liquid starting material to obtain a powdered composition.

12. The composition of claim 11, wherein the recombinant pepsinogen comprises one of SEQ ID NOs:

1-9, or an amino acid sequence with at least 90% identity thereto.

17. The composition of claim 1, wherein the composition comprises less than 5% of contaminating proteins.

18. The composition of claim 17, wherein the contaminating protein is a non-pepsin protein and/or an animal-derived protein.

13. A method of producing a high-activity stable pepsin powdered composition comprising:

a. providing a microorganism expressing a recombinant pepsinogen, wherein the expressed pepsinogen is secreted by the microorganism into the growth media;

b. harvesting the growth media and removing the cells therefrom to obtain a liquid starting material;

c. lowering the pH of the liquid starting material to less than pH 4.0 to obtain an activated pepsin composition;

d. raising the activated pepsin composition to a pH of greater than 5.4 to obtain a high-activity stable pepsin composition having a specific activity of at least 10,000 FCC units/mg total powder; and

e. isolating the stable pepsin polypeptide from protein and small molecules in the liquid starting material to obtain a powdered composition;

wherein step (e) is performed after steps (c) and (d).

14. The method of claim 13, wherein the high-activity stable pepsin composition comprises an intact and stable proteolytically inactive form of the pepsin polypeptide and a pH greater than 5.4 and wherein the composition has a specific activity of at least 30,000 FCC units/mg total protein.

15. The method of claim 13, wherein the high-activity stable pepsin composition comprises a pepsin polypeptide having an amino acid sequence of a sheep, pig, cow, human, zebu, yak, Central European red deer, or goat pepsin.

16. The method of claim 13, wherein the microorganism is a Pichia species, a yeast, a filamentous fungus, a Saccharomyces species, a bacteria, a Trichoderma species, or an Aspergillus species.

19. The method of claim 16, wherein the composition comprises less than 5% of contaminating proteins.

20. The method of claim 19, wherein the contaminating protein is a non-pepsin protein and/or an animal-derived protein.