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