COMPOSITIONS COMPRISING DIGESTIVE ENZYMES
Provided herein are compositions with enhanced protein specific activity, protein combinations and methods for the preparation thereof.
1 . A method for producing a consumable composition comprising combining a high-activity stable pepsin powdered composition with one or more consumable ingredients comprising steps of:
(a) providing a microorganism expressing a recombinant pepsinogen, wherein the expressed pepsinogen is secreted by the microorganism into its growth media;
(b) harvesting the growth media and removing the microorganismal 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 5.4 or higher to obtain a high-activity stable pepsin composition; and
(e) isolating the stable pepsin polypeptide from protein and small molecules in the liquid starting material to obtain the high-activity stable pepsin powdered composition having a specific activity from 500 FCC units/mg total protein to 50,000 FCC units/mg total protein;
(f) combining the high-activity stable pepsin powdered composition with the one or more consumable ingredients to produce a consumable composition; and
wherein step (e) is performed after steps (c) and (d).
2 . The method of claim 1 , 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.
3 . The method of claim 2 , wherein the recombinant pepsinogen comprises any one of SEQ ID NO: 1 to SEQ ID NO: 9, or an amino acid sequence with at least 90% identity thereto.
4 . The method of claim 2 , wherein the high-activity stable pepsin powdered composition comprises at least 5000 FCC units/mg total protein.
5 . The method of claim 1 , wherein the microorganism is a Pichia species, a yeast, a filamentous fungus, a Saccharomyces species, a bacterium, a Trichoderma species, or an Aspergillus species.
6 . The method of claim 1 , wherein the high-activity stable pepsin powdered composition comprises less than 5% of contaminating proteins.
7 . The method of claim 6 , wherein the contaminating protein is a non-pepsin protein and/or an animal-derived protein.
8 . The method of claim 1 , wherein the consumable composition is a food composition.
9 . The method of claim 8 , wherein the one or more consumable ingredients comprise a food additive.
10 . The method of claim 1 , wherein the consumable composition is a pharmaceutical composition.
11 . The method of claim 10 , wherein the one or more consumable ingredients is a pharmaceutically acceptable excipient.
12 . The method of claim 1 , further comprising purifying the stable pepsin polypeptide before step (f) wherein after purifying, the high-activity stable pepsin powdered composition comprises less than 5% of contaminating proteins.