METHODS OF USING THERMOSTABLE SERINE PROTEASES

• US 20190330577A1
• Filed: 12/18/2017
• Published: 10/31/2019
• Est. Priority Date: 12/21/2016
• Status: Abandoned Application

• Current Assignee: DuPont Nutrition Biosciences ApS (International Flavors & Fragrances Incorporated)
• Original Assignee: DuPont Nutrition Biosciences ApS (International Flavors & Fragrances Incorporated)
• Inventors: Cramer, Jacob Flyvholm, Kolkman, Marc Anton Bernhard, Ma, Zhen, Scheffers, Martijn, Shipovskov, Stepan, Van Brussel-Zwijnen, Marco, Yu, Shukun
• Application Number:
  US16/470,376
• Publication Number:
  US 20190330577A1
• Time in Patent Office: Days
• CPC Class Codes: A23J 3/347 of proteins from microorganisms or unicellular algae
• CPC Class Codes: A23K 20/189 Enzymes
• CPC Class Codes: C12C 5/004 Enzymes
• CPC Class Codes: C12C 7/04 Preparation or treatment of the mash
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12P 19/02 Monosaccharides 2-ketogulonic acid C12P7/60
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12P 2203/00 Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source ethanol C12P7/10
• CPC Class Codes: C12P 7/06 Ethanol, i.e. non-beverage
• CPC Class Codes: C12P 7/64 Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
• CPC Class Codes: C12Y 301/03008 3-Phytase (3.1.3.8)
• CPC Class Codes: C12Y 301/03026 4-Phytase (3.1.3.26), i.e. 6-phytase
• CPC Class Codes: C12Y 302/01001 Alpha-amylase (3.2.1.1)
• CPC Class Codes: C12Y 302/01003 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• CPC Class Codes: C12Y 302/01008 Endo-1,4-beta-xylanase (3.2.1.8)
• CPC Class Codes: C12Y 302/01041 Pullulanase (3.2.1.41)
• CPC Class Codes: C12Y 304/21 Serine endopeptidases (3.4.21)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Methods of using thermostable serine proteases are described herein.

Claims

1. A method for producing a thermostable serine protease comprising:

(a) transforming a production host with a recombinant construct encoding a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10; and

(b) culturing the production host of step (a) at a temperature above 37.5°

C. to increase expression level of the thermostable serine protease.

2. The method according to claim 1 wherein the thermostable serine protease is optionally recovered from the production host.

3. A thermostable serine protease-containing culture supernatant obtained by the method of claim 1 or 2.

4. A feed, feedstuff, feed additive composition, premix, food or grain product comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs:

3, 8, or 10 wherein said thermostable serine protease can be used alone or in combination with at least one direct fed microbial.

6. The feed additive composition of claim 4 for use in animal feed further comprising 10 and at least one component selected from the group consisting of a protein, a peptide, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methyl paraben and propyl paraben.

5. Animal feed comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NO:

1 wherein the thermostable serine protease is present in an amount from 1-20 g/ton feed and further wherein said thermostable serine protease can be used alone or in combination with at least one direct fed microbial.

7. A granulated feed additive composition for use in animal feed comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs:

3, 8, or 10, used either alone or in combination with at least one direct fed microbial, wherein the granulated feed additive composition comprises particles produced by a process selected from the group consisting of high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spray coating, spray drying, freeze drying, prilling, spray chilling, spinning disk atomization, coacervation, tableting, or any combination of the above processes.

8. A granulated feed additive composition of claim 7, wherein the mean diameter of the particles is greater than 50 microns and less than 2000 microns

9. The feed additive composition of claim 7 wherein said composition is in a liquid form.

10. The feed additive composition of claim 9 wherein said composition is in a liquid form suitable for spray-drying on a feed pellet.

11. A method for hydrolyzing starch-containing material comprising:

(a) contacting starch-containing material with a liquid to form a mash; and

(b) hydrolyzing starch in the mash to form a liquefact by contacting the mash with an enzyme cocktail comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10.

12. The method of claim 11 wherein the enzyme cocktail further comprises at least one enzyme selected from the group consisting of alpha-amylase, amyloglucosidase, phytase, pullulanase, beta-glucanase, cellulase and xylanase.

13. A method for producing fermentation products from starch-containing material comprising:

(a) liquefying the starch-containing material with an enzyme cocktail comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10;

(b) saccharifying the product of step (a);

(c) fermenting with a suitable organism; and

(d) optionally, recovering the product produced in step (c).

14. The method of claim 13 wherein steps (b) and (c) are performed simultaneously.

15. The method of claim 13 or claim 14 wherein addition of a nitrogen source is eliminated or reduced by at least 60% by using 1-20 g thermostable serine protease/MT starch-containing material having at least 80% sequence identity to SEQ ID NOs:

3, 8, or 10.

16. The method of claim 15 wherein when the fermentation product is ethanol then no acid proteolytic enzyme is needed in step (c) when using 1-20 g thermostable serine protease/MT starch-containing material having at least 80% sequence identity to SEQ ID NOs:

3, 8, or 10.

17. A method for reducing viscosity of a liquefied starch-containing material which comprises contacting a slurry of starch-containing material with a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs:

3, 8, or 10.

18. A method for extracting oil from an oilseed crop comprising:

(a) contacting an oil-containing oilseed fraction with an aqueous extractant to form an extract oilseed fraction; and

(b) separating the extracted oilseed fraction into an aqueous phase, an oil-in-water emulsion, and an insoluble phase;

(c) contacting the oil-in-water emulsion with at least a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10, either alone or in combination with at least a phospholipase, under conditions permitting enzyme activity for a time sufficient to destabilize the emulsion; and

(d) separating the destabilizing emulsion of step (c) into an aqueous phase, an oil phase, and an insoluble phase to obtain oil from the oilseed.

19. The method of claim 18 wherein the oilseed fraction is from soybean, corn seed, rape seed, palm kernel, sunflower seed, safflower seed, coconut, peanut, cotton seed, sesame seed, flax seed, poppy seed, almond, hazelnut, walnut, evening primrose seed, grape seed, hemp seed, black currant seed, red raspberry seed, carrot seed, cumin seed, blueberry seed, cranberry seed, parsley seed, onion seed, pumpkin seed, apricot kernel, mustard seed, linseed, castor seed or jatropha.

20. The process of claim 18 wherein the oilseed fraction comprises a protein fraction that is useful as a food, food ingredient, a food additive or food supplement.

21. A plant-derived oil prepared by the process of claim 18.

22. A feed, feedstuff, feed additive composition, premix, food or grain product comprising the oil of claim 21.

23. A method for preparing a wort comprising:

a) contacting starch-containing material comprising a mix of milled grain with water and a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10 to form a mash;

b) slowly heating the mash of step (a) to convert the starch to sugars; and

c) separating the heated mash of step (b) into residual grain and liquid wort

wherein the thermostable protease is present in an amount from 1-30 g/ton milled grain.

24. The method of claim 23 wherein at least one alpha-amylase is added in step (a) along with the thermostable serine protease.

25. A method for hydrolyzing at least one food or animal by-product comprising:

(a) contacting the food or animal by-product with a liquid; and

(b) hydrolyzing food or animal by-product in the mash to form a liquefact by contacting with an enzyme cocktail comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10.

26. The method of claim 25 wherein the food by-product is keratin rich material selected from the group consisting of feather, hair and wool.

27. A method for hydrolyzing proteins in lignocellulosic biomass comprising

(a) contacting the biomass with a liquid;

and

(b) hydrolyzing the proteins in the biomass by contacting the biomass with an enzyme cocktail comprising a thermostable serine protease having at least 80% sequence identity to SEQ ID NOs;

3, 8, or 10.