阅读说明：本技术 具有增加的n-末端氨基酸产率的蛋白质水解产物 (Protein hydrolysates with increased yield of N-terminal amino acids ) 是由 P·E·德根 顾晓岗 K·M·克拉格 R·A·索尔格 S·Y·巴克 S·哈宁 唐辛悦 于 2020-02-25 设计创作，主要内容包括：本发明涉及一种通过将蛋白质材料与具有脯氨酸特异性外肽酶的蛋白水解酶混合物接触来从所述材料制备蛋白质水解产物的方法。特别地,所述脯氨酸特异性外肽酶为对五个氨基酸N-末端序列X-Pro-Gln-Glv-Pro-处具有特异性的氨肽酶,其中X为任一氨基酸。本发明还涉及所述氨肽酶与第二外肽酶和内肽酶的用途。(The present invention relates to a process for preparing a protein hydrolysate from a proteinaceous material by contacting said material with a mixture of proteolytic enzymes having a proline-specific exopeptidase. In particular, the proline-specific exopeptidase is an aminopeptidase having specificity for the five amino acid N-terminal sequence X-Pro-Gln-Glv-Pro-, wherein X is any amino acid. The invention also relates to the use of said aminopeptidase with a second exopeptidase and endopeptidase.)

1. A process for preparing a protein hydrolysate from a proteinaceous material, the process comprising contacting the proteinaceous material under aqueous conditions with a combination of proteolytic enzymes comprising an exopeptidase specific for a peptide having a proline at the penultimate N-terminus.

2. A process for preparing a protein hydrolysate from a proteinaceous material according to claim 1, wherein the exopeptidase is specific for a peptide having five amino acid sequences X-Pro-Gln-Pro-as the N-terminus, wherein X is an amino terminal amino acid and may be any naturally occurring amino acid, Pro is proline and Gln is glutamine.

3. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 2, wherein the exopeptidase comprises a sequence having at least 70% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

4. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 3, wherein the exopeptidase comprises a sequence having at least 80% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

5. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 4, wherein the exopeptidase comprises a sequence having at least 85% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

6. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 5, wherein the exopeptidase comprises a sequence having at least 90% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

7. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 6, wherein the exopeptidase comprises a sequence having at least 95% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

8. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 7, wherein the exopeptidase comprises a sequence having at least 99% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

9. The method for preparing a protein hydrolysate from a proteinaceous material according to claim 8, wherein the exopeptidase comprises a sequence according to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

10. The process for preparing a protein hydrolysate according to any one of the preceding claims, wherein the mixture of proteolytic enzymes further comprises a second exopeptidase.

11. The process for preparing a protein hydrolysate according to claim 10, wherein the second exopeptidase is an aminopeptidase.

12. The method of claim 11, wherein the aminopeptidase comprises a sequence having at least 70% sequence identity to one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 28, or an aminopeptidase active fragment thereof.

13. The method of claim 12, wherein the aminopeptidase comprises a sequence having at least 80% sequence identity to one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 28, or an aminopeptidase active fragment thereof.

14. The method of claim 13, wherein the aminopeptidase comprises a sequence having at least 85% sequence identity with one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 28, or an aminopeptidase active fragment thereof.

15. The method of claim 14, wherein the aminopeptidase comprises a sequence having at least 90% sequence identity to one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 28, or an aminopeptidase active fragment thereof.

16. The method of claim 15, wherein the aminopeptidase comprises a sequence having at least 95% sequence identity to one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 28, or an aminopeptidase active fragment thereof.

17. The method of claim 16, wherein the aminopeptidase comprises a sequence having at least 99% sequence identity to one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, and SEQ ID No. 28, or an aminopeptidase active fragment thereof.

18. The method according to claim 17, wherein the aminopeptidase comprises a sequence according to one of SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No. 28 or an aminopeptidase active fragment thereof.

19. The method of claim 18, wherein the aminopeptidase comprises the sequence according to SEQ ID No. 10 or an aminopeptidase-active fragment thereof.

20. The process for preparing a protein hydrolysate according to any one of the preceding claims, wherein the mixture of proteolytic enzymes further comprises an endopeptidase.

21. The method of claim 20, wherein the endopeptidase comprises a sequence having at least 70% sequence identity with one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

22. The method of claim 21, wherein the endopeptidase comprises a sequence having at least 80% sequence identity with one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

23. The method of claim 22, wherein the endopeptidase comprises a sequence having at least 85% sequence identity with one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

24. The method of claim 22, wherein the endopeptidase comprises a sequence having at least 90% sequence identity with one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

25. The method of claim 23, wherein the endopeptidase comprises a sequence having at least 95% sequence identity with one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

26. The method of claim 24, wherein the endopeptidase comprises a sequence having at least 99% sequence identity with one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

27. The method according to claim 25, wherein the endopeptidase comprises a sequence according to one of SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26 and SEQ ID No. 27 or an endopeptidase active fragment thereof.

28. The process for preparing a protein hydrolysate according to any one of the preceding claims, wherein the proteinaceous material comprises a plant derived protein, an animal derived protein, a fish derived protein, an insect derived protein or a microbial derived protein.

29. A process for preparing a protein hydrolysate according to claim 27 wherein the proteinaceous material comprises gluten, soy protein, milk protein, egg protein, whey protein, casein, meat, hemoglobin or myosin.

30. The process for preparing a protein hydrolysate according to any one of the preceding claims, wherein the mixture of proteolytic enzymes comprises at least an exopeptidase, a second exopeptidase and an endopeptidase specific for peptides having a proline at the penultimate N-terminus.

31. A process for preparing a protein hydrolysate according to claim 29 wherein the exopeptidase specific for a peptide having a proline at the penultimate N-terminus corresponds to the exopeptidase specified in any one of claims 2 to 9, the second exopeptidase corresponds to the second exopeptidase specified in any one of claims 11 to 19 and the endopeptidase corresponds to the endopeptidase specified in any one of claims 21 to 26.

32. A process for preparing a protein hydrolysate according to claim 29 wherein the proteinaceous material is treated simultaneously with the exopeptidase specific for peptides having a proline at the penultimate N-terminus, the second exopeptidase and the endopeptidase.

33. A process for preparing a protein hydrolysate according to claim 29 wherein the proteinaceous material is treated with the exopeptidase specific for peptides having a proline at the penultimate N-terminus, the second exopeptidase and the endopeptidase at different times.

34. The process for preparing a protein hydrolysate according to any one of the preceding claims, wherein the process is for producing a protein hydrolysate having elevated levels of glutamic acid.

35. A process for preparing a protein hydrolysate according to claim 33 wherein the proteolytic enzyme mixture further comprises glutaminase.

36. The process for preparing a protein hydrolysate according to claim 34, wherein the glutaminase comprises a sequence having at least 70% sequence identity to SEQ ID No. 29 or a glutaminase-active fragment thereof.

37. The process for preparing a protein hydrolysate according to claim 35, wherein the glutaminase comprises a sequence having at least 80% sequence identity to SEQ ID No. 29 or a glutaminase-active fragment thereof.

38. The process for preparing a protein hydrolysate according to claim 36, wherein the glutaminase comprises a sequence having at least 85% sequence identity to SEQ ID No. 29 or a glutaminase-active fragment thereof.

39. The process for preparing a protein hydrolysate according to claim 37, wherein the glutaminase comprises a sequence having at least 90% sequence identity to SEQ ID No. 29 or a glutaminase-active fragment thereof.

40. The process for preparing a protein hydrolysate according to claim 34, wherein the glutaminase comprises a sequence having at least 95% sequence identity to SEQ ID No. 29 or a glutaminase-active fragment thereof.

41. The process for preparing a protein hydrolysate according to claim 34 wherein the glutaminase comprises a sequence having at least 99% sequence identity to SEQ ID No. 29.

42. The process for preparing a protein hydrolysate according to claim 34 wherein the glutaminase comprises a sequence according to SEQ ID NO:29 or a glutaminase-active fragment thereof.

43. A process for preparing a protein hydrolysate according to any one of claims 33 to 41 wherein the proteinaceous material comprises gluten.

44. A process for preparing a protein hydrolysate according to any one of claims 1 to 32 wherein the process is for producing a protein hydrolysate having an elevated level of proline.

45. A protein hydrolysate produced according to the method of any one of the preceding claims.

46. A food product comprising the protein hydrolysate of claim 44.

Technical Field

The present invention relates to a protein hydrolysate with increased yield of the N-terminal amino acid, wherein the next to N-terminal amino acid is proline. More particularly, the invention relates to the use of an aminopeptidase having specificity for proline located at the penultimate N-terminal position for producing a hydrolysate with increased yield of free amino acids.

Background

Many food products, such as soups, sauces and seasonings, contain flavoring agents obtained by hydrolyzing proteinaceous materials. Generally, protein hydrolysates are produced by hydrolyzing a proteinaceous material, such as defatted soy flour or wheat gluten, with hydrochloric acid (HCl) at elevated temperature, typically under reflux conditions. The protein hydrolysate produced by HCl is both palatable and inexpensive. However, HCl-treated proteins are also known to produce chlorohydrins, such as Monochlorodihydroxypropanol (MCDP) and Dichloropropanol (DCP), which are considered to be potential health risks to consumers. See, for example, J Velisek, J Davidek et al, New Chlorine-Containing Organic Compounds in proteins Hydrolysates [ New Chlorine-Containing Organic Compounds in Protein Hydrolysates ], J.Agric.food Chem. [ J.Agrochemical Chem ]28,1142-1144 (1980).

The possible health risks associated with chemical hydrolysis of proteins have led to the development of enzymes for the production of palatable and low cost protein hydrolysates. To ensure a high degree of hydrolysis, the enzymatic procedure for the manufacture of protein hydrolysates uses two non-specific proteases. First, non-specific endoproteases are used to perform internal cleavage in proteins or peptides. Secondly, the protein fragments generated by endoproteases can be degraded into amino acids, di-or tripeptides using exopeptidases. The non-specificity of endoproteases is important to generate as many starting points for exoproteases as possible. In this regard, amino-terminal peptidases cleave amino acids, dipeptides or tripeptides from the amino terminus of a protein or peptide. Carboxy-terminal peptidases cleave amino acids or dipeptides from the carboxy terminus. It will be appreciated in the art that non-specific exoproteases are also important, so that as many amino acids as possible are removed from the N-terminus or C-terminus.

For protein hydrolysates intended for flavoring, the presence of glutamic acid (Glu) is crucial for taste and palatability. In this respect, glutamine (Gln) is almost tasteless, whereas the corresponding Glu is palatable and provides a satisfying taste. In conventional HCl proteolysis, deamidation occurs without further steps. However, in order to carry out enzymatic proteolysis, glutaminase must be used, which converts glutamine to glutamic acid.

There is a continuing need for methods and enzymes that produce protein hydrolysates with high levels of glutamic acid.

Disclosure of Invention

According to an aspect of the present invention, there is provided a process for the preparation of a protein hydrolysate from a proteinaceous material, wherein the proteinaceous material is contacted under aqueous conditions with a combination of proteolytic enzymes having an exopeptidase specific for a peptide having a proline at the penultimate N-terminus. Optionally, the exopeptidase is specific for peptides having five amino acid sequences X-Pro-Gln-Pro-as N-terminus, wherein X is an amino terminal amino acid and can be any naturally occurring amino acid, Pro is proline and Gln is glutamine.

Optionally, the exopeptidase has a sequence or active fragment thereof having at least 70% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). Optionally, the exopeptidase has a sequence or active fragment thereof having at least 80% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). Optionally, the exopeptidase has a sequence or active fragment thereof having at least 85% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). Optionally, the exopeptidase has a sequence or active fragment thereof having at least 90% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5).

Optionally, the exopeptidase has a sequence or active fragment thereof having at least 95% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). Optionally, the exopeptidase has a sequence or active fragment thereof having at least 99% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). Optionally, the exopeptidase has a sequence according to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

Optionally, the proteolytic enzyme mixture has a second exopeptidase. Preferably, the second exopeptidase is an aminopeptidase. Optionally, the aminopeptidase has a sequence having at least 70% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Optionally, the aminopeptidase has a sequence having at least 80% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Optionally, the aminopeptidase has a sequence having at least 85% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Optionally, the aminopeptidase has a sequence having at least 90% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof.

Optionally, the aminopeptidase has a sequence having at least 95% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Optionally, the aminopeptidase has a sequence having at least 99% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Optionally, the aminopeptidase has a sequence according to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase-active fragment thereof. Optionally, the aminopeptidase has a sequence according to SEQ ID NO 10 or an aminopeptidase active fragment thereof.

Optionally, the proteolytic enzyme mixture also has an endopeptidase. Preferably, the endopeptidase has a sequence with at least 70% sequence identity to one of SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 or an endopeptidase active fragment thereof. Optionally, the endopeptidase has a sequence with at least 80% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. Optionally, the endopeptidase has a sequence with at least 85% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. Optionally, the endopeptidase has a sequence with at least 90% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. Optionally, the endopeptidase has a sequence with at least 95% sequence identity to one of SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 or an endopeptidase active fragment thereof. Optionally, the endopeptidase has a sequence with at least 99% sequence identity to one of SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 or an endopeptidase active fragment thereof. Optionally, the endopeptidase has a sequence according to one of SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 or an endopeptidase active fragment thereof.

Optionally, the protein material is a protein of plant origin, a protein of animal origin, a protein of fish origin, a protein of insect origin, or a protein of microbial origin. Optionally, the proteinaceous material comprises gluten, soy protein, milk protein, egg protein, whey protein, casein, meat, hemoglobin, or myosin.

Optionally, the proteolytic enzyme mixture has at least an exopeptidase, a second exopeptidase and an endopeptidase as described above specific for peptides having a proline at the penultimate N-terminus. Optionally, these enzymes are used to simultaneously treat the proteinaceous material. Optionally, the enzymes are used at different times.

Optionally, the method for producing a protein hydrolysate is for producing a hydrolysate having elevated levels of glutamic acid. Optionally, the proteolytic enzyme mixture has glutaminase. Optionally, the glutaminase has a sequence having at least 70% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Optionally, the glutaminase has a sequence having at least 80% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Optionally, the glutaminase has a sequence having at least 85% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Optionally, the glutaminase has a sequence having at least 90% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Optionally, the glutaminase has a sequence having at least 95% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Optionally, the glutaminase has a sequence having at least 99% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Optionally, the glutaminase has a sequence according to SEQ ID NO:29 or a glutaminase active fragment thereof. According to this aspect of the invention, the proteinaceous material is optionally gluten.

Optionally, the method for producing a protein hydrolysate is for producing a hydrolysate with elevated levels of proline.

In another aspect of the invention, there is provided a protein hydrolysate produced according to any of the methods disclosed above.

In another aspect of the invention, there is provided a food product having a protein hydrolysate as described above.

Brief description of biological sequences

SEQ ID NO 1 lists the protein sequence of full-length MalPro 11.

SEQ ID NO 2 lists the protein sequence of full-length MciPro 4.

SEQ ID NO 3 lists the protein sequence of full-length TciPro 1.

SEQ ID NO 4 lists the protein sequence of full-length FVePro 4.

SEQ ID NO 5 lists the protein sequence for full-length SspPro 2.

SEQ ID NO 6 is the DNA sequence of the additional 5' DNA fragment of pGXT-MalPro11, pGXT-MciPro4 and pGXT-TciPro 1.

SEQ ID NO 7 lists the predicted leader truncated FvePro4 protein sequence.

SEQ ID NO 8 lists the predicted leader truncated SspPro2 protein sequence.

SEQ ID NO 9 lists the protein sequence of the pentapeptide substrate.

The predicted leader truncated AcPepN2 Tri035 protein sequence is shown in SEQ ID NO 10.

SEQ ID NO 11 shows the predicted protein sequence of the leader truncated aminopeptidase Tr 031.

SEQ ID NO 12 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 032.

SEQ ID NO 13 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 033.

SEQ ID NO 14 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 034.

SEQ ID NO 15 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 036.

SEQ ID NO 16 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 037.

SEQ ID NO 17 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 038.

The protein sequence of mature subtilisin A is set forth in SEQ ID NO 18.

SEQ ID NO 19 lists the protein sequence of mature subtilisin BPN'.

SEQ ID NO:20 lists the protein sequence of the mature Subtilisin lentus (Subtilisin proteins).

SEQ ID NO 21 lists the protein sequence of mature thermolysin.

SEQ ID NO:22 lists the protein sequence of mature Bacillus lysin (Bacillus lysin).

SEQ ID NO 23 lists the protein sequence of Trichoderma pepsin (Trichoderma peppsin) from Trichoderma.

SEQ ID NO 23 lists the protein sequence of Trichoderma pepsin (Trichoderma peppsin) from Trichoderma.

SEQ ID NO:24 lists the protein sequence of mature bromelain (Bromelalin).

SEQ ID NO:25 lists the protein sequence of mature Aspergillus pepsin (Aspergillus pepsin).

SEQ ID NO 26 lists the protein sequence of mature trypsin 1.

SEQ ID NO 27 lists the protein sequence of mature chymotrypsin A.

SEQ ID NO 28 lists the predicted protein sequence of the leader truncated aminopeptidase Tr 063.

SEQ ID NO 29 shows the protein sequence of the full-length glutaminase.

Drawings

Fig. 3A depicts dose response curves for MalPro11, MciPro4, TciPro1, fveporo 4, and SspPro2 purified on Phe-Pro.

Figure 3B depicts dose response curves for MalPro11, MciPro4, TciPro1, fveporo 4, and SspPro2 purified on Ser-Pro.

Figure 4 depicts pH profiles of purified MalPro11, MciPro4, TciPro1, fveporo 4 and SspPro 2.

Fig. 5 depicts temperature profiles of purified MalPro11, MciPro4, TciPro1, fveporo 4, and SspPro 2.

Figure 6 depicts the thermal stability testing of purified MalPro11, MciPro4, TciPro1, fveporo 4 and SspPro 2.

FIG. 7 depicts Gln-Pro-Gln-Gln-Pro hydrolysis analysis of purified MalPro11, MciPro4, TciPro1, FvePro4, and SspPro 2.

Figure 8 shows the effect of different doses of SspPro2 on the formation of free glutamate from gluten prehydrolyzate after incubation with AcPepN2 and glutaminase for 19 h. Reference: contains glutelin prehydrolyzate and glutaminase. AcPepN2 contains gluten prehydrolyzate + glutaminase + AcPepN 2. The last two samples contained the same content as AcPepN2, but additionally contained 131. mu.g/mL or 392. mu.g/mL of the prehydrolyzate.

FIG. 9 is the same as FIG. 8, but after incubation for 26 h.

FIG. 10 shows the effect of different X-ProAPs on glutamate yield. Incubation with prehydrolyzate, glutaminase and the mentioned enzymes was performed for 24h at 50 ℃. In all cases, the dose of X-ProAP was 312. mu.g/mL of prehydrolyzate.

FIG. 11 shows the effect of AoX-ProAP and HX-ProAP on glutamic acid yield. Incubation with prehydrolyzate, glutaminase and the mentioned enzymes was carried out for 42h at 50 ℃. In all cases, the dose of X-ProAP was 15. mu.g/mL of prehydrolyzate.

Fig. 12 shows overlapping chromatograms of hydrolysates. Solid line: prehydrolyzate was incubated with glutaminase and AcPepN2 for 26 h. Dotted line: prehydrolyzates were incubated with glutaminase, AcPepN2, and SspPro2 for 26 h. The time interval between major elution of amino acids (AA's) and the interval between major elution of DP2 to DP5 are indicated.

Fig. 13 shows overlapping chromatograms of hydrolysates. Solid line: prehydrolyzate was incubated with glutaminase and AcPepN2 for 26 h. Dotted line: the prehydrolyzate was incubated with glutaminase, AcPepN2, and HX-ProAP for 26 h. The time interval between the major elution of amino acids (AA's) and the interval between the major elution of DP2 to DP5 is indicated in the figure

Detailed Description

The practice of the present teachings will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such techniques are explained fully in the following documents, for example,Molecular Cloning: A Laboratory Manual[ molecular cloning: laboratory manual]Second edition (Sambrook et al, 1989);Oligonucleotide Synthesis[ oligonucleotide Synthesis](m.j.gait editors, 1984);Current Protocols in Molecular Biology[ Current protocols of molecular biology](f.m. ausubel et al, editors, 1994);PCR:The Polymerase Chain Reaction[ PCR: polymerase chain reaction](Mullis et al, eds, 1994);Gene Transfer and Expression:A Laboratory Manual[ Gene transfer and expression: fruit of Chinese wolfberryLaboratory manual](Kriegler,1990), andThe Alcohol Textbook[ alcohol textbook](Ingledate et al, eds, fifth edition, 2009), andEssentials of Carbohydrate Chemistry and Biochemistry[ carbohydrate chemistry and biochemistry basis](Lindhorste,2007)。

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present teachings belong. The Singleton et al, in the case of,Dictionary of Microbiology and Molecular Biology[ dictionary of microbiology and molecular biology]Second edition, John Wiley and Sons [ John Willi father, Inc.)]New York (1994), and Hale and Markham,The Harper Collins Dictionary of Biology[ Huppe Coriolis biology dictionary]Harper Perennial permanent press]New York (1991) provides the skilled person with a general dictionary of many of the terms used in the present invention. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings.

Numerical ranges provided herein include the numbers defining the range.

Definition of

With respect to polypeptides, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polypeptide that does not comprise human substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the terms "wild-type", "parent" or "reference" refer to a naturally occurring polynucleotide that does not contain human nucleoside changes. However, it is noted that a polynucleotide encoding a wild-type, parent, or reference polypeptide is not limited to a naturally occurring polynucleotide and encompasses any polynucleotide encoding a wild-type, parent, or reference polypeptide.

Reference to a wild-type polypeptide is to be understood to include the mature form of the polypeptide. A "mature" polypeptide or variant thereof is one in which no signal sequence is present, e.g., cleaved from the immature form of the polypeptide during or after expression of the polypeptide.

With respect to polypeptides, the term "variant" refers to a polypeptide that differs from the specified wild-type, parent or reference polypeptide in that it includes one or more naturally occurring or artificial amino acid substitutions, insertions or deletions. Similarly, with respect to polynucleotides, the term "variant" refers to a polynucleotide that differs in nucleotide sequence from the specified wild-type, parent or reference polynucleotide. The nature of the wild-type, parent or reference polypeptide or polynucleotide will be apparent from the context.

The term "recombinant" when used in reference to a subject cell, nucleic acid, protein, or vector indicates that the subject has been modified from its native state. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell, or expresses a native gene at a level or under conditions different from those found in nature. The recombinant nucleic acid differs from the native sequence by one or more nucleotides and/or is operably linked to a heterologous sequence, e.g., a heterologous promoter in an expression vector. Recombinant proteins may differ from the native sequence by one or more amino acids, and/or be fused to heterologous sequences. The vector comprising the nucleic acid encoding the protease is a recombinant vector.

The terms "recovered", "isolated" and "alone" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as it occurs in nature. An "isolated" polypeptide thereof includes, but is not limited to, a culture medium containing a secreted polypeptide expressed in a heterologous host cell.

The term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.

The term "enriched" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is at about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

"pH range" in reference to an enzyme refers to the range of pH values at which the enzyme exhibits catalytic activity.

The terms "pH stable" and "pH stability" in reference to an enzyme relate to the ability of the enzyme to retain activity for a predetermined period of time (e.g., 15min., 30min., 1 hour) at a pH value within a wide range.

The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein", and "peptide" and is used interchangeably. When such amino acid sequences exhibit activity, they may be referred to as "enzymes". The amino acid sequence is represented in the standard amino-terminal-to-carboxyl-terminal orientation (i.e., N → C) using the conventional single-letter or three-letter code for amino acid residues.

The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid may be single-stranded or double-stranded, and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the compositions and methods of the invention encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5 '-to-3' orientation.

"hybridization" refers to the process by which a strand of nucleic acid forms a duplex (i.e., a base pair) with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by hybridization under the following conditions: 65 ℃ and 0.1X SSC (where 1X SSC ═ 0.15M NaCl, 0.015M trisodium citrate, pH 7.0). The hybridized double-stranded nucleic acid is characterized by a melting temperature (T)_m) Wherein half of the hybridized nucleic acids are not paired with complementary strands. Mismatched nucleotide in duplex decreases T_m. Very stringent hybridization conditions involve 68 ℃ and 0.1 XSSC.

"synthetic" molecules are produced by in vitro chemical or enzymatic synthesis and not by organisms.

The terms "transformation", "stably transformed" and "transgenic" as used with respect to a cell mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or as an episome through a multi-generation system.

The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection", "transformation" or "transduction" as known in the art.

A "host strain" or "host cell" is an organism into which has been introduced an expression vector, phage, virus or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., a protease). Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest. The term "host cell" includes protoplasts produced from a cell.

The term "heterologous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

The term "endogenous" with respect to a polynucleotide or protein refers to a polynucleotide or protein that is naturally present in the host cell.

The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

A "selectable marker" or "selectable marker" refers to a gene that can be expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include, but are not limited to, antimicrobial agents (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage (e.g., a nutritional advantage) on the host cell.

"vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.

By "expression vector" is meant a DNA construct comprising a DNA sequence encoding a polypeptide of interest, operably linked to suitable control sequences capable of effecting the expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences which control termination of transcription and translation.

The term "operatively linked" means: the specified components are in a relationship (including but not limited to a juxtaposition) that allows them to function in the intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is controlled by the regulatory sequence.

A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks a signal sequence that is cleaved off during secretion.

"biologically active" refers to a sequence having a specified biological activity, e.g., an enzymatic activity.

The term "specific activity" refers to the number of moles of substrate that can be converted to a product by an enzyme or enzyme preparation per unit time under specified conditions. The specific activity is usually expressed as unit (U)/mg protein.

As used herein, "percent sequence identity" means that a particular sequence has at least a certain percentage of amino acid residues that are identical to the amino acid residues in a designated reference sequence when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al (1994) Nucleic Acids Res. [ Nucleic Acids research ]22: 4673-one 4680. The default parameters for the CLUSTAL W algorithm are:

gap opening penalty: 10.0

Gap extension penalty: 0.05

Protein weight matrix: BLOSUM series

DNA weight matrix: IUB

Delayed divergence sequence%: 40

Vacancy separation distance: 8

DNA conversion weight: 0.50

List hydrophilic residues: GPSNDQEKR

Using a negative matrix: closing device

Switch special residue penalties: opening device

Switching hydrophilicity penalties: opening device

The end of handover gap separation penalty is off.

Deletions are considered residues that are not identical compared to the reference sequence. Including deletions occurring at either end. For example, a variant having a deletion of five amino acids from the C-terminus of a mature 617 residue polypeptide relative to the mature polypeptide will have a percent sequence identity of 99% (612/617 residues identical x 100, rounded to the nearest integer). Such variants will be encompassed by variants having "at least 99% sequence identity" to the mature polypeptide.

A "fusion" polypeptide sequence is linked, i.e., operatively linked, via a peptide bond between the two subject polypeptide sequences.

The term "filamentous fungus" refers to all filamentous forms of the subdivision Eumycotina, in particular the species Ascomycotina.

The term "about" refers to ± 5% of a reference value.

The term "peptidase" or "protease" refers to an enzyme that hydrolyzes peptide bonds in polypeptides or oligopeptides. As used herein, the term peptidase or protease includes enzymes that partition to the EC 3.4 subclass.

The term "exopeptidase" or "exoprotease" refers to peptidases that act to hydrolyze peptide bonds at the terminus (amino or carboxyl) of polypeptides or oligopeptides. Exopeptidases that act on the amino terminus of a polypeptide are referred to herein as aminopeptidases. Aminopeptidases can act to cleave or release single amino acids, dipeptides and tripeptides from the amino terminus depending on their specificity. Exopeptidases that act on the carboxy terminus are referred to herein as carboxypeptidases. Carboxypeptidases can act to cleave or release single amino acids, dipeptides and tripeptides from the carboxyl terminus depending on their specificity.

The term "endopeptidase" or "endoprotease" refers to a peptidase or protease that hydrolyzes internal peptide bonds in proteins or oligopeptides.

"hydrolysate" is the product of a reaction in which a compound is decomposed by water. Protein hydrolysates, or "protein hydrolysates", occur when protein bonds are hydrolysed with water. The hydrolysis of the protein may be increased by heat or enzymes. During hydrolysis, the protein is broken down into smaller proteins, polypeptides and free amino acids.

Other definitions are shown below.

Additional mutations

In some embodiments, the proteases of the invention further include one or more mutations that may provide additional performance or stability benefits. Exemplary performance benefits include, but are not limited to: increased thermostability, increased storage stability, increased solubility, altered pH profile, increased specific activity, modified substrate specificity, modified substrate binding, modified pH-dependent activity, modified pH-dependent stability, increased oxidative stability, and increased expression. In some cases, performance benefits are achieved at relatively low temperatures. In some cases, performance benefits are achieved at relatively high temperatures.

Furthermore, the proteases of the invention may comprise any number of conservative amino acid substitutions. Exemplary conservative amino acid substitutions are listed in the table below.

TABLE 1 conservative amino acid substitutions

The reader will appreciate that some of the foregoing conservative mutations may be generated by genetic manipulation, while others are generated by genetically or otherwise introducing synthetic amino acids into the polypeptide.

The proteases of the invention may be "precursor", "immature" or "full-length", in which case they comprise a signal sequence; or "mature", in which case they lack a signal sequence. Mature forms of the polypeptide are often the most useful. Unless otherwise indicated, amino acid residue numbering as used herein refers to the mature form of the corresponding protease polypeptide. The protease polypeptide of the present invention may also be truncated to remove the N-terminus or C-terminus, so long as the resulting polypeptide retains protease activity. In addition, the protease may be an active fragment derived from a longer amino acid sequence. Active fragments are characterized by retaining some or all of the activity of the full-length enzyme, but having deletions from the N-terminus, from the C-terminus, or within or in combination.

The protease of the invention may be a "chimeric" or "hybrid" polypeptide in that it includes at least a portion of a first protease polypeptide and at least a portion of a second protease polypeptide. The protease of the invention may further comprise a heterologous signal sequence, i.e. an epitope that allows tracking or purification etc. Exemplary heterologous signal sequences are from bacillus licheniformis (b.licheniformis) amylase (LAT), bacillus subtilis (AmyE or AprE), and Streptomyces (Streptomyces) CelA.

Production of variant proteases

The protease of the invention may be produced in a host cell, for example by secretion or intracellular expression. After secretion of the protease into the cell culture medium, a cultured cell material (e.g., whole cell culture fluid) containing the protease can be obtained. Optionally, the protease may be isolated from the host cell, or even from the cell culture broth, depending on the desired purity of the final protease. The gene encoding the protease can be cloned and expressed according to methods well known in the art. Suitable host cells include bacteria, fungi (including yeast and filamentous fungi), and plant cells (including algae). Particularly useful host cells include Aspergillus niger, Aspergillus oryzae or Trichoderma reesei. Other host cells include bacterial cells such as Bacillus subtilis or Bacillus licheniformis (b.licheniformis), and Streptomyces (Streptomyces), escherichia coli (e.coli).

The host cell may also express a nucleic acid encoding a homologous or heterologous protease (i.e., a protease of a different species than the host cell) or one or more other enzymes. The protease may be a variant protease. In addition, the host may express one or more coenzymes, proteins, peptides.

Carrier

A DNA construct comprising a nucleic acid encoding a protease can be constructed for expression in a host cell. Due to the well-known degeneracy in the genetic code, variant polynucleotides encoding the same amino acid sequence can be designed and prepared using conventional techniques. Optimization of codons for a particular host cell is also well known in the art. The nucleic acid encoding the protease may be incorporated into a vector. The vectors may be transferred into host cells using well-known transformation techniques, such as those disclosed below.

The vector may be any vector that can be transformed into a host cell and replicated in the host cell. For example, a vector comprising a nucleic acid encoding a protease can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector may also be transformed into an expression host such that the encoding nucleic acid may be expressed as a functional protease. Host cells for use as expression hosts may include, for example, filamentous fungi. The strain catalog of the american fungal genetics inventory center (FGSC) lists vectors suitable for expression in fungal host cells. See FGSC, strain catalogue, university of missouri, website www.fgsc.net (latest modification time of 2007, 1 month, 17 days). A representative vector is pJG153, which is a promoterless Cre expression vector that can replicate in a bacterial host. See Harrison et al, (6.2011) Applied environ. microbiol [ Applied and environmental microbiology ]77: 3916-22. pJG153 may be modified by conventional techniques to contain and express nucleic acid encoding a protease.

The nucleic acid encoding the protease may be operably linked to a suitable promoter that allows transcription in the host cell. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Exemplary promoters for directing transcription of a DNA sequence encoding a protease, particularly in a bacterial host, are the promoter of the lac operon of Escherichia coli, the promoter of the Streptomyces coelicolor agarase gene dagA or celA, the promoter of the Bacillus licheniformis alpha-amylase gene (amyL), the promoter of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoter of the Bacillus amyloliquefaciens alpha-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes, and the like. Examples of useful promoters for transcription in a fungal host are promoters derived from the genes encoding Aspergillus oryzae (Aspergillus oryzae) TAKA amylase, Rhizobium mibehii (Rhizomucor miehei) aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger glucoamylase, Rhizobium miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae trisaccharide phosphate isomerase, or Aspergillus nidulans (A.nidulans) acetamidase. When the gene encoding the protease is expressed in a bacterial species such as E.coli, a suitable promoter may be selected from, for example, phage promoters including the T7 promoter and the phage lambda promoter. Examples of suitable promoters for expression in yeast species include, but are not limited to, the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae (Saccharomyces cerevisiae) and the Pichia pastoris AOX1 or AOX2 promoters. cbh1 is an endogenous inducible promoter from trichoderma reesei (t. See Liu et al (2008) "Improved heterologous gene expression in Trichoderma reesei by cellulobiohydroslase I gene (cbh1) promoter optimization [ cellobiohydrolase I gene (cbh1) promoter optimization improves heterologous gene expression in Trichoderma reesei ]," Acta Biochim. Biophys. sin (Shanghai) [ biochem and biophysics (Shanghai) ]40(2): 158-65.

The coding sequence may be operably linked to a signal sequence. The DNA encoding the signal sequence may be a DNA sequence naturally associated with the protease gene to be expressed or from a different genus or species. The signal sequence and promoter sequence comprising the DNA construct or vector may be introduced into the fungal host cell and may be derived from the same source. For example, the signal sequence is the cbh1 signal sequence operably linked to the cbh1 promoter.

The expression vector may also contain a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding the variant protease. The termination sequence and polyadenylation sequence may suitably be derived from the same sources as the promoter.

The vector may further comprise a DNA sequence enabling the vector to replicate in a host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUB110, pE194, pAMB1, and pIJ 702.

The vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, e.g., a dal gene from B.subtilis or B.licheniformis, or a gene conferring antibiotic resistance (e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance). In addition, the vector may comprise Aspergillus selection markers, such as amdS, argB, niaD and xxsC, markers that cause hygromycin resistance, or selection may be achieved by co-transformation (as known in the art). See, for example, international PCT application WO 91/17243.

Intracellular expression may be advantageous in certain aspects, for example, when using certain bacteria or fungi as host cells to produce large amounts of protease for subsequent enrichment or purification. Extracellular secretion of proteases into culture media can also be used to prepare cultured cell material comprising an isolated protease.

Expression vectors typically include components of a cloning vector, such as, for example, elements that allow the vector to replicate autonomously in the host organism of choice and one or more phenotypically detectable markers for selection purposes. Expression vectors typically comprise control nucleotide sequences, such as a promoter, operator, ribosome binding site, translation initiation signal, and optionally a repressor gene or one or more activator genes. In addition, the expression vector may comprise a sequence encoding an amino acid sequence capable of targeting the protease to a host cell organelle (e.g., peroxisome) or to a particular host cell compartment. Such targeting sequences include, but are not limited to, the sequence SKL. For expression under the direction of the control sequence, the nucleic acid sequence of the protease is operatively linked to the control sequence in a suitable manner for expression.

The procedures used to ligate the protease-encoding DNA construct, the promoter, the terminator and other elements separately and insert them into a suitable vector containing the information required for replication are well known to those skilled in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, 1989, and 3 rd edition, 2001).

Transformation and culture of host cells

Isolated cells comprising the DNA construct or expression vector are advantageously used as host cells for the recombinant production of proteases. The cell may conveniently be transformed with the enzyme-encoding DNA construct by integrating the DNA construct (in one or more copies) into the host chromosome. This integration is generally considered to be advantageous because the DNA sequence is more likely to be stably maintained in the cell. The integration of the DNA construct into the host chromosome may be carried out according to conventional methods, for example, by homologous or heterologous recombination. Alternatively, the cells may be transformed with expression vectors as described above in connection with different types of host cells.

Examples of suitable bacterial host organisms are gram-positive bacterial species such as Bacillus (Bacillus), including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis, and Bacillus species such as Lactobacillus sp, Lactobacillus strains, Lactobacillus sp, and Lactobacillus sp, and Lactobacillus sp, and Lactobacillus sp, and Lactobacillus sp, and Lactobacillus sp, preferably Lactobacillus sp, preferably Lactobacillus sp, preferably, such as Lactobacillus sp, preferably, such as Lactobacillus sp, preferably, Lactobacillus sp, preferably, such as Lactobacillus sp, preferably, such as Lactobacillus sp, or Lactobacillus sp, preferably, Lactobacillus sp, preferably, Lactobacillus sp, preferably, such as Lactobacillus sp, preferably, Lactobacillus sp, such as Lactobacillus sp, preferably, such as Lactobacillus sp, preferably, Lactobacillus sp, preferably, such as Lactobacillus sp, Lactobacillus sp (Pediococcus sp.); and Streptococcus species (Streptococcus sp.). Alternatively, strains of gram-negative bacterial species belonging to the family Enterobacteriaceae (including escherichia coli) or pseudomonas (pseudomonas adaceae) may be selected as host organisms.

Suitable yeast host organisms may be selected from biotechnologically relevant yeast species, such as, but not limited to, yeast species such as Pichia species (Pichia sp.), Hansenula species (Hansenula sp.) or Kluyveromyces species (Kluyveromyces), yarrowia species (yarrowia), Schizosaccharomyces species or Saccharomyces species including Saccharomyces cerevisiae (Saccharomyces cerevisiae), or species belonging to the Schizosaccharomyces genus, e.g. Schizosaccharomyces pombe (s. The methylotrophic yeast species strain pichia can be used as host organism. Alternatively, the host organism may be a hansenula species. Suitable host organisms in filamentous fungi include species of the genus Aspergillus (Aspergillus), for example Aspergillus niger, Aspergillus oryzae, Aspergillus tubingensis (Aspergillus tubigenis), Aspergillus awamori (Aspergillus awamori) or Aspergillus nidulans (Aspergillus nidulans). Alternatively, strains of Fusarium (Fusarium) species, such as Fusarium oxysporum (Fusarium oxysporum) or rhizobium (rhizobium) species, such as rhizobium miehei, may be used as host organisms. Other suitable strains include thermophilic (Thermomyces) and Mucor species. In addition, Trichoderma sp may be used as a host. Suitable procedures for transforming an aspergillus host cell include, for example, the procedures described in EP 238023. The protease expressed by the fungal host cell may be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different than that present in the wild-type protease. The type and/or extent of glycosylation may alter the enzymatic and/or biochemical properties.

Deletion of the gene from the expression host is advantageous, where gene defects can be cured by the transformed expression vector. Known methods can be used to obtain fungal host cells having one or more inactivated genes. Gene inactivation may be accomplished by complete or partial deletion, by insertional inactivation, or by any other means that renders the gene inoperative for its intended purpose such that the gene is prevented from expressing a functional protein. Any gene cloned from a Trichoderma species or other filamentous fungal host may be deleted, for example, cbh1, cbh2, egl1 and egl2 genes. Gene deletion can be accomplished by inserting the form of the desired gene to be inactivated into a plasmid by methods known in the art.

Introduction of the DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction; transfection, such as lipofection-mediated and DEAE-dextrin-mediated transfection; incubating with calcium phosphate DNA precipitate; bombarding with DNA coated particles at high speed; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al (2001), supra. Expression of heterologous proteins in trichoderma is described, for example, in U.S. patent No. 6,022,725. For the transformation of Aspergillus strains, reference is also made to Cao et al (2000) Science 9: 991-. Genetically stable transformants can be constructed using vector systems whereby the nucleic acid encoding the protease is stably integrated into the host cell chromosome. Transformants are then selected and purified by known techniques.

The preparation of Trichoderma species for transformation may for example involve the preparation of protoplasts from fungal mycelia. See Campbell et al (1989) curr. Genet. [ contemporary genetics ]16: 53-56. The mycelium may be obtained from germinated vegetative spores. The mycelium is treated with an enzyme that digests the cell wall, producing protoplasts. Protoplasts are protected by the presence of osmotic stabilizers in the suspension medium. These include sorbitol, mannitol, potassium chloride, magnesium sulfate, and the like. Typically the concentration of these stabilizers varies between 0.8M and 1.2M, for example a 1.2M solution of sorbitol can be used in the suspension medium.

Depending on the calcium ion concentration, the DNA is taken up into the host Trichoderma species strain. Typically, between about 10mM and 50mM CaCl is used in the uptake solution₂. Additional suitable compounds include buffer systems such as TE buffer (10mM Tris, pH 7.4; 1mM EDTA) or 10mM MOPS (pH 6.0) and polyethylene glycol. It is believed that the polyethylene glycol fuses the cell membranes, allowing the contents of the culture medium to be delivered into the cytoplasm of the trichoderma species strain. This fusion often leaves multiple copies of the plasmid DNA integrated into the host chromosome.

Typically, a Trichoderma species is transformed with protoplasts or cells that have been osmotically treated, typically at 10⁵To 10⁷Per mL, in particular 2X 10⁶Density per mL. Can be used forAdd 100. mu.L of the mixture in a suitable solution (e.g., 1.2M sorbitol and 50mM CaCl)₂) The protoplasts or cells of (1) are mixed with the desired DNA. Typically, PEG is added to the uptake solution at high concentrations. From 0.1 to 1 volume of 25% PEG 4000 may be added to the protoplast suspension; however, it is useful to add about 0.25 volumes to the protoplast suspension. Additives such as dimethyl sulfoxide, heparin, spermidine, potassium chloride, and the like may also be added to the uptake solution to facilitate the conversion. Similar procedures can be used for other fungal host cells. See, for example, U.S. patent No. 6,022,725.

Expression of

Methods of producing a protease may comprise culturing a host cell as described above under conditions conducive to production of the enzyme, and recovering the enzyme from the cell and/or culture medium.

The medium used to culture the cells may be any conventional medium suitable for the growth of the host cell in question and to obtain expression of the protease. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).

The enzyme secreted from the host cell can be used in the whole broth preparation. In the methods of the present invention, the preparation of the spent whole fermentation broth of the recombinant microorganism, resulting in the expression of the protease, may be achieved using any cultivation method known in the art. Thus, fermentation is understood to include shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the protease to be expressed or isolated. The term "spent whole fermentation broth" is defined herein as the unfractionated content of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is to be understood that the term "spent whole fermentation broth" also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.

The enzyme secreted from the host cell may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating the proteinaceous components of the medium by means of a salt (e.g. ammonium sulphate), followed by the use of chromatographic procedures, such as ion exchange chromatography, affinity chromatography and the like.

The polynucleotide encoding the protease in the vector may be operably linked to control sequences capable of providing for the expression of the coding sequence by a host cell, i.e., the vector is an expression vector. The control sequence may be modified, for example, by the addition of other transcriptional regulatory elements, to make the transcriptional level directed by the control sequence more responsive to the transcriptional regulator. The control sequence may in particular comprise a promoter.

The host cell may be cultured under suitable conditions that allow for expression of the protease. Expression of these enzymes may be constitutive, such that they are produced continuously, or inducible, requiring stimulation to initiate expression. In the case of inducible expression, protein production can be initiated when desired, for example by adding an inducing substance, such as dexamethasone or IPTG or sophorose, to the culture medium. The polypeptide may also be in an in vitro cell-free system (e.g., TNT)^TM(Promega) rabbit reticulocyte system).

The expression host may also be cultured under aerobic conditions in a medium suitable for the host. A combination of shaking or agitation and aeration may be provided, wherein production occurs at a temperature appropriate for the host (e.g., from about 25 ℃ to about 75 ℃ (e.g., 30 ℃ to 45 ℃), depending on the needs of the host and the production of the desired protease). Incubation may occur for from about 12 to about 100 hours or more (and any hour values in between, e.g., from 24 to 72 hours). Typically, the pH of the culture broth is from about 4.0 to about 8.0, again depending on the culture conditions required for the host relative to the production of the protease.

Method for enriching and purifying protease

Fermentation, isolation and concentration techniques are well known in the art and conventional methods can be used to prepare solutions containing protease polypeptides.

After fermentation, a fermentation broth is obtained, and the microbial cells and various suspended solids (including residual crude fermentation material) are removed by conventional separation techniques to obtain a protease solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction or chromatography, or the like is typically used.

It is desirable to concentrate the solution containing the protease polypeptide to optimize recovery. The use of an unconcentrated solution requires increased incubation time to collect the enriched or purified enzyme precipitate.

The enzyme-containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme-containing solution can be achieved by any of the techniques discussed herein. Exemplary methods of enrichment and purification include, but are not limited to, rotary vacuum filtration and/or ultrafiltration.

The enzyme solution is concentrated to a concentrated enzyme solution until the enzyme activity of the concentrated protease polypeptide-containing solution reaches a desired level.

The enriched or purified enzyme can be made into a final product that is either liquid (solution, slurry) or solid (granules, powder).

PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

According to one aspect of the invention, it was found that some aminopeptidases stagnate or only slowly digest peptides or proteins having proline in the penultimate N-terminal position. In particular, it was found that these aminopeptidases do not digest proteins having peptides of the N-terminal sequence X-Pro-Gln-Gln-Pro-de (wherein X is any amino acid). The use of such aminopeptidases in the production of protein hydrolysates will result in hydrolysates with low amounts of X amino acids, since such peptides are resistant to digestion.

Glutamate in the form of monosodium glutamate (MSG) is a commonly used flavour enhancer. It is responsible for salty or umami taste. MSG can be produced by enzymatic hydrolysis of proteins. In this regard, gluten is present in high amounts in glutamine and can be a source of MSG (glutaminase can be used to convert glutamine to glutamic acid). According to one aspect of the invention, it was found that gluten contains a significant amount of the sequence X-Pro-Gln-Pro-, which substantially limits the amount of glutamine that can be released from gluten.

According to an aspect of the present invention, there is provided a process for the preparation of a protein hydrolysate from a proteinaceous material, wherein the proteinaceous material is contacted under aqueous conditions with a combination of proteolytic enzymes having an exopeptidase specific for a peptide having a proline at the penultimate N-terminus. In a preferred embodiment, the exopeptidase is specific for peptides having the five amino acid sequences X-Pro-Gln-Gln-Pro-as the N-terminus, wherein X is the amino terminal amino acid and can be any naturally occurring amino acid, Pro is proline and Gln is glutamine.

Preferably, the exopeptidase has a sequence having at least 70% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5), or an active fragment thereof. More preferably, the exopeptidase has a sequence or an active fragment thereof having at least 80% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). Still more preferably, the exopeptidase has a sequence or an active fragment thereof having at least 85% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). In yet a more preferred embodiment, the exopeptidase has a sequence or active fragment thereof having at least 90% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5).

Still more preferably, the exopeptidase has a sequence having at least 95% sequence identity with one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5), or an active fragment thereof. In still more preferred embodiments, the exopeptidase has a sequence or active fragment thereof having at least 99% sequence identity to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO: 5). In a most preferred embodiment, the exopeptidase has a sequence according to one of MalPro11(SEQ ID NO:1), MciPro4(SEQ ID NO:2), TciPro1(SEQ ID NO:3), FvePro4(SEQ ID NO:4), and SspPro2(SEQ ID NO:5) or an active fragment thereof.

In a preferred embodiment of the invention, the proteolytic enzyme mixture has a second exopeptidase. Preferably, the second exopeptidase is an aminopeptidase. More preferably, the aminopeptidase has a sequence having at least 70% sequence identity with one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Still more preferably, the aminopeptidase has a sequence having at least 80% sequence identity with one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Still more preferably, the aminopeptidase has a sequence having at least 85% sequence identity with one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Still more preferably, the aminopeptidase has a sequence having at least 90% sequence identity to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof.

In still more preferred embodiments, the aminopeptidase has a sequence having at least 95% sequence identity with one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28, or an aminopeptidase active fragment thereof. Still more preferably, the aminopeptidase has a sequence having at least 99% sequence identity with one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase active fragment thereof. Still more preferably, the aminopeptidase has a sequence according to one of SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17 and SEQ ID NO 28 or an aminopeptidase-active fragment thereof. In a most preferred embodiment, the aminopeptidase has the sequence according to SEQ ID NO 10 or an aminopeptidase-active fragment thereof.

In a further preferred embodiment of the invention, the proteolytic enzyme mixture also has an endopeptidase. Preferably, the endopeptidase has a sequence with at least 70% sequence identity to one of SEQ ID NO 18, 19, 20, 21, 22, 23, 24, 25, 26 and 27 or an endopeptidase active fragment thereof. More preferably, the endopeptidase has a sequence with at least 80% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. Still more preferably, the endopeptidase has a sequence with at least 85% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. Still more preferably, the endopeptidase has a sequence with at least 90% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. In still more preferred embodiments, the endopeptidase has a sequence with at least 95% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. Still more preferably, the endopeptidase has a sequence with at least 99% sequence identity to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof. In a most preferred embodiment, the endopeptidase has a sequence according to one of SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26 and SEQ ID NO 27 or an endopeptidase active fragment thereof.

In a preferred embodiment of the present invention, the protein material is a protein of plant origin, a protein of animal origin, a protein of fish origin, a protein of insect origin, or a protein of microbial origin. Preferably, the proteinaceous material comprises gluten, soy protein, milk protein, egg protein, whey protein, casein, meat, hemoglobin or myosin.

In other preferred embodiments, the mixture of proteolytic enzymes has at least an exopeptidase, a second exopeptidase and an endopeptidase as described above which are specific for peptides having a proline at the penultimate N-terminus. Preferably, these enzymes are used to simultaneously treat proteinaceous material. In other preferred embodiments, the enzymes are used at different times.

In a preferred embodiment of the invention, the method for producing a protein hydrolysate is for producing a hydrolysate with elevated levels of glutamic acid. According to this aspect of the invention, the proteolytic enzyme mixture has glutaminase. Preferably, the glutaminase has a sequence having at least 70% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. More preferably, the glutaminase has a sequence having at least 80% sequence identity to SEQ ID NO 29 or a glutaminase active fragment thereof. Still more preferably, the glutaminase has a sequence having at least 85% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. In yet a more preferred embodiment, the glutaminase has a sequence having at least 90% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. Still more preferably, the glutaminase has a sequence having at least 95% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. In yet a more preferred embodiment, the glutaminase has a sequence having at least 99% sequence identity to SEQ ID NO. 29 or a glutaminase active fragment thereof. In a most preferred embodiment, the glutaminase has a sequence according to SEQ ID NO:29 or a glutaminase active fragment thereof.

According to this aspect of the invention, the proteinaceous material is gluten.

In other preferred embodiments, the method for producing a protein hydrolysate is for producing a hydrolysate with elevated levels of proline.

In another aspect of the invention, there is provided a protein hydrolysate produced according to any of the methods disclosed above.

In another aspect of the invention, there is provided a food product having a protein hydrolysate as described above.

Examples of the invention

EXAMPLE 1 cloning of fungal X-Pro protease

Two fungal strains, Leuconostoc thermosphakii (Melanocarpus albomyces) CBS177.67(GICC #2522192) and Cladosporium camphoratum (Malbranchea cinamonea) CBS 343.55(GICC #2518670) were selected as potential sources of enzymes for a variety of industrial applications. Calcilomyces thermosphaeus CBS177.67 and Mycoplasma camphoratus CBS 343.55 were purchased from CBS-KNAW Fungal Biodiversity center (CBS-KNAW Fungal Biodiversity Centre) (Uppsalaan 8, 3584CT Utrecht, Netherlands). Chromosomal DNA was sequenced using Illumina's next generation sequencing technology and two fungal X-Pro proteases were identified after annotation: MalPro11 was identified from Leuconostoc thermosphakii CBS177.67, and MciPro4 was identified from Mycoplasma camphorata CBS 343.55. The full-length protein sequences of MalPro11 and MciPro4 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

Three fungal strains listed in the JGI database (https:// genome. JGI. doe. gov/portal /) TUCIM 6016, Fusarium verticillium (Fusarium verticillium) 7600, and Microsporum species (Stagonospora sp.) SRC1lsM3a were selected as potential sources of enzymes for a variety of industrial applications. A BLAST search (Altschul et al, J Mol Biol [ J. Mol. Biol., 215: 403-: identification of TciPro1 from trichoderma citrinoviride TUCIM 6016, fvepor 4 from fusarium verticillioides 7600, and SspPro2 from cuprococcus species SRC1lsM3 a. The full-length protein sequences of TciPro1(JGI strain ID: Trici4, protein ID: 1136694), FvePro4(JGI strain ID: Fusve2, protein ID: 4472), and SspPro2(JGI strain ID: Stasp1, protein ID: 303285) are set forth as SEQ ID NO 3, SEQ ID NO 4, and SEQ ID NO 5, respectively.

EXAMPLE 2 expression of the identified fungal X-Pro protease

The DNA sequence encoding full length MalPro11, MciPro4 or TciPro1 (after an additional 5' DNA fragment (SEQ ID NO: 6)) was chemically synthesized and inserted into the Trichoderma reesei expression vector pGXT (identical to the pTTTpyr2 vector as described in published PCT application WO 2015/017256, which is incorporated herein by reference). The resulting plasmids were labeled pGXT-MalPro11, pGXT-MciPro4 and pGXT-TciPro 1. Protoplast transformation (Te' o et al (2002) J. Microbiol. methods [ journal of microbial methods ]51:393-99) was used and then each individual expression vector was transformed into the appropriate strain of Trichoderma reesei (described in published PCT application WO 05/001036). Transformants were selected on medium containing acetamide (as sole nitrogen source). After 5 days of growth on acetamide plates, transformants were harvested and fermented in 250mL shake flasks in defined medium containing a mixture of glucose and sophorose.

DNA sequences encoding truncated FvePro4(SEQ ID NO:7) and truncated SspPro2(SEQ ID NO:8) were chemically synthesized and inserted into the Bacillus subtilis expression vector p2JM103BBI (Vogtentanz, Protein Expr Purif [ Protein expression and purification ],55:40-52,2007) to generate plasmids pGXB-FvePro4 and pGXB-SspPro2, respectively. Each individual expression vector was transformed into the appropriate Bacillus subtilis strain and the transformed cells were spread onto Luria agar plates supplemented with 5ppm chloramphenicol. Colonies were selected and fermented in 250mL shake flasks with defined medium of MOPS medium.

To purify MalPro11, MciPro4 and TciPro1, each clarified culture supernatant was concentrated and ammonium sulfate was added to a final concentration of 1M. Loading of the solution to HiPrep^TMPhenyl FF16/10 column, pre-equilibrated with 20mM NaAc (pH 5.0) supplemented with additional 1M ammonium sulfate (buffer A). The target protein was eluted from the column with 0.25M ammonium sulfate. The corresponding fractions were combined, concentrated, and buffer exchanged to 20mM Tris (pH 8.0) (buffer B) using a VivaFlow 200 ultrafiltration device (Sartorius Stedim). The resulting solution was applied to HiPrep pre-equilibrated with buffer B^TMQ HP 16/10 column. With 0.3M NaClThe target protein is eluted from the column. The fractions containing active protein were then combined and concentrated via a 10K Amicon Ultra device and stored in 40% glycerol at-20 ℃ until use.

To purify fveporo 4 and SspPro2, each clarified culture supernatant was concentrated and ammonium sulfate was added to a final concentration of 1M. Loading of the solution to HiPrep^TMPhenyl FF16/10 column, pre-equilibrated with 20mM NaPi (pH 7.0) supplemented with additional 1M ammonium sulfate (buffer A). The target protein flows through the column. The solutions were combined, concentrated, and buffer exchanged to 20mM Tris (pH 8.0) (buffer B) using a VivaFlow 200 ultrafiltration unit (sartorius). The resulting solution was applied to HiPrep pre-equilibrated with buffer B^TMQ HP 16/10 column. The target protein was eluted from the column with 0.2M NaCl. The active fractions were combined and ammonium sulfate was added to a final concentration of 1.2M. Loading of the solution to HiPrep^TMPhenyl HP 16/10 column, the column was pre-equilibrated with 20mM NaPi (pH 7.0) supplemented with additional 1.2M ammonium sulfate. The target protein was eluted from the column using a gradient elution mode (from 1.2M to 0.6M ammonium sulfate). The fractions containing active protein were then combined and concentrated via a 10K Amicon Ultra device and stored in 40% glycerol at-20 ℃ until use.

EXAMPLE 3 proteolytic Activity of purified fungal X-Pro protease

Proteolytic activity of proteases (MalPro11, MciPro4, TciPro1, FvePro4 and SspPro2) purified in 50mM Tris-HCl buffer (pH 7.5) using phenylalanine-proline (Phe-Pro) (Gill Biochem, Shanghai) or serine-proline (Ser-Pro) (Gill Biochem, Shanghai) as substrates. Before the reaction, the enzyme was diluted with water to a specific concentration. The dipeptide substrate (Phe-Pro or Ser-Pro) was dissolved in 50mM Tris-HCl buffer (pH7.5, supplemented with 0.05mM CoCl₂) To a final concentration of 10 mM. To start the reaction, 90 μ L of 10mM dipeptide (Phe-Pro or Ser-Pro) was added to non-binding 96-MTP (Corning Life Sciences), #3641) and incubated in a thermostated mixer (Thermomixer) at 50 ℃ and 600rpm for 5min, followed by addition of 10 μ L of diluted enzyme sample (or water alone as the only addition to the reaction mixture)Blank control). After incubation at 50 ℃ and 600rpm for 20min in a thermostatic mixer, the protease reaction was stopped by heating at 95 ℃ for 10 min.

Free Pro resulting from hydrolysis of the dipeptide (Phe-Pro or Ser-Pro) as detected by ninhydrin reaction was used to show proteolytic activity. Prior to the reaction, ninhydrin (sigma, #151173) was dissolved in 100% ethanol to a final concentration of 5% (w/v). To start the ninhydrin reaction, 40. mu.L of 1M sodium acetate (pH 2.8) was first mixed with 10. mu.L of a 5% ninhydrin solution in a 96-MTP PCR plate (Axygen, PCR-96M2-HS-C), and then 50. mu.L of the above protease reaction solution was added. The whole mixture was then incubated in a thermocycler (BioRad) at 95 ℃ for 15min. After addition of 100. mu.L of 75% ethanol, the mixture was purified at 440nm (A) using SpectraMax 190₄₄₀) The absorbance of the resulting solution was measured. By A from enzyme samples₄₄₀Minus A of blank control₄₄₀To calculate net A₄₄₀And then plotted against different protein concentrations (from 0.3125ppm to 20 ppm). The results are shown in fig. 3A and 3B. Each value is the mean of duplicate determinations with a variance of less than 5%. Thus proteolytic activity is shown as neat A₄₄₀. Proteolytic assays using Phe-Pro (FIG. 3A) or Ser-Pro (FIG. 3B) as substrates indicated that MalPro11, MciPro4, TciPro1, FvePro4, and SspPro2 are all active proteases.

EXAMPLE 4 pH Profile of purified fungal X-Pro protease

The pH curves of the purified proteases (MalPro11, MciPro4, TciPro1, FvePro4 and SspPro2) were studied in 25mM Bis-tris propane buffers at different pH values (ranging from pH 6 to 10) using Phe-Pro dipeptide as substrate. Prior to the assay, 45. mu.L of 50mM Bis-tris propane buffer (supplemented with 0.1mM CoCl) having a specific pH was first added₂) Mixed with 45 μ L of 20mM Phe-Pro (dissolved in water) in 96-MTP, then 10 μ L of enzyme diluted with water (12.5ppm MalPro11, 25ppm MciPro4, 12.5ppm TciPro1, 12.5ppm FvePro4, 6.25ppm SspPro2, or water alone as blank) was added. The reaction was carried out and analyzed as described in example 3. Enzyme activity at each pH is reported as relative activity, with enzyme activity at the optimum pHThe sex was 100%. The tested pH values were 6, 6.5, 7, 7.5, 8, 8.5, 9.5 and 10. Each value is the mean of duplicate determinations with a variance of less than 5%. As shown in fig. 4, the optimal pH of MalPro11, MciPro4, TciPro1, fvepiro 4, or SspPro2 is 8, 8.5, 8, or 8, respectively.

EXAMPLE 5 temperature Profile of purified fungal X-Pro protease

The temperature profile of the purified proteases (MalPro11, MciPro4, TciPro1, FvePro4 and SspPro2) was analyzed in 50mM Tris-HCl buffer (pH 7.5) using Phe-Pro dipeptide as substrate. Before reaction, the solution was dissolved in 50mM Tris-HCl buffer (pH7.5, supplemented with 0.05mM CoCl)₂) 90 μ L of 10mM Phe-Pro dipeptide in (E) was added to a 200 μ L PCR tube, which was then incubated in a thermal cycler (Bio Rad) at the desired temperature (i.e., 30 ℃ to 80 ℃) for 5min. After incubation, 10 μ L of enzyme diluted with water (12.5ppm MalPro11, 25ppm MciPro4, 12.5ppm TciPro1, 12.5ppm fvepiro 4, 6.25ppm SspPro2 or water alone as blank) was added to the substrate solution to start the reaction. After incubation for 20min in a thermocycler at different temperatures, the reaction was quenched and analyzed as shown in example 3. The reported activity is relative activity, where the activity at the optimal temperature is set as 100%. The temperatures tested were 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C and 80 deg.C. Each value is the mean of duplicate determinations with a variance of less than 5%. As shown in FIG. 5, the optimum temperature for MalPro11, MciPro4, TciPro1, FvePro4 or SspPro2 is 55 deg.C, 50 deg.C, 45 deg.C or 50 deg.C, respectively.

EXAMPLE 6 thermostability of purified fungal X-Pro protease

Prior to the thermostability assay, the Phe-Pro dipeptide substrate was dissolved in 50mM Tris-HCl buffer (pH7.5, supplemented with 0.05mM CoCl)₂) To a final concentration of 10 mM. The purified proteases (MalPro11, MciPro4, TciPro1, FvePro4 and SspPro2) were diluted in 0.2mL of water to a final concentration of 200ppm, followed by incubation at different temperatures (4 ℃,55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃) for 5min. After incubation, each enzyme solution was further diluted with water to a specific concentration (12.5ppm of MalPro11,25ppm MciPro4, 12.5ppm TciPro1, 12.5ppm FvePro4, 6.25ppm SspPro2 or addition of water alone as blank). To measure proteolytic activity, 10. mu.L of the resulting enzyme solution was mixed with 90. mu.L of substrate solution; and reacted and analyzed as described in example 3. Activity was reported as residue activity, where the activity of the enzyme sample incubated at 4 ℃ was set to 100%. Each value is the mean of duplicate determinations with a variance of less than 5%. As shown in FIG. 6, all proteases lost their activity after incubation at 70 ℃, 75 ℃ and 80 ℃ for 5 min; and all other four proteases also lost their activity after incubation for 5min at 65 ℃ except for MciPro 4.

EXAMPLE 7 pentapeptide hydrolysis assay of purified fungal X-Pro protease

Proteolytic activity of the proteases (MalPro11, MciPro4, TciPro1, FvePro4 and SspPro2) purified in 50mM Tris-HCl buffer (pH 7.5) against the pentapeptide Gln-Pro-Gln-Gln-Pro (Gill Biochemical Co., Ltd., Shanghai) (SEQ ID NO: 9). Prior to the reaction, the enzyme was diluted with water to 200 ppm. The pentapeptide substrate was dissolved in 50mM Tris-HCl buffer (pH7.5, supplemented with 0.05mM CoCl)₂) To a final concentration of 10 mM. To start the reaction, 90 μ Ι of 10mM pentapeptide solution was added to non-binding 96-MTP (corning life sciences, #3641) and incubated at 50 ℃ and 600rpm for 5min in a thermostated mixer followed by 10 μ Ι of diluted enzyme sample (or water alone as blank). After incubation for 1 hour at 50 ℃ and 600rpm in a thermostatic mixer, the protease reaction was stopped by heating at 95 ℃ for 10 min.

The ninhydrin reaction to detect primary amines was used to demonstrate pentapeptide hydrolysis. Prior to the reaction, a ninhydrin solution was prepared containing 2% ninhydrin (w/v), 0.5M sodium acetate, 40% ethanol, and 0.2% fructose (w/v). To start the reaction, 90. mu.L of ninhydrin solution was mixed with 10. mu.L of the above protease reaction solution in a 96-MTP PCR plate. The whole mixture was then incubated in a thermocycler at 95 ℃ for 15min. After addition of 100. mu.L of 75% ethanol, the mixture was purified at 570nm (A) using SpectraMax 190₅₇₀) The absorbance of the resulting solution was measured. The results are shown in fig. 7. Each value is the mean of duplicate determinations with a variance of less than 5%. Those protease sample phasesA compared to blank control₅₇₀The increments indicated that all purified proteases were able to hydrolyze the pentapeptide Gln-Pro-Gln-Gln-Pro.

Example 8: preparation and analysis of gluten prehydrolyzate

Substrates comprising water-soluble gluten peptides and amino acids are prepared by a modified version of the methods described in Schlicherle-Cerny and Amad OA (2002). The following ingredients were mixed in a 100mL screw cap bottle: 6.4g of gluten (Sigma-Aldrich, Copenhagen, Denmark), 0.123g of AcPepN2, 0.6g of glutaminase SD-C100S (Tianye (Amano), Nippon Kogyo), 63mg of glutenAlkaline protease (A)Industrial biosciences, brabband, denmark), 1.73g NaCl (analytical grade, fisher Scientific, rosskole, denmark), and 24.3g water. The vial was incubated in a heat block while magnetically stirring at 600rpm and 55 ℃ for 18 hours. The enzyme was then inactivated by heating to 95 ℃ for 10min, centrifuged at 4600rpm for 5min, and the supernatant filtered through a 0,45 μm syringe filter.

For the N-terminal sequencing of the residual peptide, the gluten prehydrolyzate was filtered through a 0,2 μm syringe filter, and 2 μ L of this was loaded onto the PPSQ-31B protein sequencer from Shimadzu, Japan. A mixture of 25pmol of all 20 common amino acids was prepared and used as a standard. The retention time and peak area of these amino acids in the standard were used to identify and quantify the amino acids released after each step of the Edman (Edman) cycler. From these results, a consensus sequence at the N-terminus of the residual peptide can be derived. This co-sequence is: XPQQP, where X is any amino acid, P is proline and Q is glutamine. In addition, the results showed that 73% of the residual peptide had proline at the secondary terminal position.

Using a docked to Orbitrap Fusion Mass spectrometer (Semmer technology bulletin)Department) of Dionex3000RSLCnano LC (Seimer science and technology company) for nano LC-MS/MS analysis. mu.L of each sample was loaded with a traction transmitter onto a 2cm trap column (100 μm i.d., 375 μm o.d., C18, 5 μm inverse particles) connected to a 15cm analytical column (75 μm i.d., 375 μm o.d., filled with Rerosil C18, 3 μm inverse particles) (Dr. Maisch GmbH, Amerbuch-Entringen, Germany). Using 5% -53% solvent B (H)₂O/CH₃CN/TFE/HCOOH (100/800/100/1) v/v/v/v) at a flow rate of 300nL/min for entering a nanoelectrospray ion source (Sammer technologies). The Orbitrap Fusion instrument runs in data dependent MS/MS mode. Peptide masses were measured by Orbitrap (MS scan obtained at m/z 200 with a resolution of 120.000) and as many ions as possible were selected from the strongest peptide m/z and fragmented within 1.6 seconds using (high energy collision dissociation) HCD in a linear ion trap (LTQ). Dynamic exclusion was initiated, the list size was 500 masses, the duration was 40 seconds, and the excluded mass width relative to the masses on the list was ± 10 ppm.

RAW (RAW) files were processed using a protome scanner 2.0 and a local mascot server and searched against Uniprot Green Plants. The area of all identified peptides was estimated using the built-in region detection module in the protome discovery 2.0.

An important tool to assess the amount of bound Gln in the residual peptide from gluten hydrolysis is the Q-area. Q-area ═ Q_nArea, wherein Q_nIs the number of Gln residues in the peptide and the area under the curve of the chromatographic peak produced by the particular peptide.

The results show a specific sequence or "motif" of amino acids, XPQQP, which is common to most of the peptides examined. Based on the Q area, it is estimated that peptides carrying this sequence motif at the N-terminus contain approximately 60% residual glutamine.

And (4) conclusion: two independent analytical techniques showed that the N-terminus of the residual peptide in the gluten prehydrolyzate had the consensus sequence XPQQP.

Example 9: testing of gluten prehydrolyzates by X-ProAP

General procedure: the reaction mixture consisted of 250. mu.L of gluten prehydrolyzate, 11.8. mu.L of 50mg/mL glutaminase, 10.2. mu.L AcPepN2, and 98. mu. g X-ProAP. MilliQ water was added to a total volume of 310. mu.L or 415. mu.L. The total volume was constant in each experiment, but varied from experiment to experiment with the protein concentration of X-ProAP used. The reference sample contained glutaminase but neither AcPepN2 nor X-ProAP. The total volume was the same as the other samples in the experiment.

All reaction mixtures were prepared in Eppendorf tubes. The tubes were incubated in an Eppendorf Mixer at 50 ℃ and 800 rpm. At a specific time point, an 80 μ Ι aliquot was removed and mixed with 20 μ Ι of 2.5M TCA (fisher technologies, ross, denmark) to stop further reaction. The concentration of glutamic acid in the hydrolysate was quantified using the enzyme L-glutamic acid kit (from Bayer AG (R-BIOPHARM), Dammstadt, Germany). The method is reduced for use with 96-well plates, otherwise according to manufacturer instructions. Prior to analysis, the TCA/sample mixture was further diluted 400 times in MilliQ water (total dilution factor 500).

The Degree of Hydrolysis (DH) was determined based on the o-phthalaldehyde (OPA; Feishell technologies, Rosusler, Denmark) assay according to the method described by Nielsen et al (Nielsen, Petersen et al 2001). The mean MW of amino acids was determined by total amino acid analysis (performed in eurofine, waine, denmark). Based on this, h is_totCalculated as 7.6mmol/g gluten protein.

Amino acid and peptide profiles were analyzed using Size Exclusion Chromatography (SEC). The system used is from Saimer Feishel technologies Inc. ((R))Denmark) and is an autosampler with a Dionex UltiMate3000 solvent holder, pump, and aerosol detector (CAD) with Dionex corpa ultrars charging, Superdex^TMPeptide 10/300GL column (fromMerck (Merck), Copenhagen, Denmark).Version 7.2 is used for instrument control and data processing. The mobile phase consisted of 20% Acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA; Feishell scientific, Roschelle, Denmark) in MilliQ water. Prior to injection, all samples were diluted 10 times in the mobile phase and filtered using 0.2 μm PVDF filter plates (material #3504, corning, kennebank, maine, usa). The injection volume was 10 μ L and the flow rate was 0.500mL/min for 55 min.

All the reference samples included in the experiments contained gluten prehydrolyzate and glutaminase. It was exposed to the same treatment as all other samples. To facilitate comparison between the different runs, the reference sample was set to contain 100% glutamic acid (formed during the prehydrolysis step). All other results are given in% relative to the reference sample. Other samples contained the same components as the reference sample, but with the addition of AcPepN2 and/or X-ProAP.

Figure 8 shows the effect of increasing doses of SspPro2 on glutamate yield. Two doses of SspPro2 were tested: 131. mu.g/mL and 392. mu.g/mL of prehydrolyzate. This resulted in an increase of 16% and 34% in glutamic acid, respectively, relative to the reference sample. Under the given conditions, only AcPepN2 did not give any increase in glutamate levels.

Figure 9 shows the results from the same samples as figure 8 but after incubation for 26 h. In this case, 131. mu.g/mL and 392. mu.g/mL of TciPro1 resulted in 25% and 71% increases in glutamic acid, respectively, relative to the reference sample. In this case, AcPepN2 alone also gave a 16% increase in glutamate relative to the reference sample.

FIG. 10 shows the effect of different X-ProAPs on glutamate yield. The incubation time was 24 h. In this case, AcPepN2 alone gave an 8% increase in glutamate levels relative to the reference sample. In combination with AcPepN2, MalPro11, MciPro4, TciPro1, PchSec117, and SspPro2 gave increases of 40%, 44%, 25%, 28%, and 64%, respectively. In contrast, when MalPro11, MciPro4, and SspPro2 were tested alone (without AcPepN2), no increase in glutamate levels was observed (no more than experimental error). The results show that AcPepN2 and the X-ProAP tested act synergistically to release glutamic acid from the residual peptide in the prehydrolyzate. Because of the limited amount of material, TciPro1 and PchSec117 were not tested without AcPepN 2.

Fig. 11 shows results from two additional tested X-proaps. They gave only negligible response after incubation for 19h and 26 h. The results after 42 hours of incubation are shown in figure 11. In this case, AcPepN2 alone gave a 9% increase in glutamate levels. AoX-ProAP and HX-ProAP gave 15% and 6% increases, respectively. The difference between AcPepN2 alone and HX-ProAP is within experimental error. Because of the limited materials, the dose of X-ProAP in this case was only 15. mu.g/mL of prehydrolyzate.

The hydrolysis curves were determined on samples from the same experiment used for the glutamic acid results in fig. 8 to 11. Two examples are given below. In FIG. 12, the hydrolysis curves of the sample of AcPepN2 in 392. mu.g/mL prehydrolyzate (solid line) and the curves of the sample containing AcPepN2+ SspPro2 (dashed line) are compared. The peak area of the amino acid-containing peak of the hydrolysate produced with AcPepN2+ SspPro2 was 1.5 times higher than that of the hydrolysate produced with only AcPepN 2. Meanwhile, the DP2-5 area of the hydrolysate of AcPepN2+ SspPro2 was reduced by 1.3 times compared to the hydrolysate of AcPepN2 alone. The decrease in the area of DP2-5 was not proportional to the increase in the area of the amino acid, since the response factor for CAD is not equivalent to the amino acid and DP2-5 peptide. FIG. 13 shows a similar comparison of the hydrolysis curves of the AcPepN2 sample and the sample containing HX-ProAP. The increase in amino acids by HX-ProAP is very modest. Consistent with the observations, this treatment did not increase Gln levels.

Example 10: testing of X-ProAP on slurry of gluten protein

The production of glutamic acid from gluten proteins does not require a prehydrolysis product. The SspPro2 was tested in a setup where all components (including enzymes) were mixed at the beginning of the experiment.

The methods described by schlichhery-Cerny and Amad oa (2002) are usedA reduced-scale version of (a). The following ingredients were mixed in a 20mL Wheaton bottle (Wheaton via): 2.13g gluten, 33mg AcPepN2, 21mgAlkaline protease, 0.2g glutaminase, 1mg SspPro2, 0.58g NaCl and about 8g water. The amount of water was adjusted so that the total weight of all ingredients was equal to 10.5 g. The Wheaton bottles were incubated in a heat block with magnetic stirring at 600rpm and 55 ℃ for up to 48 hours. At different time points, 160. mu.L aliquots were taken and the reaction was stopped with 40. mu.L of 2.5M TCA. The samples were further diluted 400 times and assayed for glutamate as described in example 9 (all chemical and enzyme suppliers were the same as in example 8 and example 9).

After 24h incubation, the sample containing SspPro2 formed 22% more glutamate compared to the reference sample without X-ProAP. It is to be noted that the reference sample in this case contains active AcPepN2, in contrast to the reference sample in the gluten prehydrolysis product experiment, where the prehydrolysis product was prepared with AcPepN2+ other enzymes (which were subsequently inactivated). In the gluten slurry experiments, the reference sample without AcPepN2 was not meaningful.