TECHNICAL FIELD

The present invention relates to proline specific endopeptidases. More particularly, the present invention relates to the use of proline specific endopeptidases for reduction or elimination of beer haze, production of protein hydrolysates and detoxification of gluten proteins, including amelioration of gluten intolerance and Celiac disease.

BACKGROUND

Beer-haze, a cloudy appearance in beer, is caused by the aggregation of hydrophobic proteins, e.g. hordeins from barley, and polyphenols, resulting in a beer with an undesirable cloudy appearance or haze. See, e.g., Asano, K.; Shinagawa, K.; Hashimoto, N. Characterization of haze-forming proteins of beer and their roles in chill haze formation. J Am. Soc. Brew. Chem. 1982, 40, 147-154. The same phenomenon is also called chill-haze and similar haze formation may also occur in wine and fruit juices.

It has been suggested that acid proteases such as papain can be used to degrade beer proteins and hence prevent haze formation. However, broad spectrum proteases such as papain have been found to impair beer foam formation and stability. See, e.g, Posada, J.; Almenar, J.; Garcia Galindo, J. A practical approach on protein stabilizers. Proc. - Eur. Brew. Corn. 1971, 13, 379-391. For this reason, more selective proteases such as proline specific endopeptidases have been employed to reduce beer haze. However, there is a continuing need for proteases that can be used to reduce beer-haze, because present commercial offerings are overly expensive and do not provide complete beer-haze removal. Moreover, there is a concern that some of the current beer haze proteases survive the brewing process and are present in the final beer product. Hence, there is also a need for proteases that can be used to prevent chill haze but which do not survive the brewing process.

Celiac disease, also known as gluten-sensitive enteropathy, is a widespread, autoimmune disease of the small intestine induced in patients having susceptible genetic backgrounds by the intake of gluten proteins from common sources such as wheat, rye and barley. In susceptible patients, exposure of the small intestine to gluten induces a CD4+ T cell mediated immune response. Celiac disease normally appears in early childhood and includes such severe symptoms as chronic diarrhea, fatigue, weight loss, and abdominal distension. Left untreated, Celiac disease increases the risk of infertility, bone disorders and intestinal malignancies.

Gluten is a composite of storage proteins found in many cereal grains such as wheat, rye, oats, barley, maize and rice. Recent molecular and genetic studies have strongly implicated gliadin proteins as the immunogenic component of wheat gluten. A 33-mer peptide from alpha- 2 gliadin and a 26-mer peptide from gamma- gliadin have been identified as the primary initiators of the inflammatory response of gluten in Celiac Sprue patients (Shan et al., (2005) J Proteome Res., 4(5): 1732-1741). Both 26-mer and 33-mer peptides contain multiple copies of antigenic epitopes. They are very rich in proline and reported to be resistant to pepsin, trypsin and chymotrypsin (Bethune and Khosla, (2012) Methods Enzymol, 502: 241-271).

There is a continuing need for proteases that are capable of degrading the immunogenic proline rich protein sequences in wheat gliadins and similar proteins from barley, rye, oats and maize.

SUMMARY OF THE INVENTION

In accordance with an aspect of the instant invention, an isolated polypeptide is described having proline specific endopeptidase activity having a polypeptide which is at least 70% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or a fragment thereof. Optionally, the polypeptide has at least 80% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or a fragment thereof. Optionally, the polypeptide has at least 90% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or a fragment thereof. Optionally, the polypeptide has at least 95% sequence identity to one of SEQ ID NO: 4, SEQ ID NO:6 and SEQ ID NO:8 or a fragment thereof. Optionally, the polypeptide has at least 99% sequence identity to one of SEQ ID NO: 4, SEQ ID NO:6 and SEQ ID NO:8. Optionally, the polypeptide is a sequence according to one of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 or a fragment thereof. In accordance with an aspect of the present invention, a method for the reduction or prevention of haze in a beverage is presented having the step of adding an isolated polypeptide having proline specific endopeptidase as described above to the beverage. Optionally, the beverage contains at least one protein. Optionally, the protein comprises hordein. Optionally, the beverage further comprises polyphenols.

Optionally, the beverage has a pH of less than 7.

Optionally, the beverage is a fruit juice. Optionally, the beverage is a wine. Optionally, the beverage is a beer. Optionally, the isolated polypeptide is added to a mash.

Optionally, the isolated polypeptide is added before haze formation. Optionally, the isolated polypeptide is added after haze formation.

Optionally, the method of haze reduction has the further step of adding a second isolated polypeptide having proline specific endopeptidase activity as described above wherein the second isolated polypeptide is different than the isolated polypeptide. Optionally, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

In another aspect of the present invention, a method for forming a protein hydrolysate is presented having the step of adding to a protein substrate an isolated polypeptide having endopeptidase as described above. Optionally, the method includes the further step of adding a protease wherein the protease is different than the isolated polypeptide. Optionally, the protease is selected from the group consisting of a serine protease, a cysteine protease, an endopeptidase, and an exopeptidase.

Optionally, the protease is a serine protease. Optionally, the serine protease is a subtilisin.

Optionally, the protease is an endopeptidase.

Optionally, the endopeptidase is a second isolated polypeptide having proline specific endopeptidase activity as described above. Optionally, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO:8 or a fragment thereof.

Preferably, the protease is an exopeptidase. Optionally, the exopeptidase is a tripeptidyl aminopeptidase. Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 70% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof .

Optionally, the polypeptide has at least 70% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the polypeptide has at least 80% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the polypeptide has at least 80% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the polypeptide has at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the polypeptide has at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the polypeptide has at least 95% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the polypeptide has at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the polypeptide has at least 99% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 ,

SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 ,

SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35,

SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 ,

SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 ,

SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 ,

SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 ,

SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 ,

SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase has at least 99% sequence identity to

SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the polypeptide is a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: l9 , SEQ ID NO:20 , SEQ ID

NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the polypeptide is a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 or a fragment thereof.

Optionally, in the method of making a hydrolysate, in addition to the isolated polypeptide having proline specific endopeptidase and the polypeptide having tripeptidyl amino peptidase activity a second isolated polypeptide having proline specific endopeptidase activity as described above is added wherein the second isolated polypeptide is different than the isolated polypeptide having proline specific endopeptidase activity.

Optionally, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

Optionally, the protein substrate is derived from milk. Optionally, the protein substrate is derived from wheat.

In another aspect of the present invention, a method for degrading gluten in food is presented having the step of contacting gluten-containing food with an isolated polypeptide having proline specific endopeptidase activity as described above.

Optionally, the food is bread or beer.

In another aspect of the present invention, a method for treating gluten intolerance, celiac disease, dermatitis herpetiformis and/or gluten sensitivity in a patient in need of such treatment, wherein the treatment reduces exposure of the patient to an immunogenic gluten peptide, having the step of orally administering to the patient a therapeutically effective dose of an isolated polypeptide having proline specific endopeptidase activity as described above contemporaneously with the ingestion of a food that may contain gluten.

In another aspect of the present invention, the use is presented of an isolated polypeptide having proline specific endopeptidase activity as described above for the manufacture of a dietary supplement or medicament. Optionally, the isolated polypeptide having proline specific endopeptidase activity as described above digests gluten fragments that are resistant to normal digestive enzymes.

Optionally, the isolated polypeptide having proline specific endopeptidase activity as described above is admixed with food.

Optionally, the isolated polypeptide having proline specific endopeptidase activity as described above is stable to acid conditions.

In another aspect of the present invention, a formulation is presented having the isolated polypeptide having proline specific endopeptidase activity as described above and a pharmaceutically acceptable excipient.

In other aspect of the present invention, an enzyme blend is presented having a proline specific endopeptidase as described above and a protease wherein the proline specific endopeptidase is different than said protease. Optionally, the protease is selected from the group consisting of a serine protease, a cysteine protease, an endopeptidase, and an exopeptidase.

Optionally, the protease is a serine protease. Optionally, the serine protease is a subtilisin.

Optionally, the protease is an endopeptidase. Optionally, the endopeptidase is a second isolated polypeptide having proline specific endopeptidase activity as described above. Optionally, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

Optionally, the protease is an exopeptidase. Optionally, the exopeptidase is a tripeptidyl aminopeptidase. Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 70% sequence identity to SEQ ID NO: l5, SEQ ID NO:l6, SEQ ID NO: l7 , SEQ ID NO:l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ

ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ

ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ

ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ

ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ

ID NO:49 , SEQ ID NO:50 , SEQ ID N0:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ

ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ

ID NO:59 , SEQ ID NO:60 , SEQ ID N0:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof. Optionally, the polypeptide has at least 70% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 80% sequence identity to SEQ ID NO: 15,

SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 ,

SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 ,

SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35,

SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 ,

SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 ,

SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 ,

SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 ,

SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 ,

SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 80% sequence identity to SEQ ID NO: 15,

SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 90% sequence identity to SEQ ID NO: 15,

SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID N0:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID N0:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID N0:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID N0:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 90% sequence identity to SEQ ID NO: 15,

SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 95% sequence identity to SEQ ID NO: 15,

SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having 95% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof. Optionally, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 99% sequence identity to SEQ ID NO: 15,

SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 ,

SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 ,

SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35,

SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 ,

SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 ,

SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 ,

SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 ,

SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 ,

SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63, SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Optionally, the tripeptidyl aminopeptidase has at least 99% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof

Optionally, the tripeptidyl aminopeptidase is a polypeptide having a sequence as set forth in SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof. Optionally, the tripeptidyl aminopeptidase is a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Optionally, where an enzyme blend has a polypeptide having proline specific endopeptidase activity as described above and a tripeptidyl aminopeptidase as described above, a second isolated polypeptide having proline specific endopeptidase activity as describe above is included in the blend wherein the second isolated polypeptide is different than the isolated polypeptide having proline specific endopeptidase activity.

According to this aspect of the present invention, the isolated polypeptide is optionally a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide having proline specific endopeptidase activity is optionally a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

In another aspect of the present invention, a polynucleotide is presented having a nucleic acid sequence encoding the isolated polypeptide having proline specific endopeptidase activity as described above.

In another aspect of the present invention, a recombinant expression vector is presented having the polynucleotide.

In another aspect of the present invention, a host cell is presented having the recombinant expression vector.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of the synthesized 26-mer peptide discussed in the examples.

SEQ ID NO:2 is the amino acid sequence of the synthesized 33-mer peptide discussed in the examples.

SEQ ID NO: 3 is the nucleotide sequence of full-length MorProl gene.

SEQ ID NO: 4 is the amino acid sequence of MorProl precursor protein.

SEQ ID NO: 5 is the nucleotide sequence of full-length AflPro3 gene.

SEQ ID NO: 6 is the amino acid sequence of AflPro3 precursor protein.

SEQ ID NO: 7 is the nucleotide sequence of full-length CpoProl gene.

SEQ ID NO: 8 is the amino acid sequence of CpoProl precursor protein. SEQ ID NO: 9 is the predicted, mature amino acid sequence of MorProl, lacking the signal sequence.

SEQ ID NO: 10 is the predicted, mature amino acid sequence of AflPro3, lacking the signal sequence.

SEQ ID NO: 11 is the predicted, mature amino acid sequence of CpoProl, lacking the signal sequence.

SEQ ID NO 12 is the synthesized nucleotide sequence encoding full-length MorProl. SEQ ID NO 13 is the synthesized nucleotide sequence encoding full-length AflPro3. SEQ ID NO 14 is the synthesized nucleotide sequence encoding full-length CpoProl. SEQ ID NO 15 is the peptidase with leader sequence from Trichoderma reesei.

SEQ ID NO 16 is the peptidase with no leader sequence from Trichoderma reesei. SEQ ID NO 17 is the peptidase from Aspergillus oryzae.

SEQ ID NO 18 is the peptidase from Phaeosphaeria nodorum.

SEQ ID NO 19 is the peptidase from Trichoderma atroviride.

SEQ ID NO 20 is the peptidase from Arthroderma benhamiae .

SEQ ID NO 21 is the peptidase from Pusarium graminearum.

SEQ ID NO 22 is the peptidase from Acremonium alcalophilum.

SEQ ID NO 23 is the peptidase from Sodimomyces alkalinus.

SEQ ID NO 24 is the peptidase from Aspergillus kawachii.

SEQ ID NO 25 is the peptidase from Talaromyces stipitatus.

SEQ ID NO 26 is the peptidase from Fusarium oxysporum.

SEQ ID NO 27 is the peptidase from Trichoderma virens.

SEQ ID NO 28 is the peptidase from Trichoderma atroviride.

SEQ ID NO 29 is the peptidase from Agaricus bisporus.

SEQ ID NO 30 is the peptidase from Magnaporthe oryzae.

SEQ ID NO 31 is the peptidase from Togninia minima.

SEQ ID NO 32 is the peptidase from Bipolaris maydi.

SEQ ID NO 33 is the peptidase from Aspergillus kawachii.

SEQ ID NO 34 is the peptidase from Aspergillus nidulans.

SEQ ID NO 35 is the peptidase from Aspergillus ruber. SEQ ID NO: 36 is the peptidase from Aspergillus terreus.

SEQ ID NO: 37 is the peptidase from Penicillium digitatum. SEQ ID NO: 38 is the peptidase from Penicillium oxalicum. SEQ ID NO: 39 is the peptidase from Penicillium roquefortis . SEQ ID NO: 40 is the peptidase from Penicillium rubens.

SEQ ID NO: 41 is the peptidase from Neosartorya fischeri.

SEQ ID NO: 42 is the peptidase from Aspergillus fumigatus. SEQ ID NO: 43 is the peptidase from Trichoderma reesei.

SEQ ID NO: 44 is the peptidase from Aspergillus oryzae.

SEQ ID NO: 45 is the peptidase from Phaeosphaeria nodorum. SEQ ID NO: 46 is the peptidase from Trichoderma atroviride. SEQ ID NO: 47 is the peptidase from Arthroderma benhamiae. SEQ ID NO : 48 is the peptidase from Fusarium graminearum. SEQ ID NO: 49 is the peptidase from Acremonium alcalophilum. SEQ ID NO: 50 is the peptidase from Sodiomyces alkalinus. SEQ ID NO: 51 is the peptidase from Aspergillus kawachii. SEQ ID NO: 52 is the peptidase from Talaromyces stipitatus. SEQ ID NO: 53 is the peptidase from Fusarium oxysporum. SEQ ID NO: 54 is the peptidase from Trichoderma virens.

SEQ ID NO: 55 is the peptidase from Trichoderma atrovirde. SEQ ID NO: 56 is the peptidase from Agaricus bisporus.

SEQ ID NO: 57 is the peptidase from Magnaporthe oryzae. SEQ ID NO: 58 is the peptidase from Togninia minima.

SEQ ID NO: 59 is the peptidase from Bipolaris maydis.

SEQ ID NO: 60 is the peptidase from Aspergillus kawachii. SEQ ID NO: 61 is the peptidase from Aspergillus nidulans. SEQ ID NO: 62 is the peptidase from Aspergillus ruber.

SEQ ID NO: 63 is the peptidase from Aspergillus terreus.

SEQ ID NO: 64 is the peptidase from Penicillium digitatum. SEQ ID NO: 65 is the peptidase from Penicillium oxalicum. SEQ ID NO: 66 is the peptidase from Penicillium roqueforti.

SEQ ID NO: 67 is the peptidase from Penicillium rubens.

SEQ ID NO: 68 is the peptidase from Neosartorya fischeri.

SEQ ID NO: 69 is the peptidase from Aspergillus fumigatus.

DESCRIPTION OF FIGURES

FIG. 1 shows the plasmid map of pGX256(Trex3gM-MorProl).

FIG. 2 shows the dose response curve of MorProl, AflPro3 and CpoProl.

FIG. 3 shows the pH profile of MorProl, AflPro3 and CpoProl.

FIG. 4 shows the temperature profile of MorProl, AflPro3 and CpoProl.

FIG. 5 shows the thermostability of MorProl, AflPro3 and CpoProl.

FIG. 6 shows the gliadin-catechin haze reduction performance of purified MorProl FIG. 7 shows the gliadin-catechin haze reduction performance of purified AflPro3. FIG. 8 shows the gliadin-catechin haze reduction performance of purified CpoProl.

PET ATT /ED DESCRIPTION OF THE INVENTION

Definitions:

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, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g. , a heterologous promoter in an expression vector.

Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences. A vector comprising a nucleic acid encoding an endopeptidase is a recombinant vector.

The terms“recovered,”“isolated,” and“separated,” refer to a compound, protein (polypeptides), 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 found in nature. An“isolated” polypeptides, thereof, includes, but is not limited to, a culture broth containing secreted polypeptide expressed in a heterologous host cell.

The term“amino acid sequence” is synonymous with the terms“polypeptide,” “protein,” and“peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an“enzyme.” The

conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation ( i.e ., N C).

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

The terms“transformed,”“stably transformed,” and“transgenic,” used with reference to a cell means that the cell contains a non-native ( e.g ., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.

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 an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., a proline specific endopeptidase) has been introduced. Exemplary host strains are microorganism cells (e.g, bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest. The term“host cell” includes protoplasts created from cells.

The term“heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

The term“endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

The term“expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.

A“selective marker” or“selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene. Examples of selectable markers include but are not limited to antimicrobials ( e.g ., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.

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

An“expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting 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 suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

The term“operably linked” means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.

A“signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell.

The mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process. As used herein,“percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. ( 1994) Nucleic Acids Res. 22:4673-4680. 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

Delay divergent sequences %: 40

Gap separation distance: 8

DNA transitions weight: 0.50

List hydrophilic residues: GPSNDQEKR

Use negative matrix: OFF

Toggle Residue specific penalties: ON

Toggle hydrophilic penalties: ON

Toggle end gap separation penalty OFF.

Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either termini are included. For example, a variant with five amino acid deletions of the C-terminus of the mature 617 residue polypeptide would have a percent sequence identity of 99% (612 / 617 identical residues x 100, rounded to the nearest whole number) relative to the mature polypeptide. Such a variant would be encompassed by a variant having“at least 99% sequence identity” to a mature polypeptide.

The term“about” refers to ± 5% to the referenced value.

The present proline specific endopeptidases may be“precursor,”“immature,” or“full-length,” in which case they include a signal sequence, or“mature,” in which case they lack a signal sequence. Unless otherwise noted, the amino acid residue numbering used herein refers to the mature forms of the respective endopeptidase polypeptides. The present endopeptidase polypeptides may also be truncated to remove the N or C-termini, so long as the resulting polypeptides retain endopeptidase activity.

The terms“protein”,‘polypeptide” and“peptide” are used interchangeable herein.

A“protease” is an enzyme that breaks down proteins and polypeptides by hydrolyzing amide bonds. The term“peptidase” is used herein interchangeably with protease.

An“exopeptidase” is a protease which cleaves the terminal amino acids of a protein or polypeptide. Typically, an exopeptidase can release one, two or three amino acids from either the N- or C- terminus of a protein or polypeptide.

An“endopeptidase” is a protease which cleaves internal amide bonds within a protein or polypeptide as opposed to an exopeptidase which cleaves the terminal (e.g. I ^st , 2 ^nd , or 3 ^rd terminal amino acid).

A“proline specific endopeptidase” or an enzyme, protein or polypeptide having such activity cuts proteins or polypeptides at or near places near proline residues.

As used herein,“beverage” means beer, wine or fruit juice. Also, beverage as used herein includes the above beverages at all stages of their production. For example, with respect to beer, beverage also can mean a wort or malt.

“Gluten” is a composite of storage proteins found in many cereal grains such as wheat, rye, oats, barley, maize and rice.

“Celiac disease”, also known as gluten-sensitive enteropathy, is a widespread, autoimmune disease of the small intestine induced in patients having susceptible genetic backgrounds by the intake of gluten proteins from common sources such as wheat, rye and barley.

Polypeptides of the invention include full length polypeptides as described herein in for example the sequence ids and variants thereof, including fragments.

“Fragments” of the polypeptides of the instant invention are shorter sequences of the polypeptides than as described in the Sequence IDs that retain activity, e.g., proline specific endopeptidase activity. Fragments include N-terminally deleted polypeptides, C-terminally deleted polypeptides, internally deleted polypeptides or any combination(s) thereof.

“Variants” may include the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains the basic biological functionality of the proline specific

endopeptidases. Variants include wild type variants such as those exhibited from similar enzymes derived from other sources and those introduced using recombinant DNA technology.

Amino acid substitutions may be made, for example from 1, 2 or from 3 to 10, 20 or 30 substitutions. The modified polypeptide will generally retain activity as a proline specific endopeptidase. Conservative substitutions may be made; such substitutions are well known in the art. Preferably substitutions do not affect the folding or activity of the polypeptide.

Production of endopeptidases

The present proline specific endopeptidases can be produced in host cells, for example, by secretion or intracellular expression. A cultured cell material ( e.g ., a whole-cell broth) comprising an endopeptidase can be obtained following secretion of the endopeptidase into the cell medium. Optionally, the endopeptidase can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final endopeptidase. A gene encoding a proline specific endopeptidase can be cloned and expressed according to methods well known in the art. Suitable host cells include bacterial, fungal (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, e.g. , Bacillus subtilis or B. licheniformis , as well as

Streptomyces, and A. Coli.

The host cell further may express a nucleic acid encoding a homologous or heterologous endopeptidase, i.e., a proline specific endopeptidase that is not the same species as the host cell, or one or more other enzymes. The endopeptidase may be a variant endopeptidase. Additionally, the host may express one or more accessory enzymes, proteins, peptides.

Vectors

A DNA construct comprising a nucleic acid encoding a proline specific endopeptidase can be constructed to be expressed in a host cell. Because of the well- known degeneracy in the genetic code, variant polynucleotides that encode an identical amino acid sequence can be designed and made with routine skill. It is also well-known in the art to optimize codon use for a particular host cell. Nucleic acids encoding endopeptidase can be incorporated into a vector. Vectors can be transferred to a host cell using well-known transformation techniques, such as those disclosed below.

The vector may be any vector that can be transformed into and replicated within a host cell. For example, a vector comprising a nucleic acid encoding a proline specific endopeptidase can be transformed and replicated in a bacterial host cell as a means of propagating and amplifying the vector. The vector also may be

transformed into an expression host, so that the encoding nucleic acids can be expressed as a functional endopeptidase. Host cells that serve as expression hosts can include filamentous fungi, for example.

A nucleic acid encoding a proline specific endopeptidase can be operably linked to a suitable promoter, which allows transcription in the host cell. The promoter may be any DNA sequence that 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 the transcription of the DNA sequence encoding a proline specific endopeptidase, especially in a bacterial host, are the promoter of the lac operon of E. coli , the Streptomyces coelicolor agarase gene dagA or celA promoters, the promoters of the Bacillus licheniformis a-amylase gene (amyL), the promoters of the Bacillus stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens a-amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral a-amylase, A. niger acid stable a-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase. When a gene encoding a proline specific endopeptidaseis expressed in a bacterial species such as E. coli , a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter. Examples of suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOX1 or AOX2 promoters cbhl is an endogenous, inducible promoter from T. reesei. See Liu et al. (2008)“Improved heterologous gene expression in Trichoderma reesei by cellobiohydrolase I gene (cbhl) promoter optimization” Acta Biochim. Biophys. Sin (Shanghai) 40(2): 158-65.

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

An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding a variant endopeptidase. Termination and polyadenylation sequences 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 the host cell. Examples of such sequences are the origins of replication of plasmids pUCl9, pACYCl77, pUBl lO, rE194, pAMBl, and pIJ702.

The vector may also comprise a selectable marker, e.g, a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis , or a gene that confers antibiotic resistance such as, e.g. , ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Furthermore, the vector may comprise Aspergillus selection markers such as amdS, argB , niaD and xxsC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art. See e.g., International PCT Application WO 91/17243.

Intracellular expression may be advantageous in some respects, e.g, when using certain bacteria or fungi as host cells to produce large amounts of endopeptidase for subsequent enrichment or purification. Extracellular secretion of endopeptidase into the culture medium can also be used to make a cultured cell material comprising the isolated endopeptidase.

The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises 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. Additionally, the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the endopeptidase to a host cell organelle such as a peroxisome, or to a particular host cell compartment. Such a targeting sequence includes but is not limited to the sequence, SKL. For expression under the direction of control sequences, the nucleic acid sequence of the endopeptidase is operably linked to the control sequences in proper manner with respect to expression.

The procedures used to ligate the DNA construct encoding a proline specific endopeptidase, the promoter, terminator and other elements, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (see, e.g, Sambrook el al, MOLECULAR CLONING: A LABORATORY MANUAL, 2 ^nd ed., Cold Spring Harbor, 1989, and 3 ^rd ed., 2001).

Transformation and Culture of Host Cells

An isolated cell, either comprising a DNA construct or an expression vector, is advantageously used as a host cell in the recombinant production of a proline specific endopeptidase. The cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g ., by homologous or heterologous recombination.

Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.

Examples of suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus,

Bacillus megaterium, and Bacillus thuringiensis ; Streptomyces species such as Streptomyces murinus ; lactic acid bacterial species including Lactococcus sp. such as Lactococcus lactis ; Lactobacillus sp. including Lactobacillus reuteri ; Leuconostoc sp.; Pediococcus sp.; and Streptococcus sp. Alternatively, strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli , or to Pseudomonadaceae can be selected as the host organism.

A suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species. A strain of the methyl otrophic yeast species, Pichia pastoris , can be used as the host organism. Alternatively, the host organism can be a Hansenula species. Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans. Alternatively, strains of a Fusarium species, e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma sp. can be used as a host. A suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023. A proline specific endopeptidase expressed by a fungal host cell can be glycosylated, i.e., will comprise a glycosyl moiety. The glycosylation pattern can be the same or different as present in the wild-type endopeptidase. The type and/or degree of glycosylation may impart changes in enzymatic and/or biochemical properties.

It is advantageous to delete genes from expression hosts, where the gene deficiency can be cured by the transformed expression vector. Known methods may be used to obtain a fungal host cell 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 a gene nonfunctional for its intended purpose, such that the gene is prevented from expression of a functional protein. A gene from a Trichoderma sp. or other filamentous fungal host that has been cloned can be deleted, for example, cbhl, cbh2 , egl l , and eg/ 2 genes. Gene deletion may be accomplished by inserting a form of the desired gene to be inactivated into a plasmid by methods known in the art.

Introduction of a DNA construct or vector into a host cell includes techniques such as transformation; electroporation; nuclear microinjection; transduction;

transfection, e.g. , lipofection mediated and DEAE-Dextrin mediated transfection; incubation with calcium phosphate DNA precipitate; high velocity bombardment with DNA-coated microprojectiles; and protoplast fusion. General transformation techniques are known in the art. See, e.g., Sambrook et al. (2001), supra. The expression of heterologous protein in Trichoderma is described, for example, in ET.S. Patent No. 6,022,725. Reference is also made to Cao et al. (2000) Science 9:991- 1001 for transformation of Aspergillus strains. Genetically stable transformants can be constructed with vector systems whereby the nucleic acid encoding a proline specific endopeptidase is stably integrated into a host cell chromosome.

Transformants are then selected and purified by known techniques.

Expression

A method of producing a proline specific endopeptidase may comprise cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.

The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of a proline specific endopeptidase. 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).

An enzyme secreted from the host cells can be used in a whole broth preparation. In the present methods, the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of a proline specific endopeptidase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large- scale fermentation (including continuous, batch, fed-batch, or solid state

fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the endopeptidase to be expressed or isolated. The term“spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is 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.

An enzyme secreted from the host cells 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 proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.

The polynucleotide encoding a proline specific endopeptidase in a vector can be operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The control sequences may be modified, for example by the addition of further

transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators. The control sequences may in particular comprise promoters.

Host cells may be cultured under suitable conditions that allow expression of a proline specific endopeptidase. Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sophorose. Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TNT™

(Promega) rabbit reticulocyte system.

Methods for Enriching and Purifying endopeptidases

Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a proline specific endopeptidase polypeptide-containing solution.

After fermentation, a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain a proline specific

endopeptidase solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultra-filtration, extraction, or chromatography, or the like, are generally used.

It is desirable to concentrate a proline specific endopeptidase polypeptide-containing solution in order to optimize recovery. Use of unconcentrated solutions requires increased incubation time in order 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 may be achieved by any of the techniques discussed herein.

Exemplary methods of enrichment and purification include but are not limited to rotary drum vacuum filtration and/or ultrafiltration.

Preferred Embodiments of the Invention

In accordance with an aspect of the instant invention, an isolated polypeptide is described having proline specific endopeptidase activity having a polypeptide which is at least 70% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or a fragment thereof. More, preferably the polypeptide has at least 80% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or a fragment thereof. Still more preferably, the polypeptide has at least 90% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 or a fragment thereof. In yet more preferred embodiments, the polypeptide has at least 95% sequence identity to one of SEQ ID NO: 4, SEQ ID NO:6 and SEQ ID NO:8 or a fragment thereof. In still more preferred embodiments, the polypeptide has at least 99% sequence identity to one of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

In the most preferred embodiments, the polypeptide is a sequence according to one of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8 or a fragment thereof

In accordance with an aspect of the present invention, a method for the reduction or prevention of haze in a beverage is presented having the step of adding an isolated polypeptide having proline specific endopeptidase as described above to the beverage. Preferably, the beverage contains at least one protein. More preferably, the protein comprises hordein. Still more preferably, the beverage further comprises

polyphenols. Preferably, the beverage has a pH of less than 7.

Preferably, the beverage is a fruit juice. In other preferred embodiments, the beverage is a wine. In yet other preferred embodiments, the beverage is a beer.

Preferably, the isolated polypeptide is added to a mash.

Preferably, the isolated polypeptide is added before haze formation. In other preferred embodiments, the isolated polypeptide is added after haze formation.

In other preferred embodiments, the method of haze reduction has the further step of adding a second isolated polypeptide having proline specific endopeptidase activity as described above wherein the second isolated polypeptide is different than the isolated polypeptide. In still more preferred embodiments, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO:8 or a fragment thereof.

In another aspect of the present invention, a method for forming a protein hydrolysate is presented having the step of adding to a protein substrate an isolated polypeptide having endopeptidase as described above. Preferably, the method includes the further step of adding a protease wherein the protease is different than the isolated polypeptide. More preferably, the protease is a second isolated polypeptide having proline specific endopeptidase activity as described above. Still more preferably, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

In other preferred embodiments, the protease is an exopeptidase. More preferably, the exopeptidase is a tripeptidyl aminopeptidase. Yet more preferably, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 70% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof .

Preferably, the polypeptide has at least 70% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

More preferably, the polypeptide has at least 80% sequence identity to SEQ ID NO: l5, SEQ ID NO: 16, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO: 54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Preferably, the polypeptide has at least 80% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

More preferably, the polypeptide has at least 90% sequence identity to SEQ ID NO: l5, SEQ ID NO: 16, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO: 54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Preferably, the polypeptide has at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Still more preferably, the polypeptide has at least 95% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: l9 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO: 54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Preferably, the polypeptide has at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

Preferably, the polypeptide has at least 99% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 ,

SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 ,

SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35,

SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 ,

SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 ,

SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 ,

SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 ,

SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 ,

SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

Still more preferably, the tripeptidyl aminopeptidase has at least 99% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In still more preferred embodiments, the polypeptide is a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 , SEQ ID NO: 18 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 ,

SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 ,

SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 ,

SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 ,

SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 ,

SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO: 65 , SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

In the most preferred embodiments, the polypeptide is a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17 or a fragment thereof

In yet other preferred embodiments of the present invention, in the method of making a hydrolysate, in addition to the isolated polypeptide having proline specific endopeptidase and the polypeptide having tripeptidyl amino peptidase activity a second isolated polypeptide having proline specific endopeptidase activity as described above is added wherein the second isolated polypeptide is different than the isolated polypeptide having proline specific endopeptidase activity.

More preferably, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

Preferably, the protein substrate is derived from milk. In other preferred embodiments, the protein substrate is derived from wheat.

In another aspect of the present invention, a method for degrading gluten in food is presented having the step of contacting gluten-containing food with an isolated polypeptide having proline specific endopeptidase activity as described above.

Preferably, the food is bread or beer.

In another aspect of the present invention, a method for treating gluten intolerance, celiac disease, dermatitis herpetiformis and/or gluten sensitivity in a patient in need of such treatment, wherein the treatment reduces exposure of said patient to an immunogenic gluten peptide, having the step of orally administering to the patient a therapeutically effective dose of an isolated polypeptide having proline specific endopeptidase activity as described above contemporaneously with the ingestion of a food that may contain gluten.

In another aspect of the present invention, the use is presented of an isolated polypeptide having proline specific endopeptidase activity as described above for the manufacture of a dietary supplement or medicament. Preferably, the isolated polypeptide having proline specific endopeptidase activity as described above digests gluten fragments that are resistant to normal digestive enzymes.

Preferably, the isolated polypeptide having proline specific endopeptidase activity as described above is admixed with food.

Preferably, the isolated polypeptide having proline specific endopeptidase activity as described above is stable to acid conditions.

In another aspect of the present invention, a formulation is presented having the isolated polypeptide having proline specific endopeptidase activity as described above and a pharmaceutically acceptable excipient.

In other aspect of the present invention, an enzyme blend is presented having a proline specific endopeptidase as described above and a protease wherein the proline specific endopeptidase is different than said protease. Preferably, the protease is selected from the group consisting of a serine protease, a cysteine protease, an endopeptidase, and an exopeptidase.

More preferably, the protease is a serine protease. Still more preferably, the serine protease is a subtilisin.

In other preferred embodiments, the protease is an endopeptidase. Preferably the endopeptidase is a second isolated polypeptide having proline specific

endopeptidase activity as described above. More preferably, the isolated polypeptide is a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide is a polypeptide according to SEQ ID NO:8 or a fragment thereof.

In other preferred embodiments, the protease is an exopeptidase. Preferably, the exopeptidase is a tripeptidyl aminopeptidase. More preferably, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 70% sequence identity to SEQ ID NO: l5, SEQ ID NO:l6, SEQ ID NO: l7 , SEQ

ID NO: l8 , SEQ ID NO:l9 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO: 22 , SEQ

ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ

ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID N0:4l , SEQ ID NO:42 , SEQ

ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ

ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID N0:5 l , SEQ ID NO:52 , SEQ

ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ

ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID N0:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof. Still more preferably, the polypeptide has at least 70% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In yet more preferred embodiments, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 80% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ

ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ

ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ

ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ

ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ

ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ

ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ

ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

More preferably, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 80% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In still more preferred embodiments, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 90% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID N0:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID N0:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID N0:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID N0:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

More preferably, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 90% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In yet more preferred embodiments, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 95% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof. More preferably, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having 95% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In still more preferred embodiments, the tripeptidyl aminopeptidase is a polypeptide having tripeptidyl aminopeptidase activity having at least 99% sequence identity to SEQ ID NO: l5, SEQ ID NO: l6, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: 19 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ ID NO:22 , SEQ ID NO:23 , SEQ

ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ ID NO:27 , SEQ ID NO:28 , SEQ

ID NO:29 , SEQ ID NO:30 , SEQ ID NO:31 , SEQ ID NO:32 , SEQ ID NO:33 , SEQ

ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ ID NO:42 , SEQ ID NO:43 , SEQ

ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ ID NO:47 , SEQ ID NO:48 , SEQ

ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5 l , SEQ ID NO: 52 , SEQ ID NO: 53 , SEQ

ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ ID NO:57 , SEQ ID NO:58 , SEQ

ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63, SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

More preferably, the tripeptidyl aminopeptidase has at least 99% sequence identity to SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In still more preferred embodiments, the tripeptidyl aminopeptidase is a polypeptide having a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: l7 , SEQ ID NO: l8 , SEQ ID NO: l9 , SEQ ID NO:20 , SEQ ID NO:2l , SEQ

ID NO:22 , SEQ ID NO:23 , SEQ ID NO:24 , SEQ ID NO:25 , SEQ ID NO:26 , SEQ

ID NO:27 , SEQ ID NO:28 , SEQ ID NO:29 , SEQ ID NO:30 , SEQ ID NO:3 l , SEQ

ID NO:32 , SEQ ID NO:33 , SEQ ID NO:34 , SEQ ID NO:35, SEQ ID NO:36 , SEQ ID NO:37 , SEQ ID NO:38 , SEQ ID NO:39 , SEQ ID NO:40 , SEQ ID NO:4l , SEQ

ID NO:42 , SEQ ID NO:43 , SEQ ID NO:44 , SEQ ID NO:45 , SEQ ID NO:46 , SEQ

ID NO:47 , SEQ ID NO:48 , SEQ ID NO:49 , SEQ ID NO:50 , SEQ ID NO:5l , SEQ

ID NO:52 , SEQ ID NO:53 , SEQ ID NO:54 , SEQ ID NO:55 , SEQ ID NO:56 , SEQ

ID NO:57 , SEQ ID NO:58 , SEQ ID NO:59 , SEQ ID NO:60 , SEQ ID NO:6l, SEQ ID NO:62 , SEQ ID NO:63 , SEQ ID NO:64 , SEQ ID NO:65 , SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69 or a fragment thereof.

In the most preferred embodiments, the tripeptidyl aminopeptidase is a sequence as set forth in SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 or a fragment thereof.

In more preferred embodiments where an enzyme blend has a polypeptide having proline specific endopeptidase activity as described above and a tripeptidyl aminopeptidase as described above, a second isolated polypeptide having proline specific endopeptidase activity as describe above is included in the blend wherein the second isolated polypeptide is different than the isolated polypeptide having proline specific endopeptidase activity.

According to this aspect of the present invention, the isolated polypeptide is preferably a polypeptide according to SEQ ID NO:4 or a fragment thereof and the second isolated polypeptide having proline specific endopeptidase activity is a polypeptide according to SEQ ID NO: 8 or a fragment thereof.

In another aspect of the present invention, a polynucleotide is presented having a nucleic acid sequence encoding the isolated polypeptide having proline specific endopeptidase activity as described above.

In another aspect of the present invention, a recombinant expression vector is presented having the polynucleotide.

In another aspect of the present invention, a host cell is presented having the recombinant expression vector.

The present disclosure is described in further detail in the following examples, which are not in any way intended to limit the scope of the disclosure as claimed.

The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.

EXAMPLE 1

Cloning of MorProl, AflPro3 and CpoProl Three fungal strains ( Magnaporthe oryzae 70-15, Aspergillus flavus and

Coccidioides posadasii str.C735 delta SOWgp) were selected as potential sources of enzymes which may be useful in various industrial applications. A BLAST search (Altschul et al., J Mol Biol, 215: 403-410, 1990) led to the identification of three genes that encode proteins with homology to a fungal protease: MorProl from Magnaporthe oryzae 70-15, AflPro3 from Aspergillus flavus and CpoProl from Coccidioides posadasii str.C735 delta SOWgp.

The nucleic acid sequence of full-length MorProl gene, as identified from NCBI database (NCBI Reference Sequence: NC_0l785l. l from 2214046 to 2215835; complement), is provided in SEQ ID NO: 3. The corresponding full-length protein encoded by the MorProl gene is shown in SEQ ID NO: 4 (NCBI Reference

Sequence: XP 003716615.1). The nucleic acid sequence of full-length AflPro3 gene, as identified from Broad Institute database (Broad Institute database Reference Sequence: AFL2G 02145), is provided in SEQ ID NO: 5. The corresponding full- length protein encoded by the AflPro3 gene is shown in SEQ ID NO: 6 (NCBI Reference Sequence: XP 002374452.1). The nucleic acid sequence of full-length CpoProl gene, as identified from NCBI database (NCBI Reference Sequence:

NW_003316003.1 from 2687540 to 2689312; complement), is provided in SEQ ID NO: 7. The corresponding full-length protein encoded by the CpoProl gene is shown in SEQ ID NO: 8 (NCBI Reference Sequence: XP 003069863.1).

MorProl, AflPro3 and CpoProl have an N-terminal signal peptide as predicted by SignalP version 4.0 (Nordahl Petersen et al. (2011) Nature Methods, 8:785-786), suggestion that they are all secreted enzymes. The corresponding, predicted, mature enzyme sequence for MorProl, AflPro3 or CpoProl is provided in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, respectively.

EXAMPLE 2

Expression of MorProl, AflPro3 and CpoProl

The DNA sequence encoding full-length MorProl (SEQ ID NO: 4), AflPro3 (SEQ ID NO: 6) or CpoProl (SEQ ID NO: 8) was chemically synthesized and inserted into the Trichoderma reesei expression vector pTrex3gM (described in U.S. Published Application 2011/0136197 Al) by Generay (Shanghai, China). The synthesized nucleotide sequences for full-length MorProl, AflPro3 and CpoProl are set forth as SEQ ID NO: 12, 13 and 14, respectively. The pTrex3gM expression vector contained the T reesei cbhl-derived promoter ( cbhl) and cbhl terminator regions allowing for a strong inducible expression of the gene of interest. The A. nidulans amdS selective marker confer growth of transformants on acetamide as a sole nitrogen source.

The resulting plasmids were labeled pGX256(Trex3gM-MorProl),

pGX256(Trex3gM-AflPro3) or pGX256(Trex3gM-CpoProl), respectively. The plasmid map of pGX256(Trex3gM-MorProl) is provided in Figure 1 and the other two plasmids have similar composition except for the inserted gene encoding each fungal protease.

Each individual expression plasmid was then transformed into a quad deleted Trichoderma reesei strain (described in WO 05/001036) using biolistic method (Te'o VS et al., J Microbiol Methods, 51 :393-9, 2002). Transformants were selected on a medium containing acetamide as a sole source of nitrogen (acetamide 0.6 g/L; cesium chloride 1.68 g/L; glucose 20 g/L; potassium dihydrogen phosphate 15 g/L;

magnesium sulfate heptahydrate 0.6 g/L; calcium chloride dihydrate 0.6 g/L; iron (II) sulfate 5 mg/L; zinc sulfate 1.4 mg/L; cobalt (II) chloride 1 mg/L; manganese (II) sulfate 1.6 mg/L; agar 20 g/L; pH 4.25). Transformed colonies (about 50-100) appeared in about 1 week. After growth on acetamide plates, transformants were picked and transferred individually to acetamide agar plates. After 5 days of growth on acetamide plates, transformants displaying stable morphology were inoculated into 200 pL Glucose/Sophorose defined media in 96-well microtiter plates. The microtiter plate was incubated in an oxygen growth chamber at 28°C for 5 days. Supernatants from these cultures were used to confirm the protein expression by SDS-PAGE analysis. The stable strain with the highest protein expression was selected and subjected to fermentation in a 250 mL shake flask with Glucose/Sophorose defined media. To purify MorProl, AflPro3 and CpoProl, the crude broth from the shake flask was concentrated using a VivaFlow 200 ultra-filtration device (Sartorius Stedium). Ammonium sulfate was then added to the concentrated solution to a final

concentration of 1 M. After filtering, the resulting soluble fraction was applied to a 60 mL Phenyl-FF Sepharose column pre-equilibrated with the loading buffer containing 20 mM Tris-HCl (pH 8.0) and 1 M ammonium sulfate. The corresponding active fractions were pooled, concentrated and subsequently loaded onto a Superdex 75 gel filtration column pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.0) supplemented with additional 0.15 M NaCl and 10% glycerol. The resulting active protein fractions were then pooled and concentrated via the 10K Amicon Ultra devices, and stored in 40% glycerol at -20 °C until usage.

EXAMPLE 3

Proteolytic activity of MorProl, AflPro3 and CpoProl

The proteolytic activity of purified MorProl or CpoProl was measured in 25 mM citrate/phosphate buffer (pH 5), using Succinyl-Ala-Ala-Ala-Pro-paranitroanilide (Suc-AAAP-pNA) (GL Biochem, Shanghai) as the substrate. Prior to the reaction, the enzyme was diluted with water to specific concentrations. The Suc-AAAP-pNA substrate was dissolved in 100% Dimethylsulfoxide (DMSO) to a final concentration of 10 mM. To initiate the reaction, 5 mΐ of substrate was mixed with 85 pL of citrate/phosphate buffer in a non-binding 96-well Microtiter Plate (96-MTP) (Coming Life Sciences, #3641), and after 5 min pre-incubation at 37 °C in a Thermomixer (Eppendorf), 10 mΐ of properly diluted purified enzyme (or water as the blank) was added. After sealing the 96-MTP, the reaction was carried out in a Thermomixer at 37 °C and 600 rpm for 10 min, and the absorbance of the resulting solution was measured at 410 nm (A ₄ IO) using a SpectraMax 190. Reaction rate was subsequently calculated (Reaction rate = 6A ₄ IO / 10 (min) *1000, where 6A ₄ IO is the increase of A ₄ IO reading within the 10 min incubation time) and plotted against different protein concentrations (from 1.25 ppm to 80 ppm) to demonstrate the proteolytic activity (Figure 2). Each value was the mean of triplicate assays, with variance less than 5%. The proteolytic assay with Suc-AAAP-pNA as the substrate (shown in Figure 2) indicates that MorProl and CpoProl are active proteases.

The proteolytic activity of purified AflPro3 was measured in 25 mM

citrate/phosphate buffer (pH 5), using Benzylcarboxy-Glycine-Proline- paranitroanilide (Z-GP-pNA) (Invitrogen, Cat. No. 254295) as the substrate. Prior to the reaction, the enzyme was diluted with water to specific concentrations. The Z-GP- pNA substrate was dissolved in 100% Dimethylsulfoxide (DMSO) to a final concentration of 10 mM. To initiate the reaction, 5 pL of substrate was mixed with 85 pL of citrate/phosphate buffer in a non-binding 96-well Microtiter Plate (96-MTP) (Coming Life Sciences, #3641), and after 5 min pre-incubation at 37 °C in a

Thermomixer (Eppendorf), 10 pl of properly diluted purified enzyme (or water as the blank) was added. After sealing the 96-MTP, the reaction was carried out in a

Thermomixer at 37 °C and 600 rpm for 30 min, and the absorbance of the resulting solution was measured at 410 nm (A410) using a SpectraMax 190. Reaction rate was subsequently calculated (Reaction rate = 5A410 / 10 (min) *1000, where 5A410 is the increase of A410 reading within the 10 min incubation time) and plotted against different protein concentrations (from 1.25 ppm to 80 ppm) to demonstrate the proteolytic activity (Figure 2). Each value was the mean of triplicate assays, with variance less than 5%. The proteolytic assay with Z-GP-pNA as the substrate (shown in Figure 2) indicates that AflPro3 is an active protease.

EXAMPLE 4

pH profile of MorProl, AflPro3 and CpoProl

With Suc-AAAP-pNA as the substrate for MorProl and CpoProl, and Z-GP- pNA for AflPro3, the pH profiles of three purified fungal proteases were studied in 16.7 mM citrate/phosphate/CHES buffer with different pH values ranging from 3 to 9. To initiate the assay, 85 pl of citrate/phosphate/CHES buffer with a specific pH was first mixed with 5 mΐ of 10 mM specific substrate in a 96-MTP and pre-incubated at 37 °C for 5 min, followed by the addition of 10 mΐ of each water diluted enzyme (100 ppm) (or water alone as the blank control). The reaction was performed and analyzed as described in Example 3. Enzyme activity as each pH was reported as the relatively activity, where the activity at the optimal pH was set to be 100%. The pH vales tested were 3, 4, 5, 6, 7, 8, and 9. Each value was the mean of triplicate assays, with variance less than 5%. As shown in Figure 3, the optimal pH of MorProl, AflPro3 and

CpoProl is 5, 5 and 4, respectively.

EXAMPLE 5

Temperature profile of MorProl, AflPro3 and CpoProl

The temperature profiles of three purified fungal proteases were analyzed in 25 mM citrate/phosphate buffer (pH 5) using Suc-AAAP-pNA as the substrate for MorProl and CpoProl, and Z-GP-pNA for AflPro3. The enzyme sample and pNA substrate were prepared as in Example 3. Prior to the reaction, 85 Dl of

citrate/phosphate buffer and 5 mΐ of 10 mM pNA substrate were mixed in a 200 Dl PCR tube, which was then incubated in a Peltier Thermal Cycler (BioRad) at desired temperatures (i.e. 20-70 °C) for 5 min. After the incubation, 10 Dl of each water diluted enzyme (100 ppm) (or water alone as the blank control) was added to the solution, and the reaction was carried out in the Peltier Thermal Cycle for 10 min at different temperatures. Subsequent absorbance measurements were performed as described in Example 3. The activity was reported as the relative activity, where the activity at the optimal temperature was set to be 100%. The tested temperatures are 20, 30, 40, 50, 60, and 70°C. Each value was the mean of triplicate assays (the value varies no more than 5%). The data in Figure 4 suggest that MorProl, AflPro3 and CpoProl showed an optimal temperature at 40 °C, 30 °C and 30 °C, respectively. EXAMPLE 6

Thermostability of MorProl, AflPro3 and CpoProl

The thermostability analyses of three purified fungal proteases were performed using 50 mM acetate/phosphate buffer (pH 4.5) supplemented with additional 5% (w/w) ethanol as the incubation buffer. For remaining activity measurement, Suc- AAAP-pNA was applied as the substrate for MorProl and CpoProl, while Z-GP-pNA was applied for AflPro3. The purified enzyme was diluted in 1 mL incubation buffer to a final concentration of 1 mg/mL and subsequently incubated at 60 °C for 0, 10, 20, 30, 60 or 90 min. At the end of each incubation period, 100 pL of the enzyme-buffer mixture was transferred to a 96-MTP placed on ice. After the completion of the entire incubation, activity was measured as in Example 3. The activity was reported as the relative activity, where the activity at 0 min incubation time was set to be 100%. Each value was the mean of duplicate assays with variance less than 5%. The result in Figure 5 shows that after 20 min incubation at 60 °C, MorProl, AflPro3 and CpoProl lost 67%, 100% and 60% of its activity, respective. And after 1 hr incubation, all three proteases lost 100% of its activity.

EXAMPLE 7

Haze reduction performance of MorProl, AflPro3 and CpoProl

The haze reduction performances of three purified fungal enzymes were evaluated using the gliadin-catechin assay. Prior to the reaction, each enzyme was diluted with water to specific concentrations. And Brewers Clarex® was used as the benchmark. The gliadin substrate (Sigma, Cat. No. G3375) was dissolved in 20 mM acetate/phosphate buffer (pH 4.5) supplemented with additional 0.2% ethanol to a final concentration of 2 mg/mL and the catechin substrate (Sigma, Cat. No. C1251) was dissolved in 20 mM citrate/phosphate buffer (pH 4.5) supplemented with additional 0.2% ethanol to a final concentration of 2 mg/mL. To initiate the assay, 100 pL of gliadin solution was mixed with 5 pL of properly diluted enzyme in a 96-MTP; and after 90 min incubation at 45 °C in a Thermomixer, the resulting 96-MTP was then placed on ice for 5 min, followed by the addition of 100 pl catechin solution. Haze was developed at room temperature for 30 min. The absorbance of the developed haze at 600 nm (A ₆ oo) was measured using a SpectraMax 190 and subsequently plotted against different enzyme concentrations (from 0 to 80 ppm).

Each value was the mean of triplicate assays with variance less than 1%. The data in Figure 6, 7 and 8 indicate that MorProl, AflPro3 and CpoProl can significantly reduce the gliadin-catechin haze and all of them are more efficient than the benchmark.

EXAMPLE 8

The performances of MorProl, AflPro3 and CpoProl in degrading the immunogenic gliadin 26-mer and 33-mer peptides The 26-mer (SEQ ID NO: 1) and 33-mer peptide (SEQ ID NO: 2) test peptides were synthesized by GL Biochem (Shanghai, China).

Prior to the reaction, each purified protease was diluted with water to specific concentrations (20 ppm, 10 ppm or 5 ppm); and each peptide was dissolved in 25 mM Sodium acetate buffer (pH 4.5) to a final concentration of 1 mg/mL. The reaction was initiated by mixing 90 pL of peptide solution with lOpL of diluted enzyme in a 200 pL PCR tube; and thus the final concentration of the enzyme used in the assay was 2 ppm, 1 ppm or 0.5 ppm. The water diluted Brewers Clarex ^® or water alone was applied as the Benchmark or blank control, respectively. After 10 min incubation at 40 °C in a Peltier Thermal Cycler (BioRad), the reaction was terminated by heating at 90 °C for 10 min; and 150 pL of the reaction mixture was then filtered via a 96-well 0.22 pm filtration plate (Corning Life Sciences, #3505).

5 pL of the resulting filtrate was subjected to the HPLC analyses using an Agilent 1260 Series HPLC system equipped with a VWD detector. The reaction product was chromatographed on a C18 column (ZORBAX 300SB-C18, 5 pm, 4.6 X 150 mm, Agilent) at a flow rate of 1 mL min ¹ using a gradient from 90% solution A (100% water with 0.1% TFA(v/v))/l0% solution B (100% acetonitrile with 0.1% TFA (v/v)) to 60% solution A/40% solution B over 16 min. The substrate peptides are detected by their UV absorbance at 2l0nm; and the HPLC retention time for 26-mer or 33-mer is 9.4 min or 13.7 min, respectively. The residual amount of the substrate peptide after enzyme treatment was calculated by comparing its peak area with that of the blank control. The results were summarized in Table 1 and each value was the mean of triplicate assays, with variance less than 5%. As shown in Table 1, after 1 ppm enzyme treatment, the residual amount of 26-mer peptide for MorProl, AflPro3, CpoProl or the Benchmark is 0.07 mg/mL, 0.17 mg/mL, 0.01 mg/mL or 0.46 mg/mL, respectively; while the residue amount of 33-mer peptide for MorProl, AflPro3, CpoProl or the Benchmark is 0.26 mg/mL, 0.01 mg/mL, 0.02 mg/mL or 0.39 mg/mL, respectively. The data suggest that MorProl, AflPro3 and CpoProl are efficient in both peptide degradation.

Table 1. Residual amount of 26-mer or 33-mer peptide after protease treatment

EXAMPLE 9

The performances of MorProl, AflPro3 and CpoProl in reducing the immunogenicity of 26-mer and 33-mer peptides

26-mer immunogenicitv assay: Prior to the reaction, each purified protease was diluted with water to a final concentration of 5 ppm; and the 26-mer peptide was dissolved in 25 mM Sodium acetate buffer (pH 4.5) to a final concentration of 1 mg/mL. The reaction was initiated by mixing 45 pL of peptide solution with 5 pL of diluted enzyme in a 200 pL PCR tube. The water diluted Brewers Clarex ^® or water alone was applied as the Benchmark or blank control, respectively. After 10 min incubation at 40 °C in a Peltier Thermal Cycler (BioRad), the reaction was terminated by heating at 90 °C for 10 min; and the resulting mixture was subjected to the competitive enzyme-linked immunosorbent assays (ELISA) using the

RIDASCREEN ^® Gliadin competitive kit (R-Biopharm, Germany). Following the standard manufacturer’s instruction, the absorbance for each specific enzyme assay sample was measured at 450 nm (A ₄ so) using a SpectraMax 190. The relative A ₄ so was then calculated by dividing the A ₄₅ o of the enzyme assay sample by that of the blank control. And the data were subsequently applied to measure the corresponding residual immunogenicity (%) using the standard curve constructed from different concentrations (from 0.0625 mg/mL to 1 mg/mL) of the 26-mer peptide. The results were summarized in Table 2 and each value was the mean of triplicate assays. As shown in Table 2, after enzyme treatment, the residual immunogenicity for MorProl, AflPro3, CpoProl or Benchmark is 61.4%, 68.5%, 38.5% or 99.6%, respectively; indicating that MorProl, AflPro3 and CpoProl are effective in reducing the immunogenicity of the 26-mer peptide.

33-mer immunogenicity assay: Prior to the reaction, each purified protease was diluted with water to a final concentration of 10 ppm; and the 33-mer peptide was dissolved in 25 mM Sodium acetate buffer (pH 4.5) to a final concentration of 2 mg/mL. The reaction was initiated by mixing 45 pL of peptide solution with 5 pL of diluted enzyme in a 200 pL PCR tube. The water diluted Brewers Clarex ^® or water alone was applied as the Benchmark or blank control, respectively. After 20 min incubation at 40 °C in a Peltier Thermal Cycler (BioRad), the reaction was terminated by heating at 90 °C for 10 min; and the resulting mixture was subjected to the competitive ELISA assays using the RIDASCREEN ^® Gliadin competitive kit (R- Biopharm, Germany). Following the standard manufacturer’s instruction, the absorbance for each specific enzyme assay sample was measured at 450 nm (A ₄ so) using a SpectraMax 190. The relative A ₄ so was then calculated by dividing the A ₄₅ o of the enzyme assay sample by that of the blank control. And the data were subsequently applied to measure the corresponding residual immunogenicity (%) using the standard curve constructed from different concentrations (from 0.25 mg/mL to 2 mg/mL) of the 33-mer peptide. The results were summarized in Table 2 and each value was the mean of sextuplicate assays. As shown in Table 2, after enzyme treatment, the residual immunogenicity for MorProl, AflPro3, CpoProl or Benchmark is 57.5%, 44.6%, 16.9% or 73.7%, respectively; indicating that MorProl, AflPro3 and CpoProl are effective in reducing the immunogenicity of the 33-mer peptide.

Table 2. Residual immunogenicity of 26-mer and 33-mer peptide after protease treatment

EXAMPLE 10

The performances of MorProl, AflPro3 and CpoProl in reducing the

immunogenicity of wheat gliadins

Prior to the reaction, each purified protease was diluted with water to a final concentration of 10 ppm; and the wheat gliadin (Sigma, Cat. No. G3375) was dissolved in 20 mM citrate/phosphate buffer (pH 4.5) to a final concentration of 25 pg/mL. The reaction was initiated by mixing 45 pL of gliadin solution with 5 pL of diluted enzyme in a 200 pL PCR tube. The water diluted Brewers Clarex ^® or water alone was applied as the Benchmark or blank control, respectively. After 20 min incubation at 40 °C in a Peltier Thermal Cycler (BioRad), the reaction was terminated by heating at 90 °C for 15 min; and the resulting mixture was subjected to the competitive ELISA assays using the RIDASCREEN ^® Gliadin competitive kit (R- Biopharm, Germany). Following the standard manufacturer’s instruction, the absorbance for each specific enzyme assay sample was measured at 450 nm (A ₄ so) using a SpectraMax 190. The relative A ₄ so was then calculated by dividing the A ₄₅ o of the enzyme assay sample by that of the blank control. And the data were subsequently applied to measure the corresponding residual immunogenicity (%) using the standard curve constructed from different concentrations (from 1.5625 pg/mL to 25 pg/mL) of the wheat gliadin. The results were summarized in Table 3 and each value was the mean of sextuplicate assays. As shown in Table 3, after enzyme treatment, the residual immunogenicity for MorProl, AflPro3, CpoProl or Benchmark is 18.7%, 88.3%, 53.6% or 59.7%, respectively; indicating that all three proteases are capable of reducing the wheat gliadin immunogenicity.

Table 2. Residual immunogenicity of wheat gliadin after protease treatment

EXAMPLE 11

Chill haze reduction performance of AflPro3, CpoProl and MorProl in test tubes

The haze reduction performance of AflPro3, CpoProl and MorProl was evaluated in a Pilsener beer (from Research Brewery St. Johann brewed on 100% Pilsener malt (Fuglsang, Denmark; batch 19.03.2015.) brewed without the use of fining agents). The enzyme samples were added to 8 ml beer in 10 ml glass tubes (0.5, 2.5 and 5 ppm final enzyme protein concentrations and 0 ppm as blank control) for evaluation of haze effects. The tubes were kept at 20°C in the dark. Right after enzyme addition (day 0), after 24 hours (day 1) and on day 4 tubes were chilled for 1 hour on ice and measured according to the standard method EBC 9.29 (Analytica- EBC (1997): Method 9.29, Haze in beer: calibration of haze meters) using a

Turbimeter, Hach 2100AN. The data from haze measurements shown in Table 11.1 indicates that AflPro3, CpoProl and MorProl can reduce haze strongly. The lowest dosage (0.5 ppm) of AflPro3, CpoProl and MorProl reduces haze over time from day 1 to day 4, whereas when dosed at 2.5 and 5 ppm the enzymes give the full haze removal or a very marked effect already on day 1.

Table 11.1 Haze reduction in test tubes

EXAMPLE 12

Haze reduction performance of AflPro3, CpoProl and MorProl in beer bottles

Furthermore, the haze reduction performance of AflPro3, CpoProl and MorProl was evaluated in a Pilsener beer (from Research Brewery St. Johann brewed on 100% Pilsener malt (Fuglsang, Denmark; batch 19.03.2015.) and brewed without the use of fining agents). The enzyme samples (at 2.5 ppm enzyme protein concentrations together with 0 ppm as blank control) were added to 330 ml beer bottles, which were kept for 5 days at 20°C in the dark. Chill haze was measured after 24 h at 0°C, whereas accelerated haze was measured after an incubation schedule of 24 h at 0°C, 48 h at 60°C and 24 h at 0°C. Haze development within the bottles was measured at 0°C using a SIGRIST Lab Scat 2, instrument (from SIGRIST-PHOTOMETER AG, Ennetbiirgen, Switzerland) with a 25° or 90° angle in EBC units as described by method EBC 9.29 (Analytica-EBC (1997): Method 9.29, Haze in beer: calibration of haze meters). With all enzyme candidates strong reductions in chill haze as well as accelerated haze were measured at 25° and 90° when compared to haze in the control (Table 12.1). This indicates that the candidates are highly effective in haze reduction in beer.

Table 12.1 : Haze in Pilsener beer treated without or with 2.5 ppm AflProl, AflPro3, TreProl, CpoProl and MorProl (measured in EBC units)

EXAMPLE 13

Thermoinactivation of AflPro3 and CpoProl during beer pasteurization

In order to evaluate the inactivation of AflPro3 and CpoProl during beer pasteurisation the enzymes were incubated in Heineken beer with and without a heat treatment for 25 seconds at 75°C. The enzyme activity before and after the heat treatment was measured in an fluorometric assay using Z-Gly-Pro-AMC. As a control papain was also tested and measured before and after heat treatment with Z-Phe-Arg- AMC substrate.

The substrate used were Z-Gly-Pro-AMC (1-1145; BACHEM) or for papain Z-Phe- Arg-AMC (11160; BACHEM). A 10 mM substrate stock solution in DMSO was prepared. A 0.1 mM working substrate solution was prepared by adding 5 ul of substrate stock solution to 495 ul of buffer (0.1 M Mcllvain buffer, pH 5.0). For the assay 50 mΐ buffer and 25 mΐ 0,1 mM working substrate solution and 25 mΐ of enzyme sample diluted in buffer was used.

96 well plates (no. 265301; Thermo Scientific) were used for the assay and incubated at 37°C and are read over time in a SpectraMax Gemini Microplate

Spectrofluorometer using excitation at 355 nm and emission at 460 nm with a cutoff at 455 nm.

Table 13.1. Residual activity after pasteurization in beer measured with the substrates indicated

As seen in Table 13.1 AflPro3 shows only 8% and CpoProl no residual activity in contrast to papain which has 87% remaining activity after heat treatment. This indicates that AflPro3 and CpoProl will be highly or totally inactivated during beer pasteurization in contrast to papain.

EXAMPLE 14

Foam stability of beer treated with AflPro3, CpoProl and MorProl

The effect of AflPro3, CpoProl and MorProl on foam stability was evaluated in a Pilsener beer (from Research Brewery St. Johann brewed on 100% Pilsener malt (Fuglsang, Denmark; batch 19.03.2015.) and brewed without the use of fining agents). The enzyme samples (at 2.5 ppm enzyme protein concentrations together with 0 ppm as blank control) were added to 330 ml beer bottles, which were kept for 5 days at 20°C in the dark. Foam stability was measured using a NIBEM-T Meter according to procedure EBC 9.42 (European Brewing Convention Analytica- EBC section 9 Beer, Method 9.42 Foam Stability of beer using the NIBEM-T Meter). The beer is applied with carbon dioxide gas at a pressure of 2 bar and immediately the foam collapse time (FCT) is measured for 10, 20 and 30 mm reduction of foam. As shown in Table 14.1 treatment of beer with AflPro3, CpoProl and MorProl does not reduce FCT compared to control, on the contrary FCT is increased, meaning that foam stability is improved.

Table 14.1. Foam collapse time (FCT) in beer treated MorProl, AflPro3 and CpoProl

Although the foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, certain changes and modifications can be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference. To the extent the content of any citation, including website or accession number may change with time, the version in effect at the filing date of this application is meant. Unless otherwise apparent from the context any step, element, aspect, feature of embodiment can be used in combination with any other.