New Proteases Able to Hydrolyze Gluten Peptides and Proteins at Acidic PH, from the Actinomycete Actinoallomurus

Abstract

The invention relates to a new family of proteolytic enzymes having the ability to hydrolize at a p H between 3 and 8 gluten olygopeptides which are resistant to cleavage by gastric and pancreatic enzymes and whose presence in the intestinal lumen results in toxic effects. The enzymes have been identified as endopeptidases of the S8/S53 family and are produced by an Actinoallomurus strain. The object of the invention includes also methods for producing enzymes composition comprising the endopeptidases by cultivation of native Actinoallomurus strains, mutants thereof, or recombinant host cells comprising nucleic acids codifying for the endopeptidases. Said nucleic acids constitute a further object of the invention. The enzyme compositions comprising at least one endopeptidase of the invention are useful for the treatment and/or prevention of celiac sprue, dermatitis herpetiformis and any other disorder associated with gluten intolerance as ingredients of pharmaceutical formulations or as additives of foods and drinks.

1 . An enzyme composition comprising at least one endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from the group consisting of:
 a) endopep-140 comprising SEQ ID NO: 1, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity,   b) endopep-40 comprising SEQ ID NO: 2, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity,   c) endopep-120 comprising SEQ ID NO: 3, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity,   d) endopep-60 comprising SEQ ID NO: 4, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, and   e) endopep-41 comprising SEQ ID NO: 5, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity.   
     
     
         2 . An enzyme composition of  claim 1  wherein the endopeptidase(s) is/are selected from the group consisting of:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity. 
 
     
     
         3 . An enzyme composition as in  claim 1  wherein the endopeptidase is selected from the group consisting of:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, and 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity. 
 
     
     
         4 . An enzyme composition as in  claim 1  wherein the endopeptidase(s) is/are obtainable from an  Actinoallomurus  strain. 
     
     
         5 . An enzyme composition as in  claim 4  wherein the endopeptidase(s) is/are obtainable from  Actinoallomurus  sp. DSM24988. 
     
     
         6 . An enzyme composition as in  claim 1  which is able to hydrolyze gluten oligopeptides which are resistant to cleavage by gastric and pancreatic enzymes and whose presence in the intestinal lumen results in toxic effects. 
     
     
         7 . An enzyme composition as in  claim 1  which comprises one or more other proteolytic enzymes selected from prolyl-endoproteases, x-prolyl-dipeptidyl aminopeptidases and prolyl-aminopeptidases. 
     
     
         8 . An enzyme composition as in  claim 1  wherein the endopeptidase(s) is/are operatively fused to another polypeptide to form chimeric or tapped protein(s). 
     
     
         9 . An isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity,   b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity,   c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity,   d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and   e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity.   
     
     
         10 . An isolated endopeptidase of the S8/S53 family of  claim 9  which is selected from
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, and 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity. 
 
     
     
         11 . An enzyme composition as in  claim 1  or an isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity for use as a medicament for the treatment or prevention of the celiac sprue, dermatitis herpetiformis and/or any other disorder associated with gluten intolerance. 
 
     
     
         12 . A method for producing protein hydrolyzates used for food and drinks, said method comprising contacting gluten with an enzyme composition as in  claim 1  or at least one with one isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity. 
 
     
     
         13 . A pharmaceutical formulation which comprises as the active proteolytic ingredient an enzyme composition as in  claim 1  or at least one isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity. 
 
     
     
         14 . A pharmaceutical formulation of  claim 13  which is an oral pharmaceutical formulation. 
     
     
         15 . A food supplement which comprises as active proteolytic ingredient an enzyme composition as in  claim 1  or at least one isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity. 
 
     
     
         16 . An isolated nucleic acid encoding for at least one endopeptidase of  claim 1  which comprises at least one polynucleotide sequence selected from SEQ ID NOs: 7, 8, 9, 10 and 11, or at least one polynucleotide sequence having at least 50%, 60%, 70%, 80%, 90% or 95% of identity to any one of SEQ ID NOs: 7, 8, 9, 10 and 11. 
     
     
         17 . An isolated nucleic acid as in  claim 16  which comprises at least one polynucleotide sequence having at least 95% of identity to any one of SEQ ID NOs: 7, 8, 9, 10 and 11. 
     
     
         18 . An isolated nucleic acid as in  claim 16  which comprises at least one polynucleotide sequence selected from SEQ ID NOs: 7 and 8. 
     
     
         19 . A process for producing the enzyme composition of  claim 1  which comprises:
 A) cultivating a naturally occurring  Actinoallomurus  strain capable of producing at least one endopeptidase of the S8/S53 family in a culture medium under conditions suitable for producing the enzyme composition and recovering the enzyme composition from the cultivation batch, or 
 B) cultivating an  Actinoallomurus  strain derived by conventional mutation and/or selection procedures from a naturally occurring strain as defined under A), which maintains the capability of producing at least one endopeptidase of the S8/S53 family, in a culture medium under conditions suitable for producing the enzyme composition and recovering the enzyme composition from the cultivation batch, or 
 C) introducing into a host cell a nucleic acid encoding for at least one endopeptidase of the S8/S53 family selected from the group consisting of: 
 a) endopep-140 comprising SEQ ID NO: 1, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 cultivating the cell in a culture medium under conditions suitable for producing the endopeptidase(s) and recovering the endopeptidase(s) from the cultivation batch. 
 
     
     
         20 . A process as in  claim 19 , paragraph A) or B), wherein the  Actinoallomurus  strain is replaced by a strain belonging to the genus  Catenulispora, Ktedonobacter or Streptomyces  capable of producing at least one endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from the group consisting of:
 a) endopep-140 comprising SEQ ID NO: 1, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity,   b) endopep-40 comprising SEQ ID NO: 2, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity,   c) endopep-120 comprising SEQ ID NO: 3, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity,   d) endopep-60 comprising SEQ ID NO: 4, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, and   e) endopep-41 comprising SEQ ID NO: 5, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity.   
     
     
         21 . A process as in  claim 19  wherein the  Actinoallomurus  strain is  Actinoallomurus  sp. DSM 24988. 
     
     
         22 . A process as in  claim 19 , paragraph C), wherein the nucleic acid comprises at least one of the polynucleotide sequences selected from SEQ ID NOs: 7, 8, 9, 10 and 11, or at least one polynucleotide sequence having at least 50%, 60%, 70%, 80%, 90% or 95% of identity to any one of SEQ ID NOs: 7, 8, 9, 10 and 11. 
     
     
         23 . A process as in  claim 22 , wherein the nucleic acid comprises at least one of the polynucleotide sequences SEQ ID NOs: 7 and 8 or at least one of the polynucleotide sequence having at least 95% identity to any one of SEQ ID NOs: 7 and 8. 
     
     
         24 . A process as in  claim 19 , paragraph C), 22 and 23, wherein the host cell is a microorganism selected from  Bacillus, Streptomyces, Lactobacillus, Pyrococcus, Pseudomonas  and  Escherichia coli.    
     
     
         25 . A process as in  claim 24  wherein the host cell is  Escherichia coli BL21(DE3)Star. 
     
     
         26 . The strain  Actinoallomurus  DSM 24988. 
     
     
         27 . A method of degrading gluten oligopeptides which are resistant to cleavage by gastric and pancreatic enzymes and whose presence in the internal lumen results in toxic effects which comprises contacting said gluten oligopeptides with an enzyme composition as in  claim 1  or at least one isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from:
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity. 
 
     
     
         28 . A method as in  claim 27  for the treatment or prevention of celiac sprue, dermatitis herpetiformis and/or any other disorder associated with gluten intolerance which comprises administering to a patient in need thereof an effective amount of an enzyme composition comprising at least one endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from the group consisting of:
 a) endopep-140 comprising SEQ ID NO: 1, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 50%, 60%, 70%, 80%, 90%, or 95% of identity or at least one isolated endopeptidase of the S8/S53 family active at pH between 3 and 8 selected from: 
 a) endopep-140 comprising SEQ ID NO: 1 or a sequence having at least 95% of identity, 
 b) endopep-40 comprising SEQ ID NO: 2 or a sequence having at least 95% of identity, 
 c) endopep-120 comprising SEQ ID NO: 3 or a sequence having at least 95% of identity, 
 d) endopep-60 comprising SEQ ID NO: 4 or a sequence having at least 95% of identity, and 
 e) endopep-41 comprising SEQ ID NO: 5 or a sequence having at least 95% of identity. 
 
     
     
         29 . A method as in  claim 28  wherein the enzyme composition or the isolated endopeptidase is incorporated into a pharmaceutical formulation, a food supplement, a drink or beverage. 
     
     
         30 . A method as in  claim 27 , wherein the enzyme composition or the isolated endopeptidase is used in the manufacture of food supplements. 
     
     
         31 . A method as in  claim 30 , wherein the enzyme composition or the isolated endopeptidase is used in immobilized form. 
     
     
         32 . A method as in  claim 31 , wherein the enzyme composition or the isolated endopeptidase is used for the treatment of liquid food products.