Method for the production of an enzymatic composition comprising a recombinant endopeptidase

A method of treating and/or preventing allergy or intolerance to nuts and/or peanuts to a subject in need thereof, said method comprising:

• administering to said subject an effective amount of an enzyme preparation comprising at least one recombinant Actinoallomurus endopeptidase having glutenase activity or an effective amount of an isolated recombinant Actinoallomurus endopeptidase having glutenase activity, wherein the at least one recombinant Actinoallomurus endopeptidase or the isolated Actinoallomurus endopeptidase is selected from the group consisting of:
• endopeptidase 40 (E40) comprising SEQ ID NO:
• 1;
• a biologically active fragment of E40;
• a naturally occurring allelic variant of E40;
• and an endopeptidase having at least 60%, 70%, 80%, 90% or 95% of identity to SEQ ID NO:
• 1;
• or
• administering to said subject an effective amount of a formulation comprising said enzyme preparation or said isolated recombinant Actinoallomurus endopeptidase,
• the formulation being a food or a food supplement or a pharmaceutical formulation, or a flour that has been put in contact with the enzyme preparation or with the isolated recombinant Actinoallomurus endopeptidase;
• or administering to said subject an effective amount of an enzyme preparation prepared by a method comprising:
• a) culturing a recombinant Streptomyces lividans host cell in a culture medium under fermentation conditions, said recombinant host cell comprising a recombinant expression vector for heterologous expression of at least one recombinant Actinoallomurus endopeptidase, the recombinant expression vector comprising a polynucleotide encoding for said at least one recombinant Actinoallomurus endopeptidase operably linked to regulatory sequences capable of directing the expression of said at least one recombinant Actinoallomurus endopeptidase in the recombinant host cell;
• wherein the culture medium comprises at least 30% (wt/vol) sucrose;
• b) recovering a supernatant of the culture medium comprising the at least one recombinant Actinoallomurus endopeptidase;
• and
• c) purifying from said supernatant the enzyme preparation comprising the at least one recombinant Actinoallomurus endopeptidase;
• or
• administering to said subject an effective amount of an isolated recombinant Actinoallomurus endopeptidase having glutenase activity, isolated from the supernatant of the culture medium recovered in step c);
• wherein the at least one recombinant Actinoallomurus endopeptidase or the isolated Actinoallomurus endopeptidase is selected from the group consisting of:
• endopeptidase 40 (E40) comprising SEQ ID NO:
• 1;
• a biologically active fragment of E40;
• a naturally occurring allelic variant of E40;
• and an endopeptidase having at least 60%, 70%, 80%, 90% or 95% of identity to SEQ ID NO:
• 1;
• or
• administering to said subject an effective amount of a formulation comprising as the active proteolytic ingredient the enzyme preparation prepared by said method or comprising the isolated recombinant Actinoallomurus endopeptidases isolated from said supernatant, the formulation being a food or a food supplement or a pharmaceutical formulation, or a flour that has been put in contact with the enzyme preparation or with the isolated recombinant Actinoallomurus endopeptidase.