Method of producing non-bovine chymosin and use hereof

Abstract

A method of recombinantly producing a non-bovine pre-prochymosin, prochymosin or chymosin derived from ruminant species including deer species, buffalo species, antelope species, giraffe species, ovine species and caprine species; Camelidae species such as Camelus dromedarius ; porcine species; or Equidae species. The recombinant enzymes are used in milk coagulating compositions in cheese manufacturing based on cow's milk and milk from any animal species which are used in cheese manufacturing including camel's milk.

1 . A composition comprising a non-bovine pre-prochymosin, prochymosin or chymosin produced by the method comprising the steps of
 (i) isolating or constructing a nucleic acid sequence coding for the pre-prochymosin, prochymosin or chymosin,   (ii) constructing an expression vector comprising said coding sequence and, operably linked thereto, appropriate expression signals permitting the pre-prochymosin, prochymosin or chymosin to be expressed in a host cell,   (iii) transforming said host cell with the expression vector,   (iv) cultivating the thus transformed host cell under conditions where the coding sequence is expressed, and   (v) harvesting the pre-prochymosin, prochymosin or chymosin.   
     
     
         2 . A composition according to  claim 1  where the pre-prochymosin, prochymosin or chymosin is in a substantially deglycosylated form. 
     
     
         3 . A composition according to  claim 1  comprising pre-prochymosin, prochymosin or chymosin derived from the group consisting of a  Camelidae  species, a buffalo species, an ovine species or a caprine species. 
     
     
         4 . A method of manufacturing cheese, comprising adding a milk clotting effective amount of the composition according to  claim 1  to milk and carrying out appropriate further cheese manufacturing steps. 
     
     
         5 . A method according to  claim 4  wherein the milk is selected from the group consisting of cow's milk, camel's milk, buffalo milk, goat's milk, sheep's milk and a mixture of any such milk types. 
     
     
         6 . A method according to  claim 4  wherein the yield of cheese obtained is higher than the yield obtained under identical manufacturing conditions using the same amount of bovine pre-prochymosin, prochymosin or chymosin. 
     
     
         7 . A composition according to  claim 1 , wherein:
 the coding sequence is derived from a mammalian species selected from the group consisting of a ruminant species a  Camelidae  species, a porcine species, an  Equidae  species and a primate species; and/or   the coding sequence for pre-prochymosin, prochymosin and chymosin is isolated or derived from  Camelus dromedarius ; and/or   the nucleic acid sequence codes for a fusion protein comprising pre-prochymosin, prochymosin or chymosin; and/or   the pre-prochymosin, prochymosin or chymosin, or a fusion protein thereof, is secreted over the host cell membrane; and/or   the expression vector is derived from pGAMpR as described in Ward et al., 1990 by substituting the coding sequence of that vector for bovine prochymosin with a coding sequence for the non-bovine pre-prochymosin, prochymosin or chymosin; and/or   the expression vector is pGAMpR-C as contained in the  Aspergillus niger  var.  awamori  strains deposited under the accession numbers CBS 108915 and CBS 108916; and/or   the transformed host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a mammalian cell, an insect cell and a plant cell; and/or   the host cell is  Aspergillus niger  var.  awamori  and/or;   the yield of pre-prochymosin, prochymosin or chymosin milk clotting activity is at least 25% higher than the yield of bovine pre-prochymosin, prochymosin or chymosin milk clotting activity obtained when using, under identical production conditions, the same expression vector, but with a coding sequence for bovine pre-prochymosin, prochymosin or chymosin in place of the coding sequence for the non-bovine pre-prochymosin, prochymosin or chymosin; and/or   the method comprises, as a further step, that the harvested pre-prochymosin, prochymosin or chymosin is subjected to a deglycosylation treatment; and/or   the host cell is a cell expressing a deglycosylating enzyme (such as endoH).   
     
     
         8 . The composition according to  claim 7  wherein the mammalian species is selected from the group consisting of deer species, buffalo species, antelope species, giraffe species, ovine species and caprine species. 
     
     
         9 . The composition according to  claim 7  wherein the nucleic acid sequences code for a fusion protein which also comprises glucoamylase or an enzymatically active fragment thereof. 
     
     
         10 . The composition of  claim 1  which comprises a tylopodal pre-prochymosin, prochymosin, or chymosin. 
     
     
         11 . The composition of  claim 10  which comprises a camel pre-prochymosin, prochymosin or chymosin. 
     
     
         12 . The composition of  claim 11  which comprises a  Camelus dromedaries  pre-prochymosin, prochymosin or chymosin. 
     
     
         13 . The composition of  claim 10  in which the host cell is a non-mammalian cell. 
     
     
         14 . The composition of  claim 10  in which the host cell is a microbial cell. 
     
     
         15 . The composition of  claim 10  in which the host cell is a yeast cell. 
     
     
         16 . The composition of  claim 10  in which the host cell is an  Aspergillus niger  cell. 
     
     
         17 . The composition of  claim 14  which comprises a camel pre-prochymosin, prochymosin or chymosin. 
     
     
         18 . The composition of  claim 14  which comprises a  Camelus dromedaries  pre-prochymosin, prochymosin or chymosin. 
     
     
         19 . The composition of  claim 15  which comprises a camel pre-prochymosin, prochymosin or chymosin. 
     
     
         20 . The composition of  claim 15  which comprises a  Camelus dromedaries  pre-prochymosin, prochymosin or chymosin.