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 method of producing a non-bovine pre-prochymosin, prochymosin or chymosin, 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 method according to claim I 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.  
     
     
         3 . A method according to  claim 2  wherein the ruminant species is selected from the group consisting of deer species, buffalo species, antelope species, giraffe species, ovine species and caprine species.  
     
     
         4 . A method according to  claim 1  wherein the coding sequence for pre-prochymosin, prochymosin and chymosin is isolated or derived from  Camelus dromedarius.    
     
     
         5 . A method according to  claim 1  wherein the nucleic acid sequence codes for a fusion protein comprising pre-prochymosin, prochymosin or chymosin.  
     
     
         6 . A method according to  claim 5  wherein the fusion protein comprises glucoamylase or a fragment thereof.  
     
     
         7 . A method according to any of claims  1 - 6  wherein the pre-prochymosin, prochymosin or chymosin, or a fusion protein thereof, is secreted over the host cell membrane.  
     
     
         8 . A method according to  claim 1  wherein 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.  
     
     
         9 . A method according to  claim 8  wherein 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.  
     
     
         10 . A method according to any of claims  1 - 9  wherein 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.  
     
     
         11 . A method according to  claim 10  wherein the host cell is  Aspergillus niger  var.  awamori.    
     
     
         12 . A method according to  claim 11  wherein the  Aspergillus niger  var.  awamori  host cell is selected from the group consisting of CBS 108915 and CBS 108916.  
     
     
         13 . A method according to any of claims  1 - 12  wherein 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.  
     
     
         14 . A method according to any of claims  1 - 13  comprising, as a further step, that the harvested pre-prochymosin, prochymosin or chymosin is subjected to a deglycosylation treatment.  
     
     
         15 . A method according to  claim 1  wherein the host cell is a cell expressing a deglycosylating enzyme.  
     
     
         16 . A method according to  claim 15  wherein the deglycosylating enzyme is endoH.  
     
     
         17 . A DNA construct capable of expressing non-bovine pre-prochymosin, prochymosin or chymosin, said construct comprising an expression vector comprising a nucleic acid sequence comprising a gene coding for the pre-prochymosin, prochymosin or chymosin and, operably linked thereto, appropriate expression signals permitting the pre-prochymosin, prochymosin or chymosin to be expressed in a host cell.  
     
     
         18 . A DNA construct according to  claim 17  where the construct comprises a sequence coding for a signal peptide for the pre-prochymosin, prochymosin or chymosin.  
     
     
         19 . A DNA construct according to  claim 17  where the expression signal comprises a promoter not natively associated with the coding sequence.  
     
     
         20 . A DNA construct according to  claim 17  where 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.  
     
     
         21 . A DNA construct according to  claim 20  where the ruminant species is selected from the group consisting of deer species, buffalo species, antelope species, giraffe species, ovine species and caprine species.  
     
     
         22 . A DNA construct according to  claim 20  where the coding sequence is derived from  Camelus dromedarius.    
     
     
         23 . A DNA construct according to any of claims  17 - 22  where the nucleic acid sequence codes for a fusion protein comprising the pre-prochymosin, prochymosin or chymosin.  
     
     
         24 . A DNA construct according to  claim 23  where the fusion protein comprises glucoamylase or a fragment thereof.  
     
     
         25 . A DNA construct according to any of claims  17 - 24  where 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.  
     
     
         26 . A DNA construct according to  claim 25  where 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.  
     
     
         27 . A DNA construct according to  claim 17  comprising a sequence coding for a deglycosylating enzyme.  
     
     
         28 . A DNA construct according to  claim 27  where the deglycosylating enzyme is endoH.  
     
     
         29 . A DNA construct according to any of claims  17 - 28  where the coding sequence is a naturally occurring coding sequence.  
     
     
         30 . A DNA construct according to any of claims  17 - 28  where the coding sequence is derived from a naturally occurring coding sequence by one or more silent nucleotide substitution(s).  
     
     
         31 . A host cell transformed with the DNA construct according to any of claims  17 - 30 .  
     
     
         32 . A host cell according to  claim 31  which 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.  
     
     
         33 . A host cell according to  claim 32  which is of  Aspergillus niger  var.  awamori.    
     
     
         34 . A host cell according to  claim 33  where the  Aspergillus niger  var.  awamori  host cell is selected from the group consisting of the strains desposited as CBS 108915 and CBS 108916.  
     
     
         35 . A composition comprising a non-bovine pre-prochymosin, prochymosin or chymosin produced by the method of any of claims  1 - 16 .  
     
     
         36 . A composition according to  claim 35  where the pre-prochymosin, prochymosin or chymosin is in a substantially deglycosylated form.  
     
     
         37 . A composition according to  claim 35  or  36  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.  
     
     
         38 . A method of manufacturing cheese, comprising adding a milk clotting effective amount of the composition according to claim any of claims  35 - 37  to milk and carrying out appropriate further cheese manufacturing steps.  
     
     
         39 . A method according to  claim 38  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.  
     
     
         40 . A method according to  claim 38  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.  
     
     
         41 . A method of manufacturing cheese, comprising adding a milk clotting effective amount of a non-bovine prochymosin or chymosin to the milk and carrying out appropriate further cheese manufacturing steps, the non-bovine prochymosin or chymosin having in said milk a C/P ratio in the range of 2-20.  
     
     
         42 . A method according to  claim 41  wherein the milk is cow's milk.  
     
     
         43 . A method according to  claim 41  or  42  wherein the non-bovine prochymosin or chymosin is derived from  Camelus dromedarius.    
     
     
         44 . A milk clotting composition comprising a milk clotting bovine enzyme selected from prochymosin, chymosin and pepsin and a non-bovine milk clotting enzyme selected from prochymosin, chymosin, pepsin and a microbial aspartic protease.  
     
     
         45 . A composition according to  claim 44  where the milk clotting activity ratio between the bovine and the non-bovine milk clotting enzyme is in the range of 1:99 to 99:1.  
     
     
         46 . A composition according to  claim 44  or  45  where the non-bovine milk clotting enzyme is derived from  Camelus dromedarius.    
     
     
         47 . A method of manufacturing cheese from milk, comprising adding a milk clotting effective amount of a composition according to any of claims  44 - 46  to the milk and carrying out appropriate further cheese manufacturing steps.  
     
     
         48 . A method according to  claim 47  wherein the milk is cow's milk.