High-activity phytase compositions

Abstract

A process is disclosed for preparing aqueous phytase-containing liquids involving culturing microorganisms of the genus Aspergillus or Trichoderma in a medium containing assimilable carbon and nitrogen sources (e.g., glucose and ammonium ions), filtering the medium, and subjecting the resulting filtrate to ultra-filtration to give an aqueous composition having at least 14,000 FTU/g. This aqueous liquid optimally can be used to prepare granulates that can be incorporated in animal feedstuffs.

1 . A process for the preparation of an aqueous liquid comprising a phytase, the process comprising: 
 (a) culturing in an aqueous medium a microorganism of the genus  Aspergillus  or  Trichoderma  having a heterologous phytase gene under the control of a glucoamylase (for  Aspergillus ) or cellobiohydrolase (for  Trichoderma ) promoter, under conditions that allow recombinant expression of the phytase, where the medium comprises, as a feed for the microorganism an assimilable carbon source and an assimilable nitrogen source.    (b) filtering the aqueous medium to remove the microorganisms to give an aqueous filtrate; and    (c) subjecting the filtrate from (b) to ultrafiltration to give an aqueous liquid, having a phytase concentration of at least about 14,000 FTU/g.    
     
     
         2 . The process according to  claim 1  where the microorganism is  Aspergillus niger, Aspergillus oryzae  or  Trichoderma reesei.    
     
     
         3 . The process according to  claim 1  wherein the microorganism does not possess, or does not express, a glucoamylase (AG) gene.  
     
     
         4 . The process according to  claim 1  wherein the microorganism possesses multiple copies of the phytase gene.  
     
     
         5 . The process according to  claim 1  wherein the aqueous liquid is substantially free of taka-amylase.  
     
     
         6 . The process according to  claim 1  wherein substantially all of the carbon and nitrogen sources in the medium have been consumed by the microorganisms before filtering in (b).  
     
     
         7 . The process according to  claim 1  wherein the aqueous liquid is free of the carbon and/or nitrogen sources.  
     
     
         8 . The process according to  claim 1  wherein the phytase is expressed in the microorganism with a glucoamylase signal sequence.  
     
     
         9 . The process according to  claim 1  wherein neither the aqueous filtrate or the aqueous liquid are subjected to: (a) crystallisation; (b) a colour-removal step; or (c) crystallization and a colour removal step.  
     
     
         10 . The process according to  claim 1  wherein the resulting aqueous liquid has a phytase activity of about 18,000 FTU/g or more.  
     
     
         11 . An aqueous liquid comprising a phytase at a concentration of at least about 14,000 FTU/g.  
     
     
         12 . The aqueous liquid according to  claim 11  which is derived from a culture medium in which the phytase was expressed.  
     
     
         13 - 38 . (Canceled)  
     
     
         39 . The aqueous liquid according to  claim 11  wherein the phytase is a fungal phytase.  
     
     
         40 . The aqueous liquid according to  claim 40  wherein the phytase is an  Aspergillus  phytase or a  Trichoderma  phytase.  
     
     
         41 . The aqueous liquid according to  claim 11  wherein the phytase is at a concentration of at least about 18,000 FTU/g.