Screening Method Identifying Protease Secretion-Deficient Mutants of Microorganisms

Abstract

The invention relates to a method for identifying a protease secretion deficient strain of a microorganism, wherein: a mutant of the ciliate is added to a gel; and is incubated under conditions under which the mutant of the ciliate secretes proteins; said mutant of the ciliate is separated from the gel; wherein at least one substrate for at least one secretion protease of the ciliate is contained in said gel and/or said gel itself is the substrate and/or the substrate is added later and will at least in part diffuse into the gel; and the protease activity that has acted on the substrate is measured.

1 . A method for identifying protease secretion deficient strains of a microorganism, wherein:
 mutants of the microorganism are produced;   a gel is produced in the wells of a microtitration plate;   a mutant of the microorganism is added to each of the wells onto the gel;   incubation is effected under conditions under which the mutants of the microorganism secrete proteins;   said mutants of the microorganism are separated from the gel;   
       wherein at least one substrate for at least one secretion protease of the microorganism is contained in said gel and/or said gel itself is the substrate and/or the substrate is added later and will at least in part diffuse into the gel; 
       and the protease activity that has acted on the substrate is measured. 
     
     
         2 . The method according to  claim 1 , wherein said microorganism is selected from the group consisting of ciliates, especially  Tetrahymena , yeasts, especially  Pichia pastoris , monocellular algae, especially  Chlamydomonas, Ulva  and  Euglena , and prokaryotes, especially  Bacillus  and  Escherichia.    
     
     
         3 . The method according to  claim 1  or  2 , wherein said mutants of the microorganism are produced by random mutagenesis or directed mutagenesis. 
     
     
         4 . The method according to  claim 1  or  2 , wherein said mutants of the microorganism are produced by uniparental cytogamy (UPC). 
     
     
         5 . The method according to any of  claims 1  to  4 , wherein said incubation of the mutants of the microorganism with the gel is effected in a nutrient medium. 
     
     
         6 . The method according to  claim 5 , wherein said nutrient medium contains skim milk powder, proteose peptones, yeast extract, soybean peptones, ferrous sulfate/chelate solution and/or glucose monohydrate or is a synthetic medium. 
     
     
         7 . The method according to any of  claims 1  to  6 , wherein said gel is a gelatin, agarose, agar and/or polyacrylamide gel. 
     
     
         8 . The method according to any of  claims 1  to  7 , wherein said substrate for the protease is selected from the group consisting of casein, derivatives of casein, fluorescence-labeled casein and/or gelatin. 
     
     
         9 . The method according to any of  claims 1  to  8 , wherein said incubation is effected under conditions under which said microorganism secretes proteases, and secreted proteases can diffuse into the gel. 
     
     
         10 . The method according to any of  claims 1  to  9 , wherein said microorganism is separated from the gel by pouring (decanting), pipetting, sucking and/or washing. 
     
     
         11 . The method according to any of  claims 1  to  10 , wherein the proteolysis of the substrate produces a fluorescence signal or absorption signal whose intensity is measured, and the protease activity is determined from the result. 
     
     
         12 . The method according to any of  claims 1  to  11 , wherein a layer of the gel is applied to a film, changes of the film are measured, and the protease activity is determined from these changes. 
     
     
         13 . The method according to  claim 12 , wherein said film is an X-ray film. 
     
     
         14 . The method according to any of  claims 1  to  13 , wherein said method is performed in an automated format.