Thermostable proteinases from thermophilic bacteria

Abstract

The present invention relates to the preparation of a method for the preparation of nucleic acid samples, including the steps of adding at least one non-specific thermophilic enzyme to a sample containing nucleic acid for testing, and incubating said sample for a preferred period at 65-80° C. as required to effect one or more of lysis of cells, digestion of proteins, digestion of cell-wall enzymes, via activity of said thermophilic enzyme, wherein said thermophilic enzyme is substantially stable and active at 65-80° C. but which is readily autolysed and/or denatured when said sample is incubated at or above 90° C.

1 . A method for the preparation of nucleic acid samples for testing, comprising:
 i) adding  Bacillus  EA1 thermophilic proteinase to a sample comprising nucleic acid for testing, thereby forming a system, wherein the thermophilic proteinase digests protein in the sample and is capable of auto-catalysis at or above 90° C.;   ii) closing the system;   iii) incubating the sample in the system for a period at 65-80° C. as required to effect one or more of: lysis of one or more cells, digestion of one or more proteins, or digestion of one or more cell-wall enzymes, via activity of the thermophilic proteinase;   iv) incubating the sample in the system at or above 90° C. to effect auto-catalysis of the thermophilic proteinase;   wherein the protein and thermophilic proteinase are denatured in the system prior to opening without the addition of further denaturing agents.   
   
   
       2 . The method of  claim 1 , wherein the sample is incubated for a total of 45 minutes or less. 
   
   
       3 . The method of  claim 1 , wherein the incubation temperature required to effect one or more of the lysis of cells, digestion of proteins, or digestion of cell-wall enzymes is 75° C. 
   
   
       4 . The method of  claim 1 , wherein the sample is incubated at 94° C. to effect auto-catalysis of the thermophilic proteinase. 
   
   
       5 . The method of  claim 1 , further comprising an initial step of adding at least one mesophilic enzyme with the  Bacillus  EA1 thermophilic proteinase to the sample, and wherein prior to incubation at 65-80° C. the sample is incubated for a period below 40° C. as required to effect removal of any cell walls via activity of the at least one mesophilic enzyme. 
   
   
       6 . The method of  claim 5 , wherein the at least one mesophilic enzyme is a cellulase. 
   
   
       7 . The method of  claim 5 , wherein the initial incubation temperature required to effect removal of any cell walls is 37° C.