Active Proteinase K (PROK)

Proteinase K is a typical member of the subtilisin family of proteinases .It exhibits broad substrate specificity and degrades many proteins in the native state, even in the presence of detergents.The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. Thus,the activity of recombinant Proteinase K measued by hydrolyzing casein.The reaction was performed in adding 200μl recombinant Proteinase K(dilued by 0.01mol/L Tris-HCL,pH 8.0) to 200μl 10g/L casein ,rapidly mixing,incubated for 5min at 55 ℃,stop the reaction with 400μl 0.4mol/L TCA.Clarify by filtration through a 0.45 m filter or by centrifugation. Read the supernatant an 275nm. The blank use 200μl 0.01mol/L Tris-HCL,pH 8.0 replace the Proteinase K. One unit is described as that amount of enzyme that liberates 1 µg of L- tyrosine within one minute at 55°C using casein as a substrate.