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Description
HiPure Proteinase K is an endolytic protease that cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic or hydrophobic amino acids. The HiPure Proteinase K is classified as a serine protease. The smallest peptide to be hydrolyzed by this enzyme is a tetrapeptide.

Features

Active in a wide range of reaction products

Applications

•Isolation of genomic DNA from cultured cells and tissues

•Removal of DNases and RNases when isolating DNA and RNA from tissues or cell lines

•Determination of enzyme localization

•Improving cloning efficiency of PCR products

Storage

Store the HiPure Proteinase K powder at 4ºC or -20℃. Dissolved Proteinase K solution should be stored at -20ºC.

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Quality Control

DNase Activity:None detectable enzyme activity with λ DNA after 6 hrs incubation at 37ºC.

RNase Activity: None detectable ribonuclease activity after 16 hrs incubation at 25ºC.

Each lot was tested to ensure the absence of Nucleases and DNA.

Definition of Activity Unit
One unit of the enzyme liberates Folin-positive amino acids and peptides corresponding to 1 µmol tyrosine in 1 min at 37°C , pH 7.5 using denatured hemoglobin as substrate.
Enzyme activity is assayed in the following mixture: 0.08 M potassium phosphate (pH 7.5), 5 M urea, 4 mM NaCl, 3 mM CaCl2 and 16.7 mg/ml hemoglobin.

Source
Pichia pastoris cells with a cloned gene encoding Tritirachium album endolytic protease (Proteinase K).

Molecular Weight
28.9 kDa monomer

Dilution Buffer
50 mM Tris-HCl (pH 7.5), containing 5 mM calcium chloride and 50% (v/v) glycerol.

Inhibition and Inactivation
Inhibitors: Proteinase K is not inactivated by metal chelators, by thiol-reactive reagents or by specific trypsin and chymotrypsin inhibitors. Phenylmethylsulfonyl fluoride and diisopropyl phosphorofluoridate completely inhibit the enzyme.

Inactivated by heating at 95°C for 10 minutes.

Advantage

The recombinant enzyme is a mutant to the native protease, which gains higher specific activity and yield as well as wider pH and temperature range . The large scale recombinant preparation has advantage in lot-to-lot consistency, superior purity and cost-efficiency. DNA-free nature of recombinant Proteinase K made it well-suited in isolating PCR and RT-PCR templates.

Preparation Notes

Activators: 1~5mM Ca2+

To stimulate proteinase K activity, 1-5 mM Ca2+ can be added. Optimization using activators can increase proteinase activity significantly.

Enzyme activity will be reduced by 25% when calcium is removed by addition of EDTA. Enzyme activity will be reduced by 80% if the EDTA-Ca2+ complex is removed from the enzyme solution by gel filtration, while it can be partially restored by addition of excess Ca2+.

Inhibitors: DIFP or PMSF

The enzyme is inactivated by DIFP or PMSF (PMSF used at final concentration 5 mM. ). However, it is not inhibited by EDTA , iodoacetic acid, trypsin-specific inhibitor TLCK, chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.

Note

• Optimum activity at 50-55°C.

•Rapid denaturation of enzyme occurs at temperatures above 65°C.

•The recommended working concentration for Hipure Proteinase K is 0.05-1 mg/ml. The activity of the enzyme is stimulated by 0.2-1% SDS or by 1-4 M urea.

•Stable over a wide pH range: 4.0-12.5, optimum pH 7.5-8.0.