A brief talk on the Protease K

Posted by beauty33 on February 27th, 2019

Protease K is a powerful proteolytic enzyme isolated from Candida albicans. It has high specific activity and is a key reagent for DNA extraction. It is used for the isolation of plasmid or genomic DNA and RNA. In DNA extraction, the main function is to enzymatically hydrolyze histones bound to nucleic acids, so that DNA is free in the solution, and then extracted by different methods to remove impurities and collect DNA.

The protease K is a kind of biocatalyst, and a serine protease that cleavage widely. It cleavages carboxy terminal peptide bonds of aliphatic amino acids and aromatic amino acids. The activity of RNA enzyme and DNA enzyme was purified by purification of the enzyme. Because protease K is stable in urea and SDS and has the ability to degrade natural proteins, it has been widely used, including the preparation of chromosomal DNA by pulsed electrophoresis, protein imprinting, and the removal of nucleases from DNA and RNA preparation.

In situ hybridization, protease K is usually used for pre-hybridization treatment. It can digest and surround the target DNA protein, increase the chance of binding probe to target nucleic acid, and enhance the hybridization signal. As a result, the loss of tissue slices and the disappearance of nuclei affect the results of hybridization. EDTA buffer can replace the role of protease K, solve the above problems, and achieve an ideal dyeing effect.

The protease K is such a powerful Protease that it has a wide active range, in another word, its Enzyme Activity is affected by little things. Protease K storage solution is generally 10mg/ml or 20mg/ml. Store at 20 degrees C. Protease K solution is colorless and transparent, and can not be used if precipitation occurs. The enzyme was active in a wide range of pH (4 ~ 12.5) and high temperature (50 ~ 70 C).

Protease K has been widely used in leather, fur, silk, medicine, food, brewing and so on. Protease has been widely used in the depilation and softening of leather industry, which not only saves time, but also improves labor hygiene conditions. Protease can also be used for degumming of silk, tenderness of meat and clarification of alcohol. The enzyme has two Ca2+ binding sites, which are not directly related to the catalytic mechanism. However, if Ca2+ is removed from the enzyme, it loses about 80% of its catalytic activity due to remotely structural changes, but its residual activity is usually sufficient to degrade proteins that normally contaminate acid products. Therefore, EDTA is usually added during the digestion of protease K to inhibit the action of Mg2+ dependent nucleases. However, to digest proteins that are more resistant to protease k, such as keratins, a buffer containing 1mmol/l Ca2+ may be required without EDTA. After digestion and before nucleic acid purification, EGTA (ph8.0) should be added to the final concentration of 2 mmol/l to chelate Ca2 +.

It can be used as medicine in clinic, such as pepsin for dyspepsia, acid protease for bronchitis, elastase for vasculitis, trypsin and chymotrypsin for surgical purulent wound cleaning and pleural serosa adhesion. Enzymatic detergent powder is a new product in detergent. It contains alkaline protease. It can remove blood stains and protein dirt on clothes. But it should not touch the skin when used, so as to avoid damaging the protein on the skin surface and causing skin rash, eczema and other allergic phenomena.