Blog: Biological Research Record by Rosie
by rosieliu

Applications of proteinase K

Proteinase K has lots of applications, and this article will show you the details.

Date:   11/24/2020 7:17:26 AM   ( 7 mon ) ... viewed 65 times

Proteinase K, derived from Limbert Candida albicans, belongs to the class of serine proteases. Proteinase K has extremely high enzyme activity and wide substrate specificity, and can preferentially decompose the ester bonds and peptide bonds adjacent to the C-terminus of hydrophobic amino acids, sulfur-containing amino acids, and aromatic amino acids. It is active from pH4.0 to pH12.0, the optimum pH is between 7.5-8.0, and the optimum temperature is between 50-55℃. In the presence of 0.2-1% SDS or 10 mM urea, proteinase K showed higher activity, while the presence of commonly used concentrations of EDTA, Triton X-100, and guanidine hydrochloride had little effect on the activity of proteinase K.

 

Proteinase K is usually used for pre-hybridization in the in situ hybridization technology. It has the function of digesting and surrounding target DNA protein to increase the chance of probe binding to target nucleic acid and increase the hybridization signal. However, the concentration of proteinase K is too high, digestion When the time is too long or the incubation temperature is too high, it will damage the structure of the cell to a certain extent, causing the tissue section to fall off and the cell nucleus to disappear, thereby affecting the hybridization result. EDTA buffer can replace the role of proteinase K, solve the above-mentioned problems, and achieve the ideal dyeing effect.

 

 

The following are specific applications of proteinase K.

 

Application in biological detection such as SARS-CoV-2

For SARS-CoV-2 (COVID-19), the processing, extraction, and amplification of nucleic acid amplification tests are high risks. Therefore, it is necessary to inactivate the virus before amplification. Proteinase K is an important component of the virus sampling solution, which can cleavage the virus and release nucleic acid. Eliminate RNase to prevent virus RNA degradation. And it can inactivate the virus, denature the virus protein, lose its activity and "dead", no longer be infectious, and improve the safety of the transportation and detection stage. The Proteinase K used in the SARS-CoV-2 detection is usually Proteinase K Recombinant Protein.

 

Application in DNA and RNA extraction process

Proteinase K can degrade proteins bound to nucleic acids, promote the separation of nucleic acids, and has great advantages in extracting high molecular weight nucleic acids. Proteinase K is also often used to inhibit and degrade RNase during RNA extraction. The quality of RNA is critical to the success of experiments such as Northern blot and hybridization analysis, cDNA synthesis and in vitro translation. RNA is extremely unstable and easily degraded by widely existing RNase. Therefore, the use of protease to create an RNase-free environment and strictly prevent RNase contamination is the key to successful RNA extraction.

 

Application in situ hybridization

Proteinase K is usually used in the processing of pre-hybridization specimens in the in situ hybridization technology. It has the function of digesting and surrounding target DNA proteins to facilitate the penetration of detection probes and improve the sensitivity of detection.

 

Application of bacteria in inactivating domestic and industrial water

The outer structure of the virus is generally dominated by the capsid protein, and proteinase K can destroy the viral capsid protein, thereby interfering with the combination of the virus and the host cell. Under certain conditions, the inactivation rate of virus in pure water and domestic sewage reached 99.4% and 49.4% after treatment with proteinase K at a concentration of 67.51ug/mL for 1h, and the inactivation rate for 3h is >99.9% and 81.8%, respectively. The effect of inactivating the virus is obvious. The advantage of using proteinase K for virus inactivation is that the disinfectant itself is harmless and does not produce harmful disinfection products, so this is an efficient and environmentally friendly method.

 

Application in meat tenderization

Proteinase K can decompose muscle fibers in meat, proteins in connective tissues, even elastin and collagen, so that the spatial structure of proteins at all levels changes, some peptide bonds are broken, the meat absorbs water and expands, and the texture becomes tender. Class quality requirements.

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