Protease is a general term for a class of enzymes that hydrolyze protein peptide chains. According to the way they degrade polypeptides, they are divided into two categories: endopeptidases and telopeptidases. Endopeptidase can cut the large molecular weight polypeptide chain from the middle to form the smaller molecular weight prions and peptones. Telopeptidases can be further divided into carboxypeptidases and aminopeptidases, which hydrolyze the peptide chain one by one to generate amino acids from the free carboxyl-terminal or free amino-terminal of the polypeptide, respectively.
According to the protease active center and the optimum pH value, proteases can be divided into serine proteases, thiol proteases, metalloproteases and aspartic proteases. According to the optimum pH value of its reaction, it is divided into acidic protease, neutral protease and alkaline protease. The proteases used in industrial production are mainly endopeptidases.
Proteases are widely found in animal offal, plant stems and leaves, fruits and microorganisms. Microbial proteases are mainly produced by molds and bacteria, followed by yeast and actinomycetes.
There are many types of enzymes that catalyze protein hydrolysis, mainly including pepsin, trypsin, cathepsin, papain and subtilisin. Proteases have strict selectivity for the reaction substrates they act on. A protease can only act on certain peptide bonds in protein molecules, such as the peptide bonds formed by the hydrolysis of basic amino acids catalyzed by trypsin. Proteases are widely distributed, mainly in the digestive tract of humans and animals, and are abundant in plants and microorganisms. Due to limited animal and plant resources, the industrial production of protease preparations is mainly prepared by fermentation of microorganisms such as Bacillus subtilis and Aspergillus terrestris.
The role of trypsin is to hydrolyze intercellular proteolysis to dissociate the cells. Different tissues or cells have different responses to the action of trypsin. The activity of the cells dispersed by trypsin is also related to its concentration, temperature and action time. When the pH is 8.0 and the temperature is 37°C, the pancreatin solution has the strongest effect. When using trypsin, the concentration, temperature and time should be carefully controlled to avoid cell damage caused by excessive digestion. Because Ca2+, Mg2+, serum and protein can reduce the activity of pancreatin, BSS without Ca2+ and Mg2+ should be used when preparing pancreatin solution, such as D-Hanks solution. When the digestion is terminated, the action of trypsin on the cells can be stopped with a culture medium containing serum or a trypsin inhibitor.
Weigh trypsin: According to the trypsin concentration of 0.25%, accurately weigh the powder with an electronic balance and dissolve it into double distilled water (if double distilled water is used, the pH needs to be adjusted to about 7.2) or PBS solution. Stir to mix and place at 4°C overnight.
Suction filtration and sterilization with a syringe filter: The prepared pancreatin solution should be sterilized by suction filtration with a syringe filter (0.22 micron microporous membrane) in an ultra-clean bench. Then aliquot into vials and store at -20°C until use.
Trypsin catalyzes the hydrolysis of specific peptide bonds in proteins. This catalytic process requires no energy, does not inactivate the enzyme, does not change shape and hydrolyze itself. The binding between the substrate and the active center of the enzyme is reversible. This binding makes the specific peptide bond of the protein activated due to bending deformation, and is more easily attacked by water molecules, forming amino and carboxyl groups and breaking them to obtain small molecular polypeptides or amino acids. Different proteases can act on peptide bonds formed by connecting different amino acids, so trypsin cannot act on all peptide bonds.
Protease is the most important industrial enzyme preparation, which can catalyze the hydrolysis of proteins and polypeptides, and widely exists in animal offal, plant stems and leaves, fruits and microorganisms. Proteases are used extensively in cheese production, meat tenderization and vegetable protein modification. In addition, pepsin, chymotrypsin, carboxypeptidase and aminopeptidase are all proteases in the human digestive tract. Under the action of these proteases, the protein ingested by the human body is hydrolyzed into small molecular peptides and amino acids.