Protease is a general term for a class of enzymes that hydrolyze protein peptide chains. According to their way of degrading polypeptides, they are divided into two types: endopeptidases and telopeptidases. The former can cut off the polypeptide chain of large molecular weight from the middle to form protein and peptone with smaller molecular weight; the latter can be divided into carboxypeptidase and aminopeptidase, which separate the peptide from the free carboxyl terminal or free amino terminal of the polypeptide one by one. chain hydrolysis to amino acids.
Proteases widely exist in animal viscera, plant stems and leaves, fruits and microorganisms. Microbial proteases are mainly produced by molds and bacteria, followed by yeasts and actinomycetes.
Introduction
Enzymes that catalyze the hydrolysis of proteins. There are many types, the important ones are pepsin, trypsin, cathepsin, papain and subtilisin. Protease has strict selectivity to the reaction substrate it acts on. A protease can only act on certain peptide bonds in protein molecules, such as the peptide bond formed by trypsin catalyzing the hydrolysis of basic amino acids. Protease is widely distributed, mainly exists in human and animal digestive tract, and is abundant in plants and microorganisms. Due to the limited resources of animals and plants, the industrial production of protease preparations is mainly prepared by fermentation of microorganisms such as Bacillus subtilis and Aspergillus terreus.
Classification
Currently known proteases can be divided into the following six categories:
Serine proteases
Threonine proteases
Cysteine proteases
Aspartic acid proteases
Metalloproteases
Glutamic acid proteases
A general term for a class of enzymes that hydrolyze peptide bonds in proteins. According to their way of hydrolyzing polypeptides, they can be divided into two types: endopeptidases and exopeptidases. Endopeptidase cuts off the inside of protein molecules to form peptones and peptones with smaller molecular weights. The exopeptidase hydrolyzes the peptide bonds one by one from the free amino or carboxyl end of the protein molecule to release amino acids. The former is aminopeptidase and the latter is carboxypeptidase. According to its active center and optimum pH value, proteases can be divided into serine proteases, sulfhydryl proteases, metalloproteases and aspartic acid proteases. According to the optimum pH value of its reaction, it is divided into acid protease, neutral protease and alkaline protease. Proteases used in industrial production are mainly endopeptidases.
Content
The depilation and softening of the leather industry has made extensive use of protease, which not only saves time, but also improves labor hygiene conditions. Protease can also be used for silk degumming, meat tenderization, wine clarification. Clinically, it can be used as medicine, such as treating dyspepsia with pepsin, treating bronchitis with acid protease, treating vasculitis with protease, and using trypsin and chymotrypsin to purify surgical suppurative wounds and interthoracic interstitial plasma. Treatment of membrane adhesions. Enzyme laundry detergent is a new product in detergents. It contains alkaline protease, which can remove blood stains and protein stains on clothes, but be careful not to touch the skin when using it, so as not to damage the protein on the skin surface and cause allergic phenomena such as rashes and eczema. .
Proteases widely exist in animal viscera, plant stems and leaves, fruits and microorganisms. Microbial protease, mainly produced by mold and bacteria, followed by yeast and actinomycetes
Application
Protease is the most important industrial enzyme preparation, which can catalyze the hydrolysis of proteins and polypeptides, and widely exists in animal viscera, 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 their action, the protein ingested by the human body is hydrolyzed into small molecule peptides and amino acids.
Proteases currently used in the baking industry include mold proteases, bacterial proteases and plant proteases. The application of protease in bread production can change the properties of gluten, and its mode of action is different from the role of force in bread preparation and the chemical reaction of reducing agent. The role of protease is not to destroy disulfide bonds, but to break the three-dimensional network structure that forms gluten. The role of protease in bread production is mainly manifested in the dough fermentation process. Due to the action of protease, the protein in the flour is degraded into peptides and amino acids to supply yeast carbon source and promote fermentation.
Effect
The function of trypsin is to hydrolyze the protein between cells to separate the cells. Different tissues or cells respond differently to the action of trypsin. The activity of trypsin to disperse cells is also related to its concentration, temperature and action time. When the pH is 8.0 and the temperature is 37°C, the action of trypsin solution is the strongest. When using trypsin, the concentration, temperature and time should be properly controlled to avoid cell damage caused by excessive digestion. Because Ca2+, Mg2+, serum and protein can reduce the activity of trypsin, BSS without Ca2+ and Mg2+ should be used when preparing trypsin solution, such as D-Hanks solution. When terminating the digestion, the action of trypsin on the cells can be terminated with serum-containing culture medium or trypsin inhibitor.
- Weigh trypsin: According to the concentration of trypsin solution at 0.25%, use an electronic balance to accurately weigh the double distilled water (if using double distilled water, adjust the pH to about 7.2) or PBS (D- Hanks ) liquid. Stir to mix well and place at 4°C overnight.
- Suction filtration disinfection with syringe filter: The prepared trypsin solution should be sterilized by suction filtration with a syringe filter (0.22 micron microporous membrane) in the ultra-clean bench. Then aliquot into vials and store at -20°C for use.
Trypsin can catalyze the hydrolysis of specific peptide bonds in proteins. This catalytic process does not require energy, does not inactivate the enzyme, and does not change shape and hydrolyze itself. The combination of the substrate and the active center of the enzyme is reversible. This combination makes the specific peptide bond of the protein activated due to bending deformation, making it easier to be attacked by water molecules, forming amino and carboxyl groups respectively, and breaking to obtain small molecular polypeptides or amino acids. . Different proteases can act on peptide bonds composed of different amino acids, so trypsin cannot act on all peptide bonds.