What Is an Endopeptidase?

Peptidase is an enzyme capable of hydrolyzing peptide chains, and is a general term recommended by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) as a proteolytic enzyme Can be divided into two major types, namely endopeptidases (EC3.4.21 ~ 3.4.99) and exopeptidases (EC3.4.11 ~ 3.4.19). The endopeptidase acts on specific peptide bonds inside the polypeptide chain, and the exopeptidase acts on the N-terminus or C-terminus of the polypeptide substrate, catalyzing the production of free amino acids and small peptides.

Peptidase is a general term for enzymes that can decompose short peptides of proteins. Most of them exist in microbial cells. When the cells are autolysed during the fermentation process, peptidases are released to hydrolyze peptides to form amino acids, which serve as flavor precursors. They are an enzyme necessary for the survival of all living things, and the gene encoding peptidase accounts for 2% of all protein codes. according to

Endopeptidases, also commonly known as proteases, mainly act on peptide bonds inside protein polypeptide chains to break down the long chains of proteins into short peptide fragments. Proteases are divided into different families based on structural homology, and subclasses are further divided into different families based on the degree of sequence homology.

When protease hydrolyzes a protein, the site of action varies depending on the type of peptide bond. For example, the cut point of trypsin is that the carboxyl side is a basic amino acid (arginine, lysine) peptide bond; pepsin requires aromatic amino acids at both ends of the cut point; the carboxyl side of the cut point of Bacillus subtilis requires hydrophobicity Aromatic amino acids (tryptophan, tyrosine, phenylalanine). The specificity of this protease to the cleavage point is called the substrate specificity of the protease. The substrate specificity of protease can be used to obtain peptides with special structures ^[3]

Exopeptidases are widely distributed in animals, plants, microorganisms, and soils, and can play an important role in normal physiological and pathophysiological processes of organisms, mainly involving signal transduction, regulation of peptide hormones, protein binding and digestion, etc. . The most studied aminopeptidase is lactobacillus aminopeptidase. More than 100 aminopeptidases have been isolated and purified from lactic acid bacteria, but there are relatively few reports on other microbial exopeptidases related to food fermentation ^[2]

Effect on food flavor formation

cocoa

During the fermentation of cocoa beans, one dominant endopeptidase (aspartate endopeptidase, optimal pH 3.5) and one carboxypeptidase (optimal pH 5.8) are secreted. Coco carboxypeptidase cannot hydrolyze Arg, Lys, and Pro residues at the carboxyl terminus, and is more suitable for hydrophobic amino acids. These two enzymes produce hydrophobic free amino acids and hydrophilic peptides to produce cocoa-specific aroma precursors.

beef

In meat production, electrical stimulation causes changes in the activity of exopeptidases in muscle, and the types and contents of free amino acids produced will affect the quality of meat. During the maturation of beef, low-voltage stimulation of the carcass can activate the activity of dipeptidyl peptidase in muscle and inhibit the activity of leucine aminopeptidase, methionine aminopeptidase, and pyroglutamine aminopeptidase. It has no effect on the activity of alanine aminopeptidase and arginine aminopeptidase.

Salted meat

The aminopeptidase activity in dry-cured ham was 25% to 75% of the original fresh ham, and the contents of Glu, Leu, Arg, Ala, Tyr and Lys increased significantly. The increase in free amino acids during dry curing is caused by the action of aminopeptidase. The composition of protease in fresh pork has a great influence on the quality of dried cured ham products. The bitterer the sample, the more proteolytic, cathepsin B, and dipeptidyl peptidase activity. In contrast, less bitter fresh ham and cured mature muscle have higher aminopeptidase activity. Met and Asp are higher in bitter ham. During the whole process of pickling, LAP activity is always high. The alanine aminopeptidase isolated from porcine skeletal muscle has high specificity for Ala at the 4- and 5-positions, but lacks specificity for Ala at the 2-position. Therefore, dipeptides and free amino acids are produced during meat processing and have an impact on the final flavour of the meat. A new method to promote the formation of flavor is to add microorganisms isolated from traditional pickled brines. The lipolytic enzymes and proteolytic enzymes produced by these bacteria are related to the formation of flavor.

beer

Malt contains at least 8 different exopeptidases, of which 3 are carboxypeptidases, the optimum pH is 4.8 to 5.7; 5 are aminopeptidases, the optimum pH is 5.8 to 8.6. Peptidase activity was stable at 45 ° C. During saccharification, the temperature rose from 45 ° C to 70 ° C, and these exopeptidases were quickly inactivated. After incubating at 55 ° C for 30 min, the five aminopeptidases were inactivated at different rates, while the carboxypeptidase activity was still 70% to 100%. Among them, one carboxypeptidase was incubated at 70 ° C for 60 min and there was 40% residual enzyme activity. Therefore, carboxypeptidase has higher thermal stability than aminopeptidase in malt saccharification. This characteristic is extremely important for the generation of free amino acids during saccharification, because the free amino acids formed during the saccharification process account for 50% to 75% of the total free amino acid release. These three carboxypeptidases have similar molecular masses, with slightly different ionic characteristics and specificities. Malt carboxypeptidase is more suitable for peptides containing aromatic amino acid residues and Pro. Carboxypeptidase can reduce the viscosity of -glucan extracted from the endosperm cell wall and improve the solubilization of endosperm by hydrolyzing the peptides in the cells. Optimum pH and thermal stability indicate that carboxypeptidase can act not only on barley prolamin, but also on peptides formed during germination and saccharification, and promote the release of free amino acids.

cheese

In addition to microbial cells, intracellular and extracellular peptidase activity is also present in cheese extracts. The exo-peptidase released after microbial autolysis plays an important role in the degradation of casein oligopeptides. When soft cheese is made with Penicillium carmenii and Geotrichum candidum, the maturation of the cheese is mainly controlled by proteolytic enzymes. Capsule-treated recombinant aminopeptidase can shorten the cheddar cheese fermentation process and increase the sensory evaluation score of cheese.

Effect on Debittering of Protein Hydrolysate

Food proteins from animal sources (milk, meat, fish, etc.) and plant sources (beans, grains) undergo a certain degree of hydrolysis during food processing. The hydrolysis is mainly through two ways: one is the fermentation of bacterial or fungal endopeptidases and exopeptidases, and the second is the direct processing of protein hydrolysates. The presence of bitter peptides is a defect of most protein hydrolysates. After the action of proteins by endopeptidases, the hydrolysates formed usually have a bitter taste. Due to the different cut points of different endopeptidases, the bitter peptides formed are also different ^.

The majority of commercial exopeptidases are aminopeptidases, which are mainly used in the production of food protein hydrolysates, with the aim of improving palatability, functionality and nutritional properties. Exopeptidases are often used in cheese production to degrade casein and reduce bitterness. Normal commercial enzymes have optimum pH values in the neutral range. Enzyme activity is indicated in the instructions according to leucine aminopeptidase, so it is difficult to compare the activity of commercial exopeptidases. Some enzyme preparations are exopeptidases, such as Protease R, Protease M, Flavourzyme, and CorolaseLAP. For example, Imperial Biotechnology offers two exo-peptidase mixed preparations for removing bitterness, Accelase and Debirase. Accelase is the aminopeptidase of Lactococcus lactis, which is an aminopeptidase extract of Lactococcus lactis and A. oryzae. Some are a mixture of endopeptidases and exopeptidases ^[4] .