PROTEASES

CAS NUMBER: 9001-92-7

A protease (also called a peptidase or proteinase) is an enzyme that catalyzes (increases reaction rate or "speeds up") proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products.
They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in many biological functions, including digestion of ingested proteins, protein catabolism (breakdown of old proteins), and cell signaling.

In the absence of functional accelerants, proteolysis would be very slow, taking hundreds of years.
Proteases can be found in all forms of life and viruses. 
They have independently evolved multiple times, and different classes of protease can perform the same reaction by completely different catalytic mechanisms.

Hierarchy of proteases
Based on catalytic residue
Proteases can be classified into seven broad groups:
Serine proteases - using a serine alcohol
Cysteine proteases - using a cysteine thiol
Threonine proteases - using a threonine secondary alcohol
Aspartic proteases - using an aspartate carboxylic acid
Glutamic proteases - using a glutamate carboxylic acid
Metalloproteases - using a metal, usually zinc
Asparagine peptide lyases - using an asparagine to perform an elimination reaction (not requiring water)
Proteases were first grouped into 84 families according to their evolutionary relationship in 1993, and classified under four catalytic types: serine, cysteine, aspartic, and metallo proteases.

Proteases threonine and glutamic-acid proteases were not described until 1995 and 2004 respectively. 
Proteases mechanism used to cleave a peptide bond involves making an amino acid residue that has the cysteine and threonine (proteases) or a water molecule (aspartic acid, metallo- and acid proteases) nucleophilic so that it can attack the peptide carbonyl group. 
One way to make a nucleophile is by a catalytic triad, where a histidine residue is used to activate serine, cysteine, or threonine as a nucleophile. 
This is not an evolutionary grouping, however, as the nucleophile types have evolved convergently in different superfamilies, and some superfamilies show divergent evolution to multiple different nucleophiles.

Peptide lyases
A seventh catalytic type of proteolytic enzymes, asparagine peptide lyase, was described in 2011. 
Proteases proteolytic mechanism is unusual since, rather than hydrolysis, it performs an elimination reaction.
During this reaction, the catalytic asparagine forms a cyclic chemical structure that cleaves itself at asparagine residues in proteins under the right conditions. Given its fundamentally different mechanism, its inclusion as a peptidase may be debatable.

Evolutionary phylogeny
An up-to-date classification of protease evolutionary superfamilies is found in the MEROPS database.
In this database, proteases are classified firstly by 'clan' (superfamily) based on structure, mechanism and catalytic residue order (e.g. the PA clan where P indicates a mixture of nucleophile families). 
Within each 'clan', proteases are classified into families based on sequence similarity. 
Each family may contain many hundreds of related proteases.

Currently more than 50 clans are known, each indicating an independent evolutionary origin of proteolysis.

Classification based on optimal pH
Alternatively, proteases may be classified by the optimal pH in which they are active:

Acid proteases
Neutral proteases involved in type 1 hypersensitivity. 
Here, it is released by mast cells and causes activation of complement and kinins.
This group includes the calpains.
Basic proteases (or alkaline proteases)
Enzymatic function and mechanism

The Role of Protease
Compared to lipase and amylase, which break down fats and carbohydrates, respectively, the protease family has more extensive roles.  
Yes, protease helps break down protein in food into amino acids, which the body can then use for energy, but where proteases stand apart is the fact that they also play a number of other roles in essential processes, such as:
Blood clotting
Cell division
Recycling of proteins
Immune support
In some cases, enzymes are directly responsible for activating these processes, and in other cases, they speed them up to the point where they have a notable effect.  
Studies are also showing that getting added protease may have some potential health benefits.  
Here are some standout findings.

Digestive support: We mentioned that protease helps the body absorb essential amino acids, but by helping the digestive process, protease enzymes may help people who experience indigestion symptoms like loss of appetite, bloating, and abdominal discomfort.

Muscle soreness: Athletes consider protein to be a major part of their health regimen, and protease may factor in as well.  
In one study, a protease enzyme blend reduced muscle tenderness and soreness post-workout over placebo.

Wound healing: One small study showed that swelling and discomfort sensations were reduced in post-dental surgery patients after taking the protease enzyme serrapeptase.
Choosing the Appropiate Protease 
So now that we know all that proteases can do, where can you get them from?  
As mentioned earlier, both plants and animals have proteases, and in some cases, incorporating plant enzymes is a great option.  
Two popular proteases that come from plant sources are papain from papayas and bromelain from pineapples.  
Both of these have been used for their ability to break down proteins for centuries, but as a meat tenderizer, not for health reasons.
These are two of the most popular food sources, but there are others as well, such as ginger, asparagus, kiwifruit and kimchi.  
Another option is getting proteases from supplements for a variety of health support functions.  
For example, using protease in a plant-based digestive formula, will help with nutrient absorption while supporting digestive function; However, proteases are also used to help address excessive mucus due to allergies or temperature changes. 

One should note that there are many proteases, so it is important to choose the appropriate protease for a particular issue.  
When it comes to getting more proteases through supplemental means, it’s important to find the right fit for your client.  
There are two options to choose from, as proteases are available in digestive or systemic / therapeutic formulations.  
Proteases former is taken with food and the latter, in most cases, is taken away from food.  
Please contact your Enzyme Science Representative for more information about the options that are available.

A comparison of the two hydrolytic mechanisms used for proteolysis. 
Enzyme is shown in black, substrate protein in red and water in blue. 
Proteases top panel shows 1-step hydrolysis where the enzyme uses an acid to polarise water, which then hydrolyses the substrate. 
Proteases bottom panel shows 2-step hydrolysis where a residue within the enzyme is activated to act as a nucleophile (Nu) and attack the substrate. 
Proteases forms an intermediate where the enzyme is covalently linked to the N-terminal half of the substrate. 
In a second step, water is activated to hydrolyse this intermediate and complete catalysis. 
Other enzyme residues (not shown) donate and accept hydrogens and electrostatically stabilise charge build-up along the reaction mechanism.
See also: Catalytic triad
Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. 
Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).

Catalysis
Catalysis is achieved by one of two mechanisms:

Aspartic, glutamic, and metallo-proteases activate a water molecule, which performs a nucleophilic attack on the peptide bond to hydrolyze it.
Serine, threonine, and cysteine proteases use a nucleophilic residue (usually in a catalytic triad). 
Proteases residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. 
Proteases covalent acyl-enzyme intermediate is then hydrolyzed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme

Specificity
Proteolysis can be highly promiscuous such that a wide range of protein substrates are hydrolyzed. 
This is the case for digestive enzymes such as trypsin, which have to be able to cleave the array of proteins ingested into smaller peptide fragments. Promiscuous proteases typically bind to a single amino acid on the substrate and so only have specificity for that residue. 
For example, trypsin is specific for the sequences ...K... or ...R... (''=cleavage site).

Conversely some proteases are highly specific and only cleave substrates with a certain sequence. 
Blood clotting (such as thrombin) and viral polyprotein processing (such as TEV protease) requires this level of specificity in order to achieve precise cleavage events. 
This is achieved by proteases having a long binding cleft or tunnel with several pockets that bind to specified residues. 
For example, TEV protease is specific for the sequence ...ENLYFQS... (''=cleavage site).

Degradation and autolysis
Proteases, being themselves proteins, are cleaved by other protease molecules, sometimes of the same variety. 
This acts as a method of regulation of protease activity. Some proteases are less active after autolysis (e.g. TEV protease) whilst others are more active (e.g. trypsinogen).

Uses
Main article: Proteases (medical and related uses)
The field of protease research is enormous. 
Since 2004, approximately 8000 papers related to this field were published each year.
Proteases are used in industry, medicine and as a basic biological research tool.

Digestive proteases are part of many laundry detergents and are also used extensively in the bread industry in bread improver. 
A variety of proteases are used medically both for their native function (e.g. controlling blood clotting) or for completely artificial functions (e.g. for the targeted degradation of pathogenic proteins). 
Highly specific proteases such as TEV protease and thrombin are commonly used to cleave fusion proteins and affinity tags in a controlled fashion.

Inhibitors
Main articles: Protease inhibitor (biology) and Protease inhibitor (pharmacology)
Proteases activity of proteases is inhibited by protease inhibitors.
One example of protease inhibitors is the serpin superfamily. It includes alpha 1-antitrypsin (which protects the body from excessive effects of its own inflammatory proteases), alpha 1-antichymotrypsin (which does likewise), C1-inhibitor (which protects the body from excessive protease-triggered activation of its own complement system), antithrombin (which protects the body from excessive coagulation), plasminogen activator inhibitor-1 (which protects the body from inadequate coagulation by blocking protease-triggered fibrinolysis), and neuroserpin.

Natural protease inhibitors include the family of lipocalin proteins, which play a role in cell regulation and differentiation. 
Lipophilic ligands, attached to lipocalin proteins, have been found to possess tumor protease inhibiting properties. 
Proteases natural protease inhibitors are not to be confused with the protease inhibitors used in antiretroviral therapy. 
Some viruses, with HIV/AIDS among them, depend on proteases in their reproductive cycle. 
Thus, protease inhibitors are developed as antiviral therapeutic agents.

Other natural protease inhibitors are used as defense mechanisms. 
Common examples are the trypsin inhibitors found in the seeds of some plants, most notable for humans being soybeans, a major food crop, where they act to discourage predators. 
Raw soybeans are toxic to many animals, including humans, until the protease inhibitors they contain have been denatured.

Proteases use of chemicals around the globe in different industries has increased tremendously, affecting the health of people. 
Proteases modern world intends to replace these noxious chemicals with environmental friendly products for the betterment of life on the planet. 
Establishing enzymatic processes in spite of chemical processes has been a prime objective of scientists. 
Various enzymes, specifically microbial proteases, are the most essentially used in different corporate sectors, such as textile, detergent, leather, feed, waste, and others. 
Proteases with respect to physiological and commercial roles hold a pivotal position. 

As they are performing synthetic and degradative functions, proteases are found ubiquitously, such as in plants, animals, and microbes. 
Among different producers of proteases, Bacillus sp. are mostly commercially exploited microbes for proteases. 
Proteases are successfully considered as an alternative to chemicals and an eco-friendly indicator for nature or the surroundings. 
The evolutionary relationship among acidic, neutral, and alkaline proteases has been analyzed based on their protein sequences, but there remains a lack of information that regulates the diversity in their specificity. 
Researchers are looking for microbial proteases as they can tolerate harsh conditions, ways to prevent autoproteolytic activity, stability in optimum pH, and substrate specificity. 
The current review focuses on the comparison among different proteases and the current problems faced during production and application at the industrial level. 
Deciphering these issues would enable us to promote microbial proteases economically and commercially around the world.

Introduction
Proteases are a universal entity that is found everywhere, namely, in plants, animals, and microbes. 
The peptide bond present in the polypeptide chain of amino acids is hydrolyzed by means of proteases. 
Proteases are degradative enzymes and show specificity and selectivity in protein modification. 
In the industrial sector, Bacillus sp. are the most active and dynamic extracellular alkaline protease producer. 
Of the three largest groups of industrial enzymes, proteases are one of them, and their global market is drastically increasing annually. 
Of the 60% of enzymes marketed worldwide, proteases account for 20%. 
Proteases are an integral component of existing life on earth, such as animals, plants, and microbes. 
By a process of fermentation, proteases can be isolated and purified in a relatively shorter period of time, exhibiting high substrate specificity and catalytic activity. 
Proteases is estimated that proteases account for 1–5% of the genome of infectious organisms and 2% of the human genome. 
According to researchers, proteases control the activation, synthesis, and turnover of proteins to regulate physiological processes. 
Different physiological processes, such as formation, birth, aging, and even death are regulated by proteases. 
Proteases are vital in the imitation and spread of infectious diseases, and because of their significant role in the life cycle, they are imperative for drug discovery. 

In more than 50 human proteases, a single amino acid mutation may lead to a hereditary disease. 
Proteases are involved in normal and pathophysiological processes or conditions. 
This involvement of proteases may lead them to produce a therapeutic agent against deadly diseases, such as cancer and AIDS. 
Proteases similar in sequences and structures are grouped into clans and families, which are available in the MEROPS database. 
The proposed review highlights the proteolysis, function, and wide range of sources among different bacteria of microbial proteases. 
It also discusses the broad range of applications and upcoming advancement for the discovery of new and fresh proteases, especially alkaline proteases from bacteria.

Microbial Proteases
Proteases have been successfully produced by researchers from different microbial sources. 
Microbes account a two-thirds share of commercial protease around the globe. 
Since the advent of enzymology, microbial proteolytic proteases have been the most widely studied enzyme. 
These enzymes have gained interest not only due to their vital role in metabolic activities but also due to their immense utilization in industries. 
Proteases proteases available in the market are of microbial origin because of their high yield, less time consumption, less space requirement, lofty genetic manipulation, and cost-effectiveness, which have made them suitable for biotechnological application in the market . 
Proteolytic enzymes found in microbes and mammalian systems are small in size, dense, and structurally spherical. 
Among different producers of alkaline proteases, Bacillus sp. is of immense importance. 

The proteases isolated from these microbial sources have a large number of dilutions in various industrial sectors. 
Usually, extracellular alkaline proteases are secreted out from the producer into the liquid broth from where these proteases are simplified and purified through down streaming to produce an end product. 
Comparatively, proteases produced by plants and animals are more labor-intensive than microbially produced proteases. 
Proteases produced by microbial sources are classified into groups based on their acidic or basic properties. 
They are also classified based on the presence of functional groups and the position of peptide bond. 
Microbial proteases are the most commercially exploited enzyme worldwide. 
A large number of intracellular proteases are produced by microbes playing a vital role in differentiation, protein turnover, hormone regulation, and cellular protein pool, whereas extracellular proteases are significant in protein hydrolysis, such as in processing of photographic film , enzymatic synthesis on the basis of solvent and detergent preparation, substrate specificity, thermal tolerance , and production of zein hydrolysates .

Keratin
Keratins are proteins that are usually present in two forms, namely, hard keratins and soft keratins. 
Hard keratins mainly include the structural proteins that are prevalently present in fingernails, horns, beaks, upper layer of skin, and mainly hair. 
Fibers of the keratin proteins are self-assembled into compact follicles that make up the structure of hair. 
The process of assembling keratin proteins into a complex hair is under the control of multiple genes, cytokines, and growth factors. 
In contrast to hard keratins, soft keratins are those that are abundantly present in tissues, such as epithelial tissues. 
Proteases structure of wool keratin shows great similarity to hair keratin. 
Three types of hair keratin have been known. 
The first one is the alpha keratins; these range in size from 60 to 80 kDa. 
Having low sulfur content, these comprise mainly of alpha-helical domains. 
Overall, alpha keratins make up the structural class of proteins, as they reside in the fiber cortex of hair. 
Proteases second type is the beta keratins, which are a non-extractable, less-studied class of keratins. 
These are usually present in the hair cuticle and perform protective functions. 
Proteases third type is the gamma keratins, which have a high sulfur content; these keratins are ~15 kDa in size. 
Their size is comparatively smaller than the other classes of keratin.
These keratins help to maintain the cortical superstructure by cross-linking the disulfide bonds in the hair. 
All these types of keratins can be degraded by the enzyme keratinase, which belongs to a class of protease enzymes. 
Proteases, which account for 60% of the world's marketed enzymes, is responsible for many applications, such as detergents, food, and leather processing .

The enzyme keratinase (E.C. 3.4.99.11) is one of the serine hydrolase groups that disrupt the disulfide hydrogen bonds in the keratin proteins. 
According to UniProt results, one of the protein keratinases produced by Bacillus subtilis contains two domains. 
The first one is 59 amino acids long and encodes for inhibitor I9; the other one is 243 amino acids long and encodes for peptidase S8. 
The first domain occurs from 19 to 77 amino acid sequences and the second domain occurs from 103 to 345 amino acid sequences. 
The enzyme also has a metal ion binding site for calcium ion. 
This means that calcium ions act as cofactors for keratinases; the presence of calcium ions in the media can enhance the activity of keratinases. 
The structure of keratinase makes it very efficient in its function of degrading keratin proteins. 
Our daily green waste and animal waste includes plenty of keratins, which remain undegraded due to their complexity. 
Such insoluble keratins may lead to environmental pollution if left untreated. 
Thus, as a solution, such wastes are treated by keratinase enzymes, which convert the waste into simpler as well as biodegradable substances. 
The extracellular keratinases have been successfully isolated from several microbes by using several fermentation techniques and by optimizing conditions, such as pH, temperature, and type of nitrogen and carbon source and the choice of microbe. 
The keratinases from microbes are effective, biodegradable, and economic and provide much better results as compared to chemical treatments.

Alkaline Proteases
The genus Bacillus is vital for commercially important alkaline protease (EC.3.4.21-24.99), which is active at alkaline pH ranging between 9 and 11. 
These alkaline protease producers are distributed in water, soil, and highly alkaline conditions. 
From a variety of sources, such as detergent contamination, dried fish, sand soil, and slaughterhouses, segregation of alkaline proteases has been stated. 
The detergent industry consumes alkaline proteases most abundantly, which are serine proteases with an alkaline pH range. 
These alkaline serine proteases, which are easily inactivated by phenyl methane sulfonyl fluoride (PMSF), account for one-third of the share of the enzyme market. 
Alkaline proteases are unique in their activity and maintain a constant alkaline pH while being exploited for different formulations in pharmaceutical, food, and other related industries. 

A broad range of applications of these alkaline proteases are getting more attention from researchers with the hope of discovering new strains with unique properties and substantial activity. 
It is reported that for dehairing of animal skin and hides, Bacillus sp. provide the desired hydrolytic, elastolytic, and keratinolytic properties. 
These Bacillus strains have been commercially exploited around the globe due to the huge amounts of enzyme secreted with high enzymatic activity. 
Although alkaline proteases are produced by multiple sources, with the increasing demand of protease in the market, and for cost-effectiveness, only those strains that show greater yield with hyperactivity will be accepted in the current biotechnological advancement. 
Two essential types of alkaline proteases, such as subtilisin Carlsberg and subtilisin novo are obtained from Bacillus sp. , which can be used as an industrial enzyme to produce zein hydrolysates. 
In halophilic sources, different microbial sp. secreting serine alkaline proteases are also reported. 
The entomopathogenic bacterium Photorhabdus sp. strain EK1 (PhPrtPI) containing Ca2+ alkaline protease is categorized as a metalloprotease. 
Owing to its broad-spectrum specificity with different proteins and peptides, it is suggested that PhPrtPI provides nutrients to the nematodes by degradation of insect tissues . 

A Salinivibrio sp. strain, AF-2004, produces metallotype protease with a reasonable thermal tolerance and a broad range of pH (5.0–10.0). 
Proteases is a highly recommended strain due to its thermal and halophilic properties. 
Another strain, Bacillus clausii, is also recommended for use at a commercial scale for the production of alkaline protease with the use of peptone, Cu, and fructose as the sole source of energy. 
The optimum pH and temperature recommended is 8–9 and 37–40°C, respectively. 
A strain of Bacillus sp., MPTK 712, isolated from dairy slush producing alkaline protease exhibits a symbiotic relationship with marine shipworms. 
Very rare microbes, such as Kurthia spiroforme are also capable of producing alkaline protease. 
Some alkaline serine proteases recognized by goat skin metagenomics library shows homology to peptidases and Cryptococcus aureus shows good bioactivity with optimum temperature (45–50°C) and pH (9–10). 
Different mushrooms producing alkaline protease are also reported.

Acidic Protease
Acid proteases are stable and active between pH 3.8 and 5.6 and are frequently used in soy sauce, protein hydrolysate, and digestive aids and in the production of seasoning material. 
The optimum pH of acidic proteases is 3–4 and the isoelectric point range is between 3 and 4.5 with a molecular weight of 30–45 kDa. 
Furthermore, acid proteases are also exploited for use in clearing beer and fruit juice, improving texture of flour paste, and tenderizing the fibril muscle . 
In comparison with alkaline proteases, these extracellular acid proteases are mostly produced by fungal species, such as Aspergillus niger, Aspergillus oryzae, Aspergillus awamori, Aspergillus fumigatus, and Aspergillus saitoi. 
Most of the fungal extracellular acid proteases are known as aspergilla opepsins. Aspartic proteases are acid proteases consisting of 380–420 long chains of amino acid residues constituting the active site for catalytic activity. 
Proteases acidic proteases are endopeptidases and grouped into three families: pepsin (A1), retropepsin (A2), and enzymes from Para retroviruses (A3). 
These three families are placed in clan AA. 
Proteases is found that A1 and A2 are closely related to each other while members of the A3 family show some relatedness to families A1 and A2. 
An active site cleft of the members of the pepsin family is located between lobes of a bilobal structure. 
A great specificity of acidic proteases is exhibited against aromatic amino acid residues located on both sides of the peptide bond. These aromatic amino acid residues with peptide bonds are similar to pepsin but less stringent in action. 
Broadly, acidic proteases are divided into two groups: (i) pepsin-like enzymes and (ii) rennin-like enzymes produced by Penicillium, Aspergillus, Rhizopus, Endothia, and Mucor (Tomoda and Shimazono, 1964).

Neutral Proteases
Neutral proteases are defined as, such as they are active at a neutral or weakly acidic or weakly alkaline pH. 
Mostly neutral proteases belong to the genus Bacillus and with a relatively low thermotolerance ranging from pH 5 to 8 (Table 1). 
They generate less bitterness in hydrolysis of food proteins due to a medium rate of reaction; therefore, they are considered more valuable in the food industry. 
Neutrase is incorporated in the brewing industry due to its insensitivity to plant proteinase inhibitors. 
On the basis of high affinity toward hydrophobic amino acids, neutral proteases are identified and characterized. 
During production of food hydrolysate, it is slightly advantageous to control the reactivity of neutral proteases due to low thermotolerance. 
A divalent metal ion is required for the activity of neutral proteases belonging to the metalloprotease type

Proteases have also found more specialized applications in processes for purification of nonprotein products from animal or plant extracts including extraction of carbohydrate gums and mucopolysaccharides. 
Proteases may be used for solubilization of keratin materials to convert waste materials such as feathers to protein concentrates for use as animal feeds. 
An alkaline protease from Streptomyces species also has strong keratinolytic activity. 
The plant proteases, papain and bromelain, are effective as meat tenderization enzymes, as is the B. subtilis neutral protease. 
Other industrial applications of proteases include their use in silver recovery from conventional gelatin-containing photographic film including X-ray film, and in the liquefaction of industrial and household organic waste. 
Proteases may also be consumed by humans and animals as digestive aids.

Proteases are enzymes that break the peptide bonds of proteins; they are divided into acid, neutral, and alkaline proteases. 
These enzymes can be obtained from plants, animals, and microorganisms in several conditions, such as high salt concentrations. 
The halophilic proteases possess stable activity at high temperature and ionic strength in presence of organic solvents. 
Some protease enzymes have potential uses in detergents, the pharmaceutical industry, bioremediation processes, and food industries.

Stability to the organic solvent by different proteases has been studied, such as the tolerance of protease from the Halobacillus blutaparonensis strain M9 to ether, isooctane, and cyclohexane; and the tolerance of the Geomicrobium sp.
EMB2 to ethanol, benzene, cyclohexane, and heptane. 
In food industries, proteases play a role in fermentation processes to produce compounds with specific characteristic of flavor and aroma. 
Proteases have also been used in fish sauce preparation, in the pretreatment of leather in the tanning industry, and in the formulation of therapeutic dietary products

Proteolytic enzymes (proteases) are enzymes that break down protein. 
These enzymes are made by animals, plants, fungi, and bacteria.

Proteolytic enzymes break down proteins in the body or on the skin. 
This might help with digestion or with the breakdown of proteins involved in swelling and pain. 
Some proteolytic enzymes that may be found in supplements include bromelain, chymotrypsin, ficin, papain, serrapeptase, and trypsin.

Proteolytic enzymes are used for a long list of conditions including cleaning wounds on the skin, help with digestion, pain and swelling, and many other conditions. 
Refer to specific topics for more information on uses and effects.

proteolytic enzyme, also called protease, proteinase, or peptidase, any of a group of enzymes that break the long chainlike molecules of proteins into shorter fragments (peptides) and eventually into their components, amino acids. 
Proteolytic enzymes are present in bacteria, archaea, certain types of algae, some viruses, and plants; they are most abundant, however, in animals.

There are different types of proteolytic enzymes, which are classified according to sites at which they catalyze the cleavage of proteins. 
The two major groups are the exopeptidases, which target the terminal ends of proteins, and the endopeptidases, which target sites within proteins. 
Endopeptidases employ various catalytic mechanisms; within this group are the aspartic endopeptidases, cysteine endopeptidases, glutamic endopeptidases, metalloendopeptidases, serine endopeptidases, and threonine endopeptidases. 
The term oligopeptidase is reserved for those enzymes that act specifically on peptides.

Among the best-known proteolytic enzymes are those that reside in the digestive tract. 
In the stomach, protein materials are attacked initially by a gastric endopeptidase known as pepsin. 
When the protein material is passed to the small intestine, proteins, which are only partially digested in the stomach, are further attacked by proteolytic enzymes secreted by the pancreas. 
These enzymes are liberated in the small intestine from inactive precursors produced by the acinar cells in the pancreas. 
The precursors are called trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase. 
Trypsinogen is transformed to an endopeptidase called trypsin by an enzyme (enterokinase) secreted from the walls of the small intestine. 
Trypsin then activates the precursors of chymotrypsin, elastase, and carboxypeptidase. When the pancreatic enzymes become activated in the intestine, they convert proteins into free amino acids, which are easily absorbed by the cells of the intestinal wall. 
The pancreas also produces a protein called pancreatic secretory trypsin inhibitor, which binds to trypsin and blocks its activity. 
It is thought that in this manner the pancreas protects itself from autodigestion. 

Proteases likely arose at the earliest stages of protein evolution as simple destructive enzymes necessary for protein catabolism and the generation of amino acids in primitive organisms. For many years, studies on proteases focused on their original roles as blunt aggressors associated with protein demolition. However, the realization that, beyond these nonspecific degradative functions, proteases act as sharp scissors and catalyze highly specific reactions of proteolytic processing, producing new protein products, inaugurated a new era in protease research. 
The current success of research in this group of ancient enzymes derives mainly from the large collection of findings demonstrating their relevance in the control of multiple biological processes in all living organisms. 

Thus, proteases regulate the fate, localization, and activity of many proteins, modulate protein-protein interactions, create new bioactive molecules, contribute to the processing of cellular information, and generate, transduce, and amplify molecular signals. 
As a direct result of these multiple actions, proteases influence DNA replication and transcription, cell proliferation and differentiation, tissue morphogenesis and remodeling, heat shock and unfolded protein responses, angiogenesis, neurogenesis, ovulation, fertilization, wound repair, stem cell mobilization, hemostasis, blood coagulation, inflammation, immunity, autophagy, senescence, necrosis, and apoptosis. 
Consistent with these essential roles of proteases in cell behavior and survival and death of all organisms, alterations in proteolytic systems underlie multiple pathological conditions such as cancer, neurodegenerative disorders, and inflammatory and cardiovascular diseases. 

Accordingly, many proteases are a major focus of attention for the pharmaceutical industry as potential drug targets or as diagnostic and prognostic biomarkers. 
Proteases also play key roles in plants and contribute to the processing, maturation, or destruction of specific sets of proteins in response to developmental cues or to variations in environmental conditions. 
Likewise, many infectious microorganisms require proteases for replication or use proteases as virulence factors, which has facilitated the development of protease-targeted therapies for diseases of great relevance to human life such as AIDS. 
Finally, proteases are also important tools of the biotechnological industry because of their usefulness as biochemical reagents or in the manufacture of numerous products.

This outstanding diversity in protease functions directly results from the evolutionary invention of a multiplicity of enzymes that exhibit a variety of sizes and shapes. 
Thus, the architectural design of proteases ranges from small enzymes made up of simple catalytic units (∼20 kDa) to sophisticated protein-processing and degradation machines, like the proteasome and meprin metalloproteinase isoforms (0.7–6 MDa) (15). 
In terms of specificity, diversity is also a common rule. 
Thus, some proteases exhibit an exquisite specificity toward a unique peptide bond of a single protein; however, most proteases are relatively nonspecific for substrates, and some are overtly promiscuous and target multiple substrates in an indiscriminate manner. 
Proteases also follow different strategies to establish their appropriate location in the cellular geography and, in most cases, operate in the context of complex networks comprising distinct proteases, substrates, cofactors, inhibitors, adaptors, receptors, and binding proteins, which provide an additional level of interest but also complexity to the study of proteolytic enzymes.

This work aims at serving as a primer to a minireview series on proteases to be published in forthcoming issues of this Journal. 
This introductory article will focus on the discussion of the large and growing complexity of proteolytic enzymes present in all organisms, from bacteria to man. 
We will first show the results of comparative genomic analysis that have shed light on the real dimensions of the proteolytic space. 
The levels of protease complexity and mechanisms of protease regulation will then be addressed. Finally, we will discuss current frontiers and future perspectives in protease research.

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The Vast Proteolytic Landscape
Proteases are the efficient executioners of a common chemical reaction: the hydrolysis of peptide bonds. 
Most proteolytic enzymes cleave α-peptide bonds between naturally occurring amino acids, but there are some proteases that perform slightly different reactions. 
Thus, a large group of enzymes known as DUBs (deubiquitylating enzymes) can hydrolyze isopeptide bonds in ubiquitin and ubiquitin-like protein conjugates; γ-glutamyl hydrolase and glutamate carboxypeptidase target γ-glutamyl bonds; γ-glutamyltransferases both transfer and cleave peptide bonds; and intramolecular autoproteases (such as nucleoporin and polycystin-1) hydrolyze only a single bond on their own polypeptide chain but then lose their proteolytic activity. 
Notably and under some conditions, proteases can also synthesize peptide bonds.

Proteases were initially classified into endopeptidases, which target internal peptide bonds, and exopeptidases (aminopeptidases and carboxypeptidases), the action of which is directed by the NH2 and COOH termini of their corresponding substrates. 
However, the availability of structural and mechanistic information on these enzymes facilitated new classification schemes. 
Based on the mechanism of catalysis, proteases are classified into six distinct classes, aspartic, glutamic, and metalloproteases, cysteine, serine, and threonine proteases, although glutamic proteases have not been found in mammals so far. 

The first three classes utilize an activated water molecule as a nucleophile to attack the peptide bond of the substrate, whereas in the remaining enzymes, the nucleophile is an amino acid residue (Cys, Ser, or Thr, respectively) located in the active site from which the class names derive. 
Proteases of the different classes can be further grouped into families on the basis of amino acid sequence comparison, and families can be assembled into clans based on similarities in their three-dimensional structures.
Bioinformatic analysis of genome sequences has been decisive for establishingthedimensionsofthe complexity of proteolytic systems operating in different organisms. 
The last release of MEROPS (merops.sanger.ac.uk), a comprehensive data base of proteases and inhibitors, annotates 1008 entries for human proteases and homologs, although it includes a large number of pseudogenes and protease-related sequences derived from endogenous retroviral elements embedded in our genome. 
A highly curated data base, the Degradome Database, which does not incorporate protease pseudogenes or these retrovirus-derived sequences, lists 569 human proteases and homologs classified into 68 families. 
Metalloproteases and serine proteases are the most densely populated classes, with 194 and 176 members, respectively, followed by 150 cysteine proteases, whereas threonine and aspartic proteases contain only 28 and 21 members, respectively.

 Proteolytic hydrolysis of peptide bonds is recognized as an essential and ubiquitous mechanism for the regulation of a myriad of physiological processes. 
Four main classes of proteolytic enzymes have been routinely utilized to describe proteases. 
The serine proteases are probably the best characterized. 
This class of proteases includes trypsin, chymotrypsin and elastase. 
The cysteine protease class includes papain, calpain and lysosomal cathepsins. 
Aspartic proteases include pepsin and rennin. 
Metallo-proteases include thermolysin and carboxypeptidase A. 
Explore our comprehensive offering of rigorously tested proteinases for your protein workflow needs.

PROTEASES FOR RESEARCH APPLICATIONS
In order to assist with your protease selection, our Protease Finder allows researchers to locate endo- and exopeptidases that are required for precise protein cleavage. 
Facilitating the catalytic breakdown of proteins in smaller polypeptides or single amino acids, our proteases provide a comprehensive range of offerings to meet your protein research application needs.

PROTEASES FOR PROTEOMICS RESEARCH
Proteases are commonly used tools in proteomics research and suitable for digesting proteins into small peptide fragments for mass spectrometry analysis followed by sequencing (tandem MS). 
Trypsin generates peptides in the useful mass range for mass spectrometry and is recognized as the most used protease for protein identification. 
Explore our solution-stable recombinant SOLu-Trypsin products (EMS0004) (EMS0005) for additional information. 
Additionally, there are instances where separate or sequential digestion with other proteases may be a better choice for your protein of interest. 
We offer a variety of sequencing grade proteases that are appropriate for use in mass spectrometry sample preparation.

PROTEASES FOR INDUSTRIAL AND APPLIED RESEARCH
Proteases have commercial importance in different industrial and applied sectors and have multiple applications. 
Due to their wide variety of physiological characteristics, including hydrolysis at pH extremes or elevated temperatures, they ideal for use in the pharmaceutical, diagnostic, textile, food and beverage sectors. 
Specific applications for these proteases include but are not limited to; dietary fiber testing and clean-in-place additives for contaminate removal. 
We have a wide range of purified enzymes and enzyme mixtures for researching and developing new processes, products, and assays.

PROTEASE DETECTION KITS
We offer two easy-to-use kits to detect trace amounts of protease, or to determine total protease activity, using the same methodologies utilized by our QC department for years. 
We have used these protocols to test thousands of samples for proteolytic activity.

The Protease Detection Kits are complete kits for detection of primary or trace protease activity by your choice of fluorometric or colorimetric detection. Everything you need, including control, standards, buffers, and substrate, is included. 
For convenience, the assay can be performed in either a cuvette or microplate format. 
Each kit contains enough reagents for 200 x 1 mL assays. 
For greater sensitivity when measuring low levels of protease activity, the Protease Fluorescent Detection kit (PF0100) is recommended. For more accurate quantification of primary protease activity, the Protease Colorimetric Detection kit is (PC0100) recommended.

Both the Fluorometric Detection Kit and the Colorimetric Detection Kit have been tested against representative samples of all four types of protease classes (serine, aspartic, cysteine and metalloproteases) to ensure broad application suitability.

Proteases are enzymes that hydrolyze other proteins, including other proteases, which challenges assumptions of enzyme inertness in chemical reactions, and alters predicted protease−substrate concentrations using established mass action frameworks. 
Cysteine cathepsins are powerful proteases involved in numerous diseases by cleaving substrates, but they also hydrolyze each other, requiring inclusion of as yet undefined, protease-as-substrate dynamics. 
Here, we used experimental and computational models to improve predictions of the concentrations of multiple species and intermediates generated during substrate degradation in multiprotease systems by including protease-on-protease reactions of autodigestion, inactivation, cannibalism, and distraction in proteolytic networks. 
This was made available online for others to test perturbations and predict shifts in proteolytic network reactions and system dynamics

Abstract
Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. 
However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. 
Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. 

Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. 
Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. 
During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. 
Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. 

Proteases, as also called peptidases or proteinases, are enzymes that perform proteolysis. Proteolysis is one of the most important biological reactions. Proteolytic activity has been attributed to a class of enzymes called proteases. 
These enzymes are of wide distribution, and they perform significant biological processes. 
Proteases have evolved to perform these reactions by numerous different mechanisms and different classes of protease can perform the same reaction by completely different catalytic mechanisms. 
Proteases are found in animals, plants, bacteria, archaea, and viruses. 
Proteases are involved in protein processing, regulation of protein function, apoptosis, viral pathogenesis, digestion, photosynthesis, and numerous other vital processes. 
Proteases mechanism of action classifies them as either serine, cysteine or threonine proteases (amino-terminal nucleophile hydrolases), or as aspartic, metallo and glutamic proteases (with glutamic proteases being the only subtype not found in mammals so far).

Which are the functions of proteases?
Proteases are involved in many aspects of human biology. 
For example, in the small intestine, proteases digest dietary proteins to allow absorption of amino acids. 
Other processes mediated by proteases include blood coagulation, immune function, maturation of prohormones, bone formation, programmed cell death and the recycling of cellular proteins that are no longer needed.

Proteases classified by catalytic domains
Proteases can be classified into seven broad groups:

Serine proteases
Using a serine alcohol, display a wide range of functions.

Cysteine proteases
Using a cysteine thiol, that include caspases which are involved in apoptosis and inflammation, and cathepsins which promote protein degradation.

Threonine proteases
Using a threonine secondary alcohol

Glutamic proteases
Using a glutamate carboxylic acid

Metalloproteases
Using a metal, usually zinc. 
The Metalloprotease family includes aminopeptidases and endopeptidases, which are secreted, membrane-bound, or cytosolic.

Asparagine peptide lyases
Using an asparagine to perform an elimination reaction (not requiring water)
 
Proteases were traditionally viewed as mere protein-degrading enzymes with a very restricted spectrum of substrates. 
A major expansion in protease research has uncovered a variety of novel substrates, and it is now evident that proteases are critical pleiotropic actors orchestrating pathophysiological processes. 
Recent findings evidenced that the net proteolytic activity also relies upon interconnections between different protease and protease inhibitor families in the protease web.

In this review, we provide an overview of these novel concepts with a particular focus on pulmonary pathophysiology.
We describe the emerging roles of several protease families including cysteine and serine proteases.

The complexity of the protease web is exemplified in the light of multidimensional regulation of serine protease activity by matrix metalloproteases through cognate serine protease inhibitor processing. 
Finally, we will highlight how deregulated protease activity during pulmonary pathogenesis may be exploited for diagnosis/prognosis purposes, and utilised as a therapeutic tool using nanotechnologies.

Considering proteases as part of an integrative biology perspective may pave the way for the development of new therapeutic targets to treat pulmonary diseases related to intrinsic protease deregulation.

Proteases & Other Enzymes 
Protease is a general term for a class of enzymes that hydrolyze protein peptide bonds. 
According to the manner in which the polypeptide is hydrolyzed, it can be divided into two types, an endopeptidase and an exopeptidase. 
The endopeptidase cleaves the inside of the protein molecule to form a small molecular peptide. 
The exopeptidase hydrolyzes the peptide bond one by one from the terminal of the free amino group or carboxyl group of the protein molecule, and the amino acid is released, the former being an aminopeptidase and the latter being a carboxypeptidase. 
Protease can be further divided into serine protease, thiol protease, metallo proteinase and aspartic protease according to its active center. 

According to the optimum pH value of the reaction, it is divided into acid protease, neutral protease and alkaline protease.
Protease is used in industrial production, mainly endopeptidase.
Proteases are widely found in animal viscera, plant stems, leaves, fruits and microorganisms. 
Microbial proteases are mainly produced by molds and bacteria, followed by yeasts and actinomycetes. 
Proteases have many types, and important ones are pepsin, trypsin, cathepsin, papain, and subtilisin. 
Protease has strict selectivity for the reaction substrate to be applied. 
Proteases can only act on certain peptide bonds in the protein molecules, such as peptide bonds formed by trypsin catalyzed hydrolysis of basic amino acids. 
Protease is a widely distributed protein, and is especially abundant in the digestive tract of humans and animals. 
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 oryzae.

Other Enzymes
An enzyme is a protein or RNA produced by living cells that is highly specific and highly catalytically potent to its substrate. 
Enzymes are a very important class of biocatalysts. 
According to the nature of the reaction catalyzed by the enzyme, the enzyme is divided into six categories. 

Oxidoreductase is an enzyme that promotes the redox reaction of the substrate. 
Proteases is a kind of enzyme that catalyzes the redox reaction and can be divided into two types, oxidase and reductase. 
Transferases are enzymes that catalyze the transfer or exchange of certain groups (such as acetyl, methyl, amino, phosphate, etc.) between substrates, including methyltransferase, aminotransferase, acetyltransferase, kinase and polymerase, and so on. 
Hydrolases enzymes catalyze the hydrolysis of substrates, such as amylase, protease, lipase, phosphatase, glycosidase, and the like. 
Lyases remove a group from the substrate (non-hydrolyzed) and leave a double bond or its reverse reaction, including dehydratase, decarboxylase, carbonic anhydrase, aldolase, citrate synthase, and the like. 
Many lyases catalyze a reverse reaction that creates a new chemical bond between the two substrates and eliminates the double bond of a substrate, and synthase belongs to this class. 

Isomerase converts its substrates between various isomers, geometrical isomers, or optical isomers. 
Ligase catalyzes the synthesis of a two-molecular substrate into a single molecule of compound, coupled with an ATP-linked phosphate-cleaving enzyme, such as glutamine synthetase, DNA ligase, biotin-dependent carboxylase, and the like. 
According to the principle of uniform classification of enzymes published by the International Biochemical Association, on the basis of the above six categories, and each of the main types of enzymes, as well as the characteristics of the groups or bonds acting on the substrate, it is divided into several sub-categories. 
Precisely indicating the nature of the substrate or reactant, each subclass is subdivided into several groups (sub-subclasses); each group contains several enzymes directly. 
Due to the character of the enzyme, the chemical reaction in the living body can be carried out efficiently and specifically under extremely mild conditions.

Proteases are a class of enzymes which perform proteolysis, the mechanism of digesting or cleaving long chain proteins into smaller fragments by severing the peptide bonds that hold them together. 
Trypsin, a serine protease secreted by the pancreas, is commonly used in cell tissue culture to cleave the bonds that cells use to adhere to a flask.

Proteases also play a major role in disease. HIV-1 protease is essential to the viral replication process. MMP-9, a matrix metallopeptidase, plays a role in angiogenesis and is a therapeutic target for cancer. 
Proteases have an important role in many signaling pathways, therefore it can be difficult to achieve selectivity when targeting protease active sites.

Eurofins Discovery's broad portfolio of over 50 protease targets span across the serine, cysteine, aspartic, and metalloprotease families and provides the foundation for our services in screening and profiling. 
Key protease families and targets included in our services are highlighted below.

Proteases are classified by the amino acids or ligands that catalyze the hydrolysis reaction. 
For example, serine proteases contain a serine in the active site. 
The serine is helped by a neighboring histidine and aspartic acid. 
This combination is called the catalytic triad, and is conserved in all serine proteases. 
Serine proteases work in a two step fashion; first, they form a covalent bond with the protein to be cleaved; in the second step, water comes in and releases the second half of the cleaved protein. 
Cysteine proteases use cysteine as a nucleophile just like serine proteases use serine as a nucleophile.

Serine proteases include a number of digestive enzymes, including Trypsin, Chymotrypsin, and Elastase. 
While they all contain the same three amino acids that work together to catalyze the reaction, called the catalytic triad, they differ in where they cleave proteins. 

This specificity is due to a binding pocket that contains different functional groups. 
Chymotrypsin prefers a large hydrophobic residue; its pocket is large and contains hydrophobic residues. 
In this representation of the binding pocket, the hydrophobic phenylalanine of the substrate is shown in green, and the hydrophobicity of the surrounding amino acids is shown by grey (hydrophobic) or purple (hydrophilic) balls. 
Trypsin is specific for positively charged residues like lysine, and contains a negative amino acid, aspartic acid, at the bottom of the pocket. 
Elastase prefers a small neutral residue; it has a very small pocket.

Cysteine proteases include enzymes that have a role in regulating cellular processes such as caspases and deubiquitinase.

Another class of protease is aspartate proteases. 
This family includes HIV protease. HIV produces its proteins as one long chain; HIV protease cleaves the long protein into functional units. 
Because it cleaves long proteins, it has a tunnel to accommodate the long peptide substrate, and the top "flaps" of the protein can open and closeto allow the substrate in and products out. 
Aspartate proteases include two aspartate residues in the active site, which increase the reactivity of an active site water molecule to directly cleave the substrate protein.

A third class of proteases are metalloproteases such as carboxypeptidase. 
Carboxypeptidases remove the C terminal amino acids from proteins. 
The active site contains zinc , which is bound to the protein through interactions with histidine (H), serine (S) aspartic acid (E) residues.

The virtual meeting on Serine Proteases in Pericellular Proteolysis and Signaling continues the tradition of the ASBMB special symposium on membrane-anchored serine proteases with an expanded focus on other related proteases with overlapping substrates and functions in the pericellular environment.

The conference traditionally brings together the leading national and international researchers in the field of pericellular proteolysis and provide them with a forum to present their latest findings, exchange ideas and technologies, and network to form collaborations. 
Equally important, it also provides a venue for junior investigators at the graduate student and postdoctoral level to discuss their current research, meet with experts in the field and forge new scientific interactions crucial for their future career development. 
To this end, we plan an interactive poster session and easy access to poster presenters' video recordings to increase the visibility of their work. 
Holding the meeting virtually provides a unique opportunity to make this conference even more accessible to students as well as to investigators from other fields where pericellular proteolysis is implicated.

Topics covered
Cleavage of proteins in the extracellular environment, including hormones, growth factors and their receptors, ion channels, cell adhesion molecules and structural components of extracellular matrix, plays a key role in the regulation of cell behavior. 
Among more than 500 proteolytic enzymes encoded by mammalian genomes, membrane-anchored serine proteases, which are expressed on the cell surface in all major organs, are excellently suited to mediate signal transduction across the plasma membrane and are increasingly being recognized as important regulators of organ development and homeostasis. 
At the same time, unrestrained pericellular proteolysis has been shown to contribute to epithelial and endothelial barrier dysfunction, inflammatory, cardiovascular, and respiratory diseases, as well as cancer. 
Many of the influenza- and coronavirus-type viruses, including SARS-CoV-2, are now known to also use activity of these proteases to gain entry into the target cell, making MASPs a major determinant of cell susceptibility to infection.

In addition to the roles of serine proteases in viral biology, the meeting will cover topics including biosynthesis, trafficking and post-translational modifications, endogenous and pharmacological inhibitors, developmental and other physiological functions, mechanisms of dysregulation and pathological consequences, and molecular mechanisms of protease-mediated signaling.

Proteases are enzymes that specialize in the cleavage of peptide bonds. 
Their activities may be relatively indiscriminate, breaking polypeptides down to their basic elements, or exquisitely precise, cleaving a substrate at a specific residue to alter protein activity. 
These illustrations highlight scientific concepts that rely on proteolytic activity and emphasize the importance of proteases in some of the most studied areas of cell biology.

Enzymes are biocatalysts essential for life that catalyze almost every biological process.
Since ancient times, enzymes have been used in the manufacturing of different food products, including beer, wine, vinegar, cheese, and sourdough, and in the production of commodities like leather, linen and indigo.
Biocatalysis has evolved as a necessary tool in industrial production of active pharmaceuticals, agrochemical and pharmaceutical intermediates, bulk chemicals, and food ingredients.
Although enzymes were initially not used in pure form, fermentation processes producing pure enzymes from a specific strain were eventually developed at large scale
Microorganisms have made an essential contribution in industrial biology.
Microbial enzymes play an important role as metabolic catalysts and hence are used in various industrial applications. 
Currently, most enzymes used in industrial processes are hydrolytic and are used for the degradation of various natural substances.

Protease remains the dominant type of enzyme because of its extensive use in dairy and detergent industries. 
Proteases are very important enzymes, accounting for more than 60% of total global enzyme sales.
These enzymes can be broadly divided into two major groups: endopeptidases, which cleave internal peptide bonds, and exopeptidases, which cleave C- or N- terminal peptide bonds 
Protease enzyme catalyzes the hydrolytic reaction, which brings about the breakdown of protein molecules to amino acids and peptides.
Microbes serve as a better source of proteases than plants and animals because they can be cultured in large amounts in a short duration, are relatively inexpensive, and can produce a continuous supply of the desired product.

There are three well known enzymes that go through the serine protease mechanism of action, they are: chymotrypsin, trypsin and elastase. 
We will look at the enzyme mechanism of chymotrypsin in detail.

A protease is an enzyme that hydrolyzes peptide bonds that link amino acids together in a protein. Proteases are specific for certain amino acids and can hydrolyze those amino acids on the carboxy or amino side of the peptide bond.

There are two general types of proteases:
Endoproteases (Serine Proteases):
Can cleave specific peptide bonds within the protein.

Exoproteases:
Cleave only terminal amino acid residues.
Proenzymes (Zymogens):
An enzyme synthesized initially in an inactive form, but are present and poised to act quickly when needed. 
Serine Proteases are good examples of proenzymes or zymogens.

Physiological Roles for Proenzymes such as Serine Proteases:
1) Digestion of proteins in the small intestine.
i.e. Trypsinogen (proenzyme) ---------> Trypsin (active form)
2) Blood Coagulation

Chymotrypsin:
>Used as an example of a serine protease because it's structure and mechanism are well understood.
>Catalyzes the hydrolysis of peptide bonds, on the carboxyl side of bulky aromatic side chains (Tyr, Phe, Trp).

Active Site:
1) Serine, to which the substrate binds, all serine protease active sites contain serine.
2) Histidine, ability to donate and accept protons.
3) Aspartate, ability to accept protons, final link necessary for chymotrypsin action.

Notes:
>These residues are polar (hydrophilic) so would not ordinarily be found on the "interior" of a protein and would normally be deprotonated at physiological pH, except for their critical role in catalysis.
>Though they are in close proximity in the 30 structure, they are not adjacent in the primary sequence

What is a protease?
Proteins are chains of individual amino acids that are linked by covalent bonds — called peptide bonds — during the process of protein translation. 
New proteins are constantly being translated and old proteins degraded in the cell to ensure precise concentrations of the necessary functional protein to complete various cellular processes. 
The process of protein degradation, referred to as proteolysis, breaks proteins back down into individual amino acids. Proteolysis is accomplished by proteases, a group of enzymes whose mechanisms depend on the particular class of protease.

Protease activity is highly regulated in the cell, and this regulation occurs through a variety of mechanisms. 
One example of regulated protease activation is during the initiation of apoptosis, or programmed cell death. 
Caspase activation and subsequent cleavage of downstream proteins play a significant role in apoptosis. 
Most cellular caspases exist as a procaspase (an inactive version of the protease) that must be proteolytically cleaved to exhibit protease activity. 
Proteins in the IAP (inhibitor of apoptosis) family block apoptosis by either preventing the cleavage of a procaspase or by directly inhibiting caspase activity.

The sub-cellular localization of proteases can also control their activity. Some proteases are sequestered within specific organelles such that they can only degrade proteins that are targeted to those organelles. 
For example, cathepsins are proteases predominantly localized to lysosomes.
These proteases degrade proteins that are targeted to lysosomes. 
In intact cells, proteins not localized to lysosomes do not come into contact with the cathepsins and , thus, are not proteolyzed by them.

Proteolysis in protein isolation
When cells are lysed, regulatory mechanisms are disrupted and organelles can be ruptured. 
Cellular mechanisms of protease inhibition or protease sequestration will thus be eliminated. 
Normally sequestered proteases can then act on proteins with which they don’t generally come into contact. 
Previously inactive proteases might become activated and begin cleaving a variety of proteins. 
Because of the vast number and types of proteases present in organisms commonly used for protein expression (Table 1), it is easy to imagine that proteins of interest can be subject to extensive proteolysis upon cell lysis.

Because of this potential unwanted protease activity, it is necessary to take measures to prevent proteolysis during protein isolation protocols. 
Generally, only then can full-length, functional protein be isolated for use in experimental studies. 
Protease inhibitors are typically used in these types of studies, and the type of inhibitor(s) to use depends on the types of proteases that need inhibiting.

Protease Classification
Proteases can be classified by their mechanism of action and their substrate specificity.

General classification
Broadly, proteases can be characterized as endopeptidases or exopeptidases. 
Endopeptidases cleave bonds within the protein and are usually very specific for particular amino acid sequences. 
Exopeptidases cleave a single amino acid from the terminus of a protein, and are less specific toward the amino acid that is being cleaved. 
Exopeptidases are themselves classified as those that cleave only from the C-terminus (carboxypeptidases), only from the N-terminus (aminopeptidases), or both termini (dipeptidases). 
Both endopeptidases and exopeptidases can be very problematic during protein purification. 
Endopeptidases can recognize regions in the protein of interest and perform proteolysis to digest the protein into many smaller pieces that are useless in experimental studies. 
Exopeptidases can remove just a few amino acids which may dramatically affect protein function but is often not detectable by conventional methods.

Mechanism of proteolysis
Proteases are also characterized by their mechanism of action; that is, the amino acids that are involved in the catalytic site of the enzyme. 
The catalytic site contains the amino acids that directly play a role in facilitating peptide bond hydrolysis (Figure 1). 
This process requires a nucleophile to initiate the reaction, which can either be an active site amino acid side chain or a water molecule. 
During the later stages of proteolysis, the two parts of the hydrolyzed protein are released and the protease active site returns to its initial state, ready to bind another substrate for proteolysis.

Proteases are typically grouped into four major classes based on their active site residues: serine proteases, cysteine (thiol) proteases, aspartic proteases, and metalloproteases. 
Serine and cysteine (thiol) proteases have an amino acid within the active site that performs the initial nucleophilic attack, while aspartic proteases and metalloproteases activate a water molecule to perform the initial nucleophilic attack.

Serine proteases  act through a catalytic triad composed of a serine, a histidine residue, and an aspartate residue.
Cysteine (thiol) proteases contain a catalytic dyad with a histidine residue and a cysteine residue. 
The cysteine performs the nucleophilic attack to initiate peptide bond hydrolysis.
Aspartic proteases contain a catalytic dyad with two aspartate residues.
Metalloproteases require a zinc ion (or, more rarely, a different divalent metal ion) that is coordinated to the protein by three amino acids, whose identity can vary. 
The metal ion activates the water molecule, such that it can perform a nucleophilic attack on the carbonyl carbon to break the peptide bond.
Peptide bond specificity
Endoproteases specifically recognize certain amino acids or types of amino acids. 
The amino acids that form the peptide bond, as well as neighboring residues, may also play a role in specificity of the protease. 
This recognition is mediated by a protease’s specificity pocket, a region within the protease around the active site that binds some amino acid side chains more favorably than others. Some of the most common protease include the following:
Trypsin-like proteases predominantly cleave proteins at the carboxyl side of arginine or lysine (except when that residue follows a proline).
Chymotrypsin-like proteases preferentially cleave on the carboxyl side of large aromatic residues (tryptophan, tyrosine, or phenylalanine).
Caspase-like proteases predominantly cleave on the carboxyl side of aspartate, but one caspase in Drosophila has been shown to also cleave on the carboxyl side of glutamate.
Elastase-like proteases predominantly cleave on the carboxyl side of small, aliphatic amino acids (glycine, alanine, or valine).
Protease Inhibitors

Protease inhibitors are molecules that block the activity of proteases, and typically function on classes of proteases with similar mechanisms of action. Protease inhibitors can either be in the form of proteins, peptides, or small molecules. 
Naturally occurring protease inhibitors are usually proteins or peptides. 
Protease inhibitors used in experimental studies or drug development are often synthetic peptide-like or small molecules. 
Many different protease inhibitors are commercially available to use experimentally in both in vitro and in vivo assays and during protein purification.
Mechanisms of protease inhibition
Protease inhibitors can work in many different ways to inhibit the action of proteases. 
These inhibitors can be classified by the type of proteases they inhibit and the mechanism by which they inhibit those enzyme. 
While commercial protease inhibitors are typically sold based on the class of protease they inhibit, understanding the various mechanisms by which inhibitors function is essential for a comprehensive understanding of inhibition and for developing protease inhibitors as a therapeutic strategy.

Reversible inhibitors usually bind to the protease with multiple non-covalent interactions, without any change to the inhibitor itself. These inhibitors can be removed by dilution or dialysis. 
Reversible inhibitors include competitive inhibitors, uncompetitive inhibitors, and non-competitive inhibitors.

A competitive inhibitor binds to the active site of the protease, competing with substrates for access to the active site residues. 
One example of a competitive inhibitor is aprotinin, which inhibits many serine proteases. 
Competitive inhibitors are often similar in structure to the transition state of natural substrates. 
The transition state of the substrate is an intermediate state of the structure that typically binds most tightly with the enzyme. 
Therefore, compounds mimicking this structure bind to the enzyme with greater strength than the substrate (in its initial state) can, and thus the normal enzymatic reaction cannot proceed. 

An example of this type of transition state analog is LP-130 , an HIV-1 protease inhibitor.
Uncompetitive inhibitors bind to the protease only when it is already attached to a substrate. 
Uncompetitive inhibitors have been identified for HIV-1 protease  and the NS2B-NS3 proteinase of the West Nile virus.
Non-competitive inhibitors bind to the protease with similar affinities, regardless of the presence of a bound substrate. 
These molecules inhibit protease activity through an allosteric mechanism. BBI, a trypsin inhibitor from soybeans  and aminoglycosides, inhibitors of the anthrax lethal factor protease are examples of non-competitive inhibitors.
Irreversible inhibitors function by specifically altering the active site of its specific target protease, often through the covalent bond formation. 
They can be more appropriately called inactivators. 

Upon binding to the inhibitor, a protease’s active site is altered, and it can no longer perform peptide bond hydrolysis. 
Some of such inhibitors do not actually covalently bind to the protease, but interact with such a high affinity, that they are not easily removed. 
Whether the inhibition is reversible or not has implications in using an inhibitor: for a reversible inhibitor, the concentration of the inhibitor must be maintained throughout a protocol, such as during protein purification, while for an irreversible inhibitor, once all proteases have been inactivated, there is no need to maintain the inhibitor concentration.

Suicide inhibitors, typically analogs of the substrate, are irreversible inhibitors that covalently bind to proteases. 
An example of a suicide protease inhibitor is the serpin family of proteins, which play a role in blood coagulation and inflammation. 
A loop in serpin serves as a substrate analog. 
The serine residue in the active site of trypsin forms a nucleophilic attack on a carbonyl carbon of the substrate analog, inducing a conformational change in the enzyme that renders the remainder of the peptide bond hydrolysis reaction unfavorable. 
Thus, the serpin remains covalently bound to the protease so that the enzyme is no longer available for binding to substrates.

Commercial protease inhibitors
Protease inhibitors can be purchased individually or as a concentrated cocktail containing multiple protease inhibitors in appropriate concentrations. Individual protease inhibitors are ideal for proteolytic assays of already purified proteins. 
Often times, enzymatic assays require a control to show that the enzyme of interest is performing the monitored action; thus, adding a specific protease inhibitor can adequately serve as a control. 
Purchasing individual protease inhibitors may also be useful when the protein being purified is a protease itself. 
In this case, the presence of functional proteins and the purity is often assessed by enzymatic assays. 
For this type of purification, multiple protease inhibitors can be added, excluding those that inhibit the protease of interest. 
Some of the most common protease inhibitors that are used during protein purification are listed. 
Most offer broad inhibition of one or more classes of proteases.

Protease inhibitor cocktails are often used for their reliability and reproducibility. 
Cocktails contain multiple protease inhibitors in the appropriate relative amounts, eliminating the need for trial and error in determining the required types and amounts of inhibitors to use. 
They also reduce the opportunities for human or pipetting error, by requiring only one solution versus multiple different solutions. 
These cocktails come in both liquid and solid forms.

Protease inhibitors, both individual and cocktails, are available from a number of commercial sources. 
MilliporeSigma is one of the largest providers of inhibitor cocktails and individual protease inhibitors. 
MilliporeSigma cOmplete tablets (Figure 7) are among the most commonly used protease inhibitor cocktails. 
These tablets are added to a specific volume of the buffer to inhibit the most abundant proteases. 
MilliporeSigma cOmplete tablets have been shown to successfully inhibit a broad range of proteases in lysates from many organisms, including E. coli, yeast, insects, and mammals. 
These tablets are available both with and without EDTA.

Protease inhibitors in tablet form are added to an appropriate volume of the buffer, and the tablets dissolve to bring the concentration of each protease inhibitor to the appropriate level. 
Adding protease inhibitors in tablet form is very convenient as no pipetting is involved. In cases where tiny volumes of the buffer are required, protease inhibitor cocktails in liquid form might be more suitable to avoid wasting excess buffer or inhibitors, as the tablets require the preparation of a set volume.

EMD Millipore sells various Calbiochem protease inhibitor cocktails for different purposes. 
The components of these cocktails are given on their website and can help consumers choose which product is best for their application. 
Cocktails that inhibit multiple classes of proteases and cocktails that are developed for inhibiting a broad range of proteases from the same class are available. These are typically supplied in DMSO or as a lyophilized powder that can be reconstituted to yield a concentrated stock solution.

Thermo Scientific sells protease inhibitor cocktails in both liquid and tablet form. Halt Protease Inhibitor Cocktail is a 100X solution that is stable at 4°C. 
It contains a combination of AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A dissolved in DMSO. 
A vial of EDTA is provided for the option of metalloprotease inhibition. The same combination of protease inhibitors is sold in tablet form, both with and without EDTA.

G-Biosciences sells, a protease inhibitor cocktail that is compatible with mass spectrometry and two-dimensional gel electrophoresis. 
This cocktail contains protease inhibitors exhibiting broad inhibition and includes an alternative to EDTA for inhibiting metalloproteases. 
Recom ProteaseArrest™ is a cocktail specific for the purification of histidine-tagged proteins expressed in bacteria. 
Other ProteaseArrest™ formulations are available from G-Biosciences for proteins isolated from different types of organisms, and these formulations contain varying amounts of inhibitors depending on the relative quantity of various proteases in the organisms. 
Similarly, MilliporeSigma sells protease inhibitor cocktails reportedly optimized for mammalian tissue/cell extracts, plant tissue/cell extracts, fungal/yeast cell extracts and mammalian secreted proteins.

Many other companies also sell protease inhibitors individually, as cocktails, and as sets of multiple individual inhibitors. 
These sets are useful for determining which protease inhibitors are required for a certain application. 
They can also be useful when a specific protease inhibitor included in most commercial cocktails is known to interfere with the protein of interest or downstream application. 
Sigma Aldrich, GE Healthcare, Promega, Cell Signaling Technology, Clontech, and Santa Cruz Biotechnology, Inc. all sell protease inhibitors in various formats.

Protease inhibitory antibodies
Selective suppression of microbial proteases can be achieved by application of monoclonal antibodies. 
A novel approach for the generation of protease inhibitory antibodies by the selective conversion has recently been demonstrated. 
The authors of the study used a dual-color flow cytometry to perform the selection on matrix metalloproteinase (MMP)-9 and the counter-selection on MMP-14 catalytic domains. 
The results have shown successful conversion of MMP-14 inhibitory antibodies to MMP-9 inhibitory antibodies with high selectivity.

Another study has described a functional selection of protease inhibitory antibodies, which has been based on co-expression of recombinant proteins in Escherichia coli, a protease of interest and a β-lactamase with a protease cleavable peptide sequence. 
Several antibodies suppressing different proteases, such as matrix metalloproteinases, beta-secretase 1 and cathepsin B, have been obtained. 
The protease inhibitory antibodies have been effective in both cellular assays and animal models.

Novel protease inhibitors
A recent study has described the isolation and characterization of PPF-BBI, a novel protease inhibitor obtained from the skin secretion of the Fukien gold-striped pond frog, Pelophlax plancyi fukienesis. 
The compound showed antimicrobial activity against C aureus, C albicans and E coli.

Another study has presented GP205, a new inhibitor of NS3/4Aserine protease with low nanomolar activities against several hepatitis C virus (HCV) replicons. The analysis of GP205 pharmacokinetics demonstrated prolonged plasma half-life. 
This serine protease inhibitor might be valuable for the development of new HCV treatments.

A flavonoid library has recently been screened to identify compounds able to suppress MERS-CoV 3C-like protease (3CLpro) produced by Middle East respiratory syndrome-coronavirus. 
Flavonoids herbacetin, isobavachalcone, quercetin 3-β-d-glucoside and helichrysetin have been shown to decrease the activity of 3CLpro and can therefore be applied to the development of new treatment approaches against coronavirus.

Commonly Used Protease Inhibitors in Laboratory Experiments
Labome manually surveys formal publications for the reagents used. 
lists the commonly cited protease inhibitors. Protease inhibitor cocktails are very commonly used. 
Almost half of the publications surveyed by Labome cited MilliporeSigma protease inhibitor cocktail. 
Litke JL et al, for example, included Halt protease and phosphatase inhibitor cocktail from Thermo Fisher (78440) in the RIPA buffer to lyzing Hela cells for Western blot.

Protease Inhibitors in Action
There are many things to take into consideration when choosing the appropriate protease inhibitor(s) for a particular process. 
The most important factor is the purpose of protease inhibition—whether inhibitors are needed to prevent proteolysis during protein purification, to inhibit a purified enzyme as an experimental control, or for use in living organisms to affect physiological processes. 
The following section includes some major questions and concerns to consider when using protease inhibitors.

What protease inhibitor should be used?
The application plays a significant role in the type of protease inhibitors that should be used. 
For in vitro and in vivo assays, the choice of protease inhibitor depends on the particular enzyme or physiological process being studied. 
For enzymatic assays, the inhibitor should not interfere with the method of detection. 
Although if inhibition is reversible, and another detection method (e.g., ELISA) is available, it may not matter if the target protein is inhibited during purification provided that the inhibitor is absent from the final preparation.
Proteases should be noted that some protease inhibitors are not specific to proteases. 
For example, PMSF irreversibly inhibits many, but not all, members of the serine hydrolase family. 
This family includes proteases, such as trypsin and chymotrypsin, but also includes a great many other enzymes that hydrolyze non-protein substrates (e.g., acetyl-cholinesterases, acyl-CoA hydrolases and lipases). 
For studies in living organisms, the inhibitors must be cell permeable, highly specific, and non-toxic. 
For the broad inhibition of proteases to assist in purifying a full-length, functional protein, multiple proteases are typically required.

How should protease inhibitors be prepared and stored?
Many protease inhibitors are not stable for long periods, either as a stock solution or at their working concentration. It is critical to prepare solutions according to vendor instructions, as some protease inhibitors are more stable under certain conditions than others. 
Typically, if stock solutions have been stored as suggested and working solutions prepared immediately before use, inhibitors should retain sufficient function. However, some protease inhibitors, such as PMSF, are very unstable and may need to be added multiple times during lysis and purification to ensure protease inhibition.

How can the function of protease inhibitors be confirmed?
Several companies sell reagents as a measure to check for protease function, and these can be used to confirm adequate inhibition. 
MilliporeSigma sells a Universal Protease Substrate, which utilizes an absorbance-based assay to monitor degradation of resorufin-labeled casein. 
This substrate can be used if there is evidence to suspect inadequate protease inhibition. 
Other companies, such as Calbiochem (EMD Millipore), sell similar reagents. 
Typically, it is a better use of time and energy to make a fresh protease inhibitor solution if it is suspected that proteases have not been well inhibited. However, if proteolysis is a recurring problem, these commercial substrates can help in determining more appropriate conditions for protease inhibition.

During cell lysis and protein purification, when should protease inhibitors be added?
Protease inhibitors should be added to the lysis buffer so that inhibition begins immediately upon cell lysis, when the proteases are released from their cellular compartments or their regulation is otherwise disrupted. 
These protease inhibitors should remain in the buffers used in the purification scheme until most of the contaminating proteases have been sufficiently separated from the protein of interest. 
Typically, if the first chromatographic step is stringent (e.g., affinity chromatography), protease inhibitors only need to be present through the wash step of this chromatographic procedure. 
The elution buffer and remaining purification buffers do not usually require protease inhibitors. 
This is a significant financial consideration for large-scale protein purification efforts where large quantities of buffers may be necessary; the inclusion of high concentrations of protease inhibitors throughout large-scale purifications can be prohibitively expensive.

However, many proteases are very efficient catalysts with high turnover numbers and so if even a small amount of a protease co-purifies with the target protein it can cause problems. 
This can result in clear degradation of the target protein or it may not be evident until N-terminal or mass spectroscopic analysis reveals that the purified protein preparation contains a variety of species with different N-termini. 
For many applications, such micro-heterogeneity may not be an issue, but for others it may cause significant problems. 
For example, in a structural biology context, such N-terminal heterogeneity may affect the ability to crystallize a protein and/or the quality of any crystals obtained. 
Thus, in some cases, it may be necessary to include inhibitors against one or more classes of protease into the later stages of the purification protocol. 
The use of protease assays (as discussed above) can be used to identify the protease class(es) that needs to be blocked.

What else can be done to slow down proteolysis?
Cell lysis and purification should be performed at a low temperature. 
Typically, lysis and purification are performed on ice or at 4°C. 
This not only helps with protein folding and stability but also slows down the rate of proteolysis by contaminating proteases. 
Additionally, the faster the contaminating proteases are removed from the protein of interest, the less time they have to interact with and possibly degrade the proteins of interest. 
Do not let cell lysates sit at intermediate steps for long periods, even on ice. 
Proceed through the purification steps as quickly as possible to remove as many proteases as possible, and store purified protein appropriately.

If proteolysis of a recombinant protein is a particular problem, several approaches can be considered. 
For E.coli expression it is possible that lowering the growth temperature will reduce proteolysis of the target protein that occurs before cell harvest and lysis. 
It may also be possible to reduce intracellular proteolysis of the target protein by directing expression to the periplasm and thus reducing exposure to intracellular proteases. 
Similarly, to avoid proteolysis by mammalian intracellular proteases, it may be possible to use an expression system that drives secretion of the recombinant protein into the culture medium. 
Considering the different range of proteases produced by different organisms, it may also be possible to reduce, or even eliminate, proteolysis issues by switching expression to a different host organism (e.g., E. coli to baculovirus/insect cells).

If I don’t know what protease inhibitors to use, where should I start?
The best place to start in choosing the correct combination of protease inhibitors for use during cell lysis and protein purification is to try a commonly used protease inhibitor cocktail. 
For the majority of lysates and applications, these are sufficient. 
However, if a full-length protein is not obtained or problems are experienced in downstream applications, specific protease inhibitors or protease inhibitor sets can be used to determine the most appropriate combination of protease inhibitors.

Protease Inhibitors in Clinical Practice
Protease inhibitors have been tested in pre-clinical and clinical trials. 
Suppressors of convertase subtilisin-kexin type 9 were shown to decrease the level of low-density lipoproteins and have been approved for the treatment of patients with severe hyperlipidemia. 
Dipeptidyl peptidase-4 (DPP-4) suppressors effectively lower glucose levels and are used for treating patients with type 2 diabetes. 
DPP-4 inhibitor linagliptin was demonstrated to suppress the development of diabetic retinopathy in an experimental model. 
A recombinant serine protease inhibitor (rBmTI-A) has been evaluated in a mouse model of allergic lung inflammation. 

The proteolytic activity, polymorphonuclear and eosinophilic responses and proinflammatory cytokine production have been decreased by the treatment with rBmTI-A in Balb/c mouse lungs, suggesting potential application of this protease inhibitor for asthma therapy. 
Also, the effects of protease inhibitor MG-132 on sepsis-induced lung injury have been studied using a Sprague Dawley rat model. 
The authors have found that MG-132 protects against acute lung injury through inhibition of mTOR/4EBP1/EIF4E pathway. 
In addition, the effects of secretory leukocyte protease inhibitor (SLPI) on squamous cell carcinoma (SCC) have recently been evaluated.
The study showed that SLPI suppressed human papilloma virus-mediated phenotypes in SCC cells via an upregulated NF-κB signaling pathway. Inhibitors of cysteine protease cathepsin K increase bone mineral density in individuals with postmenopausal osteoporosis. 
Cathepsin K suppressor odanacatib was shown to decrease bone resorption in both male and female patients with osteoporosis.