An exoenzyme, or extracellular enzyme, is an enzyme that is secreted by a cell and functions extracellular. Exoenzymes are produced by both prokaryotic and eukaryotic cells and have been shown to be a crucial component of many biological processes. Most often these enzymes are involved in the breakdown of larger . The breakdown of these larger macromolecules is critical for allowing their constituents to pass through the cell membrane and enter into the cell. For and other complex organisms, this process is best characterized by the digestive system which breaks down solid nutrients[ Some pathogenic species also use exoenzymes as to assist in the spread of these pathogen .][ In addition to the integral roles in biological systems, different classes of microorganism exoenzymes have been used by humans since prehistory for such diverse purposes as food processing, , textile production and in the paper industry.]
History
Very limited information is available about the original discovery of exoenzymes. According to Merriam-Webster dictionary, the term "exoenzyme" was first recognized in the English language in 1908.
Function
In bacteria and fungi, exoenzymes play an integral role in allowing the organisms to effectively interact with their environment. Many bacteria use digestive enzymes to break down nutrients in their surroundings. Once digested, these nutrients enter the bacterium, where they are used to power cellular pathways with help from .
Many exoenzymes are also used as . , both bacterial and fungal, can use exoenzymes as a primary mechanism with which to cause disease. The metabolic activity of the exoenzymes allows the bacterium to invade host organisms by breaking down the host cells' defensive outer layers or by necrotizing body tissues of larger organisms.
In eukaryotic cells, exoenzymes are manufactured like any other enzyme via protein synthesis, and are transported via the secretory pathway. After moving through the rough endoplasmic reticulum, they are processed through the Golgi apparatus, where they are packaged in vesicles and released out of the cell.
Examples of exoenzymes as virulence factors
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Necrotizing enzymes
Necrotizing enzymes destroy cells and tissue. One of the best known examples is an exoenzyme produced by Streptococcus pyogenes that causes necrotizing fasciitis in humans.
Coagulase
By binding to prothrombin, coagulase facilitates clotting in a cell by ultimately converting fibrinogen to fibrin. Bacteria such as Staphylococcus aureus use the enzyme to form a layer of fibrin around their cell to protect against host defense mechanisms.
Kinases
The opposite of coagulase, can dissolve clots. S. aureus can also produce staphylokinase, allowing them to dissolve the clots they form, to rapidly diffuse into the host at the correct time.
Hyaluronidase
Similar to collagenase, hyaluronidase enables a pathogen to penetrate deep into tissues. Bacteria such as Clostridium do so by using the enzyme to dissolve collagen and hyaluronic acid, the protein and saccharides, respectively, that hold tissues together.
Hemolysins
target erythrocytes, a.k.a. red blood cells. Attacking and lysing these cells harms the host organism, and provides the microorganism, such as the fungus Candida albicans, with a source of iron from the lysed hemoglobin.
Examples of digestive exoenzymes
Amylases
are a group of extracellular enzymes (glycoside hydrolases) that catalyze the hydrolysis of starch into maltose. These enzymes are grouped into three classes based on their amino acid sequences, mechanism of reaction, method of catalysis and their structure.
Lipoprotein lipase
Lipoprotein lipase (LPL) is a type of digestive enzyme that helps regulate the uptake of from and other low-density from fatty tissues in the body.[ Lipoprotein lipase is downregulated by high levels of insulin,][
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Pectinase
, also called pectolytic , are a class of exoenzymes that are involved in the breakdown of pectic substances, most notably pectin.
Pepsin
Discovered in 1836, pepsin was one of the first enzymes to be classified as an exoenzyme.[ The enzyme is first made in the inactive form, pepsinogen by chief cells in the lining of the stomach.]
Trypsin
Also one of the first exoenzymes to be discovered, trypsin was named in 1876, forty years after pepsin.
Bacterial assays
The production of a particular digestive exoenzyme by a bacterial cell can be assessed using plate . Bacteria are streaked across the agar, and are left to incubate. The release of the enzyme into the surroundings of the cell cause the breakdown of the macromolecule on the plate. If a reaction does not occur, this means that the bacteria does not create an exoenzyme capable of interacting with the surroundings. If a reaction does occur, it becomes clear that the bacteria does possess an exoenzyme, and which macromolecule is hydrolyzed determines its identity.
Amylase
Amylase breaks down carbohydrates into mono- and disaccharides, so a starch agar must be used for this assay. Once the bacteria is streaked on the agar, the plate is flooded with iodine. Since iodine binds to starch but not its digested , a clear area will appear where the amylase reaction has occurred. Bacillus subtilis is a bacterium that results in a positive assay as shown in the picture.[
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Lipase
Lipase assays are done using a lipid agar with a spirit blue dye. If the bacteria has lipase, a clear streak will form in the agar, and the dye will fill the gap, creating a dark blue halo around the cleared area. Staphylococcus epidermidis results in a positive lipase assay.[
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Biotechnological and industrial applications
Microbiological sources of exoenzymes including , , pectinases, , xylanases, and are used for a wide range of biotechnological and Manufacturing uses including biofuel generation, food production, paper manufacturing, and textile production.[ Optimizing the production of has been a focus of researchers in recent years and is centered around the use of to convert biomass into ethanol. The enzymes that are of particular interest in ethanol production are cellobiohydrolase which solubilizes crystalline cellulose and xylanase that hydrolyzes xylan into xylose.][ In addition to the important role it plays in biofuel production, xylanase is utilized in a number of other industrial and biotechnology applications due to its ability to hydrolyze cellulose and hemicellulose. These applications include the breakdown of agricultural and forestry wastes, working as a feed additive to facilitate greater nutrient uptake by livestock, and as an ingredient in bread making to improve the rise and texture of the bread.]
are one of the most used exoenzymes in biotechnology and Manufacturing applications. Lipases make ideal enzymes for these applications because they are highly selective in their activity, they are readily produced and secreted by bacteria and fungi, their crystal structure is well characterized, they do not require cofactors for their enzymatic activity, and they do not catalyze side reactions.[ However, perhaps the most well known use of lipases in this field is its use in the production of biodiesel fuel. In this role, lipases are used to convert vegetable oil to methyl- and other short-chain alcohol by a single transesterification reaction.]
, hemicellulases and pectinases are different exoenzymes that are involved in a wide variety of biotechnological and industrial applications. In the food industry these exoenzymes are used in the production of , fruit nectars, fruit purees and in the extraction of olive oil among many others.[ Cellulases and hemicellulases (including xylanases) are also used in the paper and pulp industry to de-ink recycled , modify coarse mechanical pulp, and for the partial or complete hydrolysis of pulp fibers.][ Cellulases and hemicellulases are used in these industrial applications due to their ability to hydrolyze the cellulose and hemicellulose components found in these materials.
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Bioremediation applications
Bioremediation is a process in which or in the environment are removed through the use of biological or their products. The removal of these often hazardous pollutants is mostly carried out by naturally occurring or purposely introduced that are capable of breaking down or absorbing the desired pollutant. The types of pollutants that are often the targets of bioremediation strategies are petroleum products (including oil and ) and .
Fungi have been shown to be viable organisms to conduct bioremediation and have been used to aid in the decontamination of a number of pollutants including polycyclic aromatic hydrocarbons (PAHs), , , , , crude oil, and many others.[ are an important oxidative enzyme that fungi secrete and use oxygen to oxidize many pollutants. Some of the pollutants that laccases have been used to treat include dye-containing from the textile industry, wastewater pollutants (chlorophenols, PAHs, etc.), and sulfur-containing compounds from coal processing.][
Bacteria are also a viable source of exoenzymes capable of facilitating the bioremediation of the environment. There are many examples of the use of bacteria for this purpose and their exoenzymes encompass many different classes of bacterial enzymes. Of particular interest in this field are bacterial as they have an intrinsic low substrate specificity and can be used for numerous pollutants including solid wastes.][ Cell-free use of microbial exoenzymes as agents of bioremediation is also possible although their activity is often not as robust and introducing the enzymes into certain environments such as soil has been challenging.][ In addition to terrestrial based microorganisms, marine based bacteria and their exoenzymes show potential as in the field of bioremediation. Marine based bacteria have been utilized in the removal of heavy metals, petroleum/diesel fuel degradation and in the removal of polyaromatic hydrocarbons among others.]
