Keratinase
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Keratinase
Keratinases are Protease, proteolytic enzymes that digest keratin. History They were initially classified as 'proteinases of unknown mechanism' by the Nomenculture Committee on the International Union of Biochemistry in 1978 with Enzyme Commission number, EC number 3.4.99 in 1983 (Owen et al., 1983). In the 1990s, they were defined as a serine proteases due to high Homology (biology), sequence homology with alkaline protease, and their inhibition by Protease inhibitor (pharmacology), serine protease inhibitors (Wang et al., 1995; Taha et al., 1998 and Bressollier et al., 1999). Function Keratinases are produced only in the presence of keratin-containing substrate. They mainly attack the disulfide (-S-S-) bond of the keratin substrate. Keratinase production has been reported in various microorganisms including fungi and bacteria, and occurs at near-alkaline pH and thermophilic temperatures. These enzymes have a broad Substrate (biochemistry), substrate specificity, degrading fibr ...
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Protease
A protease (also called a peptidase, proteinase, or proteolytic enzyme) 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 protea ...
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In Vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology and its subdisciplines are traditionally done in labware such as test tubes, flasks, Petri dishes, and microtiter plates. Studies conducted using components of an organism that have been isolated from their usual biological surroundings permit a more detailed or more convenient analysis than can be done with whole organisms; however, results obtained from ''in vitro'' experiments may not fully or accurately predict the effects on a whole organism. In contrast to ''in vitro'' experiments, '' in vivo'' studies are those conducted in living organisms, including humans, and whole plants. Definition ''In vitro'' ( la, in glass; often not italicized in English usage) studies are conducted using components of an organism that have been isolated ...
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Enzyme Activity
Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Enzyme units The quantity or concentration of an enzyme can be expressed in molar amounts, as with any other chemical, or in terms of activity in enzyme units. Enzyme activity Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on various physical conditions, ''which should be specified''. It is calculated using the following formula: :\operatorname=\operatorname=\operatorname\times\operatorname where :\operatorname= Enzyme activity :\operatorname= Moles of substrate converted per unit time :\operatorname= Rate of the reaction :\operatorname= Reaction volume The SI unit is the katal, 1 katal = 1  mol s−1 (mole per second), but this is an excessively large unit. A more practical and commonly used value is enzyme unit (U) = 1 μmol min−1 (micromole per minute). 1 U corresponds to 16.67 ...
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Molecular Mass
The molecular mass (''m'') is the mass of a given molecule: it is measured in daltons (Da or u). Different molecules of the same compound may have different molecular masses because they contain different isotopes of an element. The related quantity relative molecular mass, as defined by IUPAC, is the ratio of the mass of a molecule to the unified atomic mass unit (also known as the dalton) and is unitless. The molecular mass and relative molecular mass are distinct from but related to the molar mass. The molar mass is defined as the mass of a given substance divided by the amount of a substance and is expressed in g/mol. That makes the molar mass an average of many particles or molecules, and the molecular mass the mass of one specific particle or molecule. The molar mass is usually the more appropriate figure when dealing with macroscopic (weigh-able) quantities of a substance. The definition of molecular weight is most authoritatively synonymous with relative molecular mass; ho ...
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SDS-PAGE
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a Discontinuous electrophoresis, discontinuous electrophoretic system developed by Ulrich K. Laemmli which is commonly used as a method to separate proteins with molecular masses between 5 and 250 Kilodalton, kDa. The combined use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel allows to eliminate the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight. Properties SDS-PAGE is an electrophoresis method that allows protein separation by mass. The medium (also referred to as ′matrix′) is a polyacrylamide-based discontinuous gel. The polyacrylamide-gel is typically sandwiched between two glass plates in a slab gel. Although tube gels (in glass cylinders) were used historically, they were rapidly made obsolete with the invention of the more convenient slab gels. In addition, SDS (sodium dodecy ...
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Size Exclusion Chromatography
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are commonly composed of dextran, agarose, or polyacrylamide polymers. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers. Applications The main application of gel-filtration chr ...
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Bacillus Licheniformis
''Bacillus licheniformis'' is a bacterium commonly found in the soil. It is found on bird feathers, especially chest and back plumage, and most often in ground-dwelling birds (like sparrows) and aquatic species (like ducks). It is a gram-positive, mesophilic bacterium. Its optimal growth temperature is around 50 °C, though it can survive at much higher temperatures. The optimal temperature for enzyme secretion is 37 °C. It can exist in a dormant spore form to resist harsh environments, or in a vegetative state when conditions are good. High capacity of secretion of the alkaline serine protease has made ''B.'' ''licheniformis'' one of the most important bacteria in industrial enzyme production. Subtilisin Carlsberg () secreted by ''B. licheniformis'' is used as a detergent protease. It is sold under the name Alcalase by Novozymes. A small antisense RNA against Subtilisin Carlsberg named BLi_r0872 was discovered in an RNA-seq based study. It may have a putative ...
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Glutathione
Glutathione (GSH, ) is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources such as reactive oxygen species, free radicals, peroxides, lipid peroxides, and heavy metals. It is a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. The carboxyl group of the cysteine residue is attached by normal peptide linkage to glycine. Biosynthesis and occurrence Glutathione biosynthesis involves two adenosine triphosphate-dependent steps: *First, γ-glutamylcysteine is synthesized from L-glutamate and cysteine. This conversion requires the enzyme glutamate–cysteine ligase (GCL, glutamate cysteine synthase). This reaction is the rate-limiting step in glutathione synthesis. *Second, glycine is added to the C-terminal of γ-glutamylcysteine. This condensation is catalyzed by glutathione synthetase. While all ani ...
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Column Chromatography
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents (normal phase, reversed phase, or otherwise) can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column. A thin-layer chromatograph can show how a mixture of compounds will behave when puri ...
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Native State
In biochemistry, the native state of a protein or nucleic acid is its properly folded and/or assembled form, which is operative and functional. The native state of a biomolecule may possess all four levels of biomolecular structure, with the secondary through quaternary structure being formed from weak interactions along the covalently-bonded backbone. This is in contrast to the denatured state, in which these weak interactions are disrupted, leading to the loss of these forms of structure and retaining only the biomolecule's primary structure. Biochemistry Proteins While all protein molecules begin as simple unbranched chains of amino acids, once completed they assume highly specific three-dimensional shapes. That ultimate shape, known as tertiary structure, is the folded shape that possesses a minimum of free energy. It is a protein's tertiary, folded structure that makes it capable of performing its biological function. In fact, shape changes in proteins are the pri ...
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In Vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism. This is not to be confused with experiments done ''in vitro'' ("within the glass"), i.e., in a laboratory environment using test tubes, Petri dishes, etc. Examples of investigations ''in vivo'' include: the pathogenesis of disease by comparing the effects of bacterial infection with the effects of purified bacterial toxins; the development of non-antibiotics, antiviral drugs, and new drugs generally; and new surgical procedures. Consequently, animal testing and clinical trials are major elements of ''in vivo'' research. ''In vivo'' testing is often employed over ''in vitro'' because it is better suited for observing the overall effects of an experiment on a living subject. In ...
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Keratin
Keratin () is one of a family of structural fibrous proteins also known as ''scleroproteins''. Alpha-keratin (α-keratin) is a type of keratin found in vertebrates. It is the key structural material making up scales, hair, nails, feathers, horns, claws, hooves, and the outer layer of skin among vertebrates. Keratin also protects epithelial cells from damage or stress. Keratin is extremely insoluble in water and organic solvents. Keratin monomers assemble into bundles to form intermediate filaments, which are tough and form strong unmineralized epidermal appendages found in reptiles, birds, amphibians, and mammals. Excessive keratinization participate in fortification of certain tissues such as in horns of cattle and rhinos, and armadillos' osteoderm. The only other biological matter known to approximate the toughness of keratinized tissue is chitin. Keratin comes in two types, the primitive, softer forms found in all vertebrates and harder, derived forms found only among saur ...
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