Keratinase
Encyclopedia
Keratinases are proteolytic enzymes in nature. It was classified as proteinase of unknown mechanism as recommended by the Nomenculture Committee on the International Union of Biochemistry (1978) with EC number
3.4.99 (Owen et al., 1983). Recently, some of the worker defined keratinase as serine protease
due to its 97% sequence homology
with alkaline protease and it is also inhibited by the same inhibitor that inhibits serine protease (Wang et al., 1995; Taha et al., 1998 and Bressollier et a1., 1999).
Keratinases are produced only in the presence of keratin containing substrate. It mainly attacks on the disulfide (-S-S-) bond of the keratin substrate (Bockel et al., 1995). The keratinase productions by various microorganisms were reported by a number of workers. It was found that keratinase produce by fungi, Streptomyces and bacteria were produced in nearly at alkaline pH and almost thermophilic temperatures. These enzymes have wide range of substrate specificity
such as it can degrade other fibrous protein
fibrin, elastin, collagen and other non fibrous protein like casein, bovine serum albumin
gelatin etc. (Noval et al., 1959; Mukhapadhayay et al., 1989; Dozie et al., 1994; Lin et al., 1995; Letourneau et al., 1998; and Bressollier et al., 1999).
At first Molyneux et al. (1959) attempted to isolate some bacteria that are able to degrade keratin. He isolated organism from the contents of experimentally induced dermoid cysts from mid lateral region of sheep. Examination of wool sample showed degraded wool with numerous corticle and cyticular cells. He found disruption of wool fiber in both in vivo
and in vitro
. He showed that the organisms belong to genus Bacillus and the organism was capable of attacking native wool protein. The same year Noval et al. (1959) published another article on enzymatic decomposition of native keratin by Streptomyces fradiae. They showed extracellular enzyme secreted by this bacteria capable of degrading the human hair in its native state
.
Keratinolytic protein from keratinophilic fungi were reported by Yu et al. (1968), Asahi et al. (1985), and Willams et al. (1989). Mukhopadhay et al. (1989) reported keratinase production by Streptomyces sp. He isolated an inducible extracellular homogenous enzyme, which shows 7.5 fold increases in its activity after DEAE cellulose column chromatography
. The enzyme-activity was inhibited by reduced glutathione
, PMSF and 2-¬Mercaptaethanol.
Williams et al. (1990) continued his work on enriched feather degrading culture and characterized the organism to its species level for the first time. The microorganisms were identified as Bacillus licheniformis
. Lin et al. (1992) purified and characterized keratinase from feather degrading Bacillus licheniformis strain isolated by Williams et al. (1990) with the help of membrane ultra filtration and C-75 gel chromatography
. He purified enzyme with 70 fold increased activity. SDS-PAGE
analysis revealed that purified keratinase had a molecular weight
of 33 kDa. Dozie et al. (1994) reported a thermostable, alkaline-active, keratinolytic proteinasefrom Chrysosporium keratinophylum which was able to solubilize keratin in lactose/mineral salt medium with DMSO. Optimum pH for the enzyme activity was 9 and optimum temperature was 90oC. Wang et al. (1999) scaled up the fermentation condition of keratinase to a pilot scale fermentar. They optimized the fermentation condition to a level of 10-fold increase in enzyme production.
EC number
The Enzyme Commission number is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze....
3.4.99 (Owen et al., 1983). Recently, some of the worker defined keratinase as serine protease
Serine protease
Serine proteases are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site.They are found ubiquitously in both eukaryotes and prokaryotes...
due to its 97% sequence homology
Homology (biology)
Homology forms the basis of organization for comparative biology. In 1843, Richard Owen defined homology as "the same organ in different animals under every variety of form and function". Organs as different as a bat's wing, a seal's flipper, a cat's paw and a human hand have a common underlying...
with alkaline protease and it is also inhibited by the same inhibitor that inhibits serine protease (Wang et al., 1995; Taha et al., 1998 and Bressollier et a1., 1999).
Keratinases are produced only in the presence of keratin containing substrate. It mainly attacks on the disulfide (-S-S-) bond of the keratin substrate (Bockel et al., 1995). The keratinase productions by various microorganisms were reported by a number of workers. It was found that keratinase produce by fungi, Streptomyces and bacteria were produced in nearly at alkaline pH and almost thermophilic temperatures. These enzymes have wide range of substrate specificity
Substrate (biochemistry)
In biochemistry, a substrate is a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving the substrate. In the case of a single substrate, the substrate binds with the enzyme active site, and an enzyme-substrate complex is formed. The substrate is transformed into one or...
such as it can degrade other fibrous protein
Fibrous protein
Scleroproteins, or fibrous proteins, constitute one of the three main classes of proteins, alongside globular proteins and conjugated proteins.Keratin, collagen, elastin, and fibroin are all scleroproteins...
fibrin, elastin, collagen and other non fibrous protein like casein, bovine serum albumin
Bovine serum albumin
Bovine serum albumin is a serum albumin protein derived from cows. It is often used as a protein concentration standard....
gelatin etc. (Noval et al., 1959; Mukhapadhayay et al., 1989; Dozie et al., 1994; Lin et al., 1995; Letourneau et al., 1998; and Bressollier et al., 1999).
At first Molyneux et al. (1959) attempted to isolate some bacteria that are able to degrade keratin. He isolated organism from the contents of experimentally induced dermoid cysts from mid lateral region of sheep. Examination of wool sample showed degraded wool with numerous corticle and cyticular cells. He found disruption of wool fiber in both in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
and in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
. He showed that the organisms belong to genus Bacillus and the organism was capable of attacking native wool protein. The same year Noval et al. (1959) published another article on enzymatic decomposition of native keratin by Streptomyces fradiae. They showed extracellular enzyme secreted by this bacteria capable of degrading the human hair in its native state
Native state
In biochemistry, the native state of a protein is its operative or functional form. 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...
.
Keratinolytic protein from keratinophilic fungi were reported by Yu et al. (1968), Asahi et al. (1985), and Willams et al. (1989). Mukhopadhay et al. (1989) reported keratinase production by Streptomyces sp. He isolated an inducible extracellular homogenous enzyme, which shows 7.5 fold increases in its activity after DEAE cellulose column chromatography
Column chromatography
Column chromatography in chemistry is a method used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms up to kilograms.The main advantage of column chromatography is the relatively low cost and disposability...
. The enzyme-activity was inhibited by reduced glutathione
Glutathione
Glutathione is a tripeptide that contains an unusual peptide linkage between the amine group of cysteine and the carboxyl group of the glutamate side-chain...
, PMSF and 2-¬Mercaptaethanol.
Williams et al. (1990) continued his work on enriched feather degrading culture and characterized the organism to its species level for the first time. The microorganisms were identified as Bacillus licheniformis
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 and aquatic species ....
. Lin et al. (1992) purified and characterized keratinase from feather degrading Bacillus licheniformis strain isolated by Williams et al. (1990) with the help of membrane ultra filtration and C-75 gel chromatography
Size exclusion chromatography
Size-exclusion 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...
. He purified enzyme with 70 fold increased activity. SDS-PAGE
SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
analysis revealed that purified keratinase had a molecular weight
Molecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
of 33 kDa. Dozie et al. (1994) reported a thermostable, alkaline-active, keratinolytic proteinasefrom Chrysosporium keratinophylum which was able to solubilize keratin in lactose/mineral salt medium with DMSO. Optimum pH for the enzyme activity was 9 and optimum temperature was 90oC. Wang et al. (1999) scaled up the fermentation condition of keratinase to a pilot scale fermentar. They optimized the fermentation condition to a level of 10-fold increase in enzyme production.