Characterization Of Keratinase Research Articles

Isolation and molecular characterization of keratinase producing Bacillus species from soil

Bacillus species present in soil contain enzymes which show proteolytic properties to decompose proteins. Soil proteases are mainly provided by soil bacteria. Bacillus species have various industrial uses. The aim of this study was to isolate and molecular characterization of keratinase producing Bacillus species from soil. Bacterial strains have been isolated from soil. Biochemical and molecular characterization have been performed using different biochemical tests (Catalase, Nitrate, simmon citrate, Triple sugar iron tests) and 16S rDNA sequence analysis respectively. Moreover, keratinase test has been performed to check the presence of keratinase enzyme in isolated species. In this study, five different bacterial strains have been isolated from soil. Out of five strains, four were Bacilli and one strain is Citrobacter farmeri. Sequencing results showed that four strains were Gram positive, Bacilli and one strain was Gram negative, Citrobacter farmeri. A keratinase test was performed to see the presence of keratinase enzyme in all strains. The results showed that keratinase was present in Bacillus cereus and Bacillus thuringiensis. This study concluded the significant role of Bacillus species in the production of keratinase enzyme. A keratinase enzyme is very important in leather industry for dehairing. Keratinase enzyme is non pollutant and economical.

Purification and Characterization of Keratinase from Bacillus licheniformis dcs1 for Poultry Waste Processing.

Feather wastes-byproduct of commercial poultry processing plant is produced in large amounts. Keratinolytic enzymes produced by feather degrading bacteria can easily degrade these waste products releasing pure keratin as a residue. The aim of present study was to isolate, and characterize feather degrading bacteria as well as assess the keratinolytic potential of purified enzyme. Three feather degrading bacteria (dps3, wps1 and dcs1) were isolated from feathers of domestic chickens. Preliminary characterization of isolated bacteria revealed these isolates belonging to genus Bacillus. 16S rRNA gene sequencing identified the isolates as B. subtilis dps3 (MW255302), B. cereus wps1 (MW255303) and B. licheniformis dcs1 (MW255304). Cell free supernatant of B. licheniformis dcs1 degraded feathers completely in 14 days indicating its keratinolytic ability. Purification of keratinase enzyme from B. licheniformis dcs1 was performed using column chromatography. SDS-PAGE indicated its molecular weight as 32 KDa. Kerotinolytice activity was maximum at optimum pH of 7 and 45℃ temperature. Enzyme showed the potential to degrade keratin material such as hairs and nails of humans. Findings of current study suggested that purified enzyme possess potential to upgrade nutritional quality of poultry waste containing keratin and might play as important biotechnological tool for keratin hydrolysis.

Production and characterization of keratinase by Ochrobactrum intermedium for feather keratin utilization

A newly isolated bacterium producing 55.5 U/mL keratinase on feather meal minimal medium was identified as Ochrobactrum intermedium. Optimization of process parameters by one-variable-at-a-time (OVAT) approach (substrate concentration 0.5% w/v, inoculum size 5% w/v, pH 7.0, 200 rpm for 96 h at 40 °C) resulted in 2.1-fold increase in keratinase secretion (117 U/mL). Keratinase was optimally active at pH 9.0 and 40 °C and was stable at pH 9.0 and 60 °C for 120 min. Calcium ions enhanced keratinase activity (158%) significantly, while it was strongly inhibited by both PMSF and EDTA, indicating it to be a metallo-serine protease. Keratinase degraded native chicken feathers efficiently resulting in 97.9% weight loss along with release of 745.5 μg/mL soluble proteins and 4196.69 μg/mL amino acids. Feather hydrolysate generated by NKIS 1 exhibited significant anti-oxidant and free-radical scavenging activity (90.46%). The present study revealed that O. intermedium NKIS 1 has potential applications in the biodegradation of chicken feathers and the value-addition of poultry waste.

Purification and characterization of keratinase from hair-degrading Streptomyces albus

The keratinase waste produced in large quantities all over the world from animals and birds including human beings. As the physiological and chemical methods of keratin degradation are not easy possible, the biological method has gained importance. The present study investigated purified keratinase from Keratinolytic Streptomyces albus . The cell-bound keratinolytic enzyme was purified 32.72-fold by gel filtration chromatography. The enzyme was characterized as a serine protease with a molecular mass of 29-35kD. Optimal activity pH and Temperature was measured at 7.0 and 40 0 C furthermore, the various inhibitors had different effect on enzyme activity. PMSF and heavy metal ion HgCl 2 were the most potent inhibitors and EDTA induced the activity by more than 135%, 2-mercaptoethanol did not show any impact on the enzyme, where pCMB, KCN, 8-hydroxyquinoline and cystine inhibited activity moderately.

Purification and characterization of a thermostable keratinase from Meiothermus sp. I40

Purification and characterization of keratinase from Meiothermus sp. I40, a newly identified strain isolated from hot springs, was carried out and reported. Crude enzyme was obtained from culture medium after incubation of I40 for 2 days and was purified by 20–80% precipitation with (NH4)2SO4, and separated by hydroxyapatite and Superdex pg 200 gel filtration chromatography. The overall recovery of enzyme activity was 45% with a 30.2-fold after purification. The molecular mass of the purified enzyme was 76 kDa with Km and Vmax of 0.29 mM and 1428.6 U/mg-min, respectively. The optimum pH and temperature of the keratinase I40 was 8.0; and 70 °C, respectively. When incubated at 65 °C and 70 °C for 12 h, the enzyme retained 96.7% and 71.3% of the activity, respectively. The purified enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF), indicating that keratinase I40 is a serine protease. Keratinase I40 exhibited good stability in the presence of dimethyl sulfoxide, ethanol, isopropanol and acetonitrile. The enzyme showed the highest activity toward chicken feather, followed by dove feather, duck feather, human hair, wool, and hog bristle. Chicken feathers treated with keratinase I40 were fully degraded in one days, but no change was detected with seven common proteases. The molecular weight (MW) of the peptides released from chicken feathers after treatment with keratinase I40 ranged from 0.48 to 10 kDa with an average of 1.84 kDa. Keratinase I40 seems to be an efficient and environmentally friendly enzyme in the biodegradation of feather keratin.

Purification and Characterization of Keratinase from Feather Degrading Bacillus sp.

Purification and Characterization of Keratinase from Feather Degrading Bacillus sp.

Purification and characterization of keratinase from recombinant Pichia and Bacillus strains

The keratinase gene from Bacillus licheniformis MKU3 was cloned and successfully expressed in Bacillus megaterium MS941 as well as in Pichia pastoris X33. Compared with parent strain, the recombinant B. megaterium produced 3-fold increased level of keratinase while the recombinant P. pastoris strain had produced 2.9-fold increased level of keratinase. The keratinases from recombinant P. pastoris (pPZK3) and B. megaterium MS941 (pWAK3) were purified to 67.7- and 85.1-folds, respectively, through affinity chromatography. The purified keratinases had the specific activity of 365.7 and 1277.7 U/mg, respectively. Recombinant keratinase from B. megaterium was a monomeric protein with an apparent molecular mass of 30 kDa which was appropriately glycosylated in P. pastoris to have a molecular mass of 39 kDa. The keratinases from both recombinant strains had similar properties such as temperature and pH optimum for activity, and sensitivity to various metal ions, additives and inhibitors. There was considerable enzyme stability due to its glycosylation in yeast system. At pH 11 the glycosylated keratinase retained 95% of activity and 75% of its activity at 80 °C. The purified keratinase hydrolyzed a broad range of substrates and displayed effective degradation of keratin substrates. The K m and V max of the keratinase for the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA was found to be 0.201 mM and 61.09 U/s, respectively. Stability in the presence of detergents, surfactants, metal ions and solvents make this keratinase suitable for industrial processes.

Purification and characterization of keratinase from a new Bacillus subtilis strain

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 degrees C was 8.5 and the optimum temperature at pH 8.5 was 55 degrees C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.