Optimization of process parameters for keratinase enzyme production using statistical experimental design revathi k and viruthagiri t bioprocess laboratory, department of chemical engineering, annamalai university, annamalai nagar, tamilnadu, india. Enzyme production encyclopedia of life support systems. Optimization of keratinase production for feather degradation by bacillus subtilis article pdf available in jundishapur journal of microbiology january 20 with 155 reads how we measure reads. The objectives of this study is to develop keratinase enzyme for largescale field application with the intent of reducing the large amount of keratin waste produced by the poultry industry each year. The keratinase enzyme production was carried out in the basal medium by using shaking flask. Industrial application of keratinase and soluble proteins. Purification and characterization of keratinase from feather. About 43% of these are feed grade enzymes, 16% are feed grade proteins, and 11% are feed grade amino acids. Mechanism of keratin degradation by microbial keratinases.
Production, purification and characterization of an. Enhancing the ability of keratinase by using immobilised. Two isolates bf and bf exhibiting maximum keratinase were selected and the keratinase production was compared in three liquid media. Lakshmi abstract keratinases are a group of proteolytic enzymes that display the capability of degrading insoluble keratin substrates such as feather resulting as poultry waste. Present study showed that the optimum moisture level of 69. Applying the mutation of bacillus subtilis and the. Sathish kumar department of biotechnology, kumaraguru college of technology, coimbatore 641 006, tamil nadu, india. The optimum ph and ph stability range for the production of. The supernatant was used as the crude enzyme enzyme source. Keratinase producing microorganisms have been isolated and characterized from soil and poultry waste sites. An enzyme control was prepared by the addition of 1ml trichloroacetic before incubation. In the present study, purification and characterization of keratinase from feather degrading bacterium bti is reported. Research article efficient degradation of feather by.
Dec 07, 2006 feather was the optimal substrate for keratinase production fig. The biomass of the organism was separated by centrifugation at 5000 rpm at 4 0c. Keratinase production and enzyme assay keratinase production and enzyme assay were performed according to the protocol of gupta and singh 2014. Screening and characterisation of keratinase enzyme obtained from keratin degrading microorganism isolated from sanjan poultry waste dumping soil 987 european academic research vol. The production of keratinase by streptomyces sp were reported in 1989 6. Microbial keratinase production and application to improve. Article purification and characterization of a bacillus.
Production, characterization and application of keratinase from streptomyces gulbargensis. The enzyme was monomeric and has a mol wt of approximately 66 kda sdspage. Production and characterization of feather degrading. The highest keratinase production was observed at ph 7 and 8 by bacillus spp. Efficient degradation of feather by keratinase producing. The novel keratinase kerp has potential industrial applications, particularly in the treatment of keratinous waste. Us3096253a us698880a us69888057a us3096253a us 3096253 a us3096253 a us 3096253a us 698880 a us698880 a us 698880a us 69888057 a us69888057 a us 69888057a us 3096253 a us3096253 a us 3096253a authority us united states prior art keywords atcc sulfur keratin amino acids disulfide bonds prior art date 19571125 legal status the legal status is. Keratin degrading microbial keratinase as a tool for. Production, partial optimization and characterization of keratinase. Isolation of keratinolytic bacteria from feather dumping site of gh. Moreover cost effective production of keratinase can be achieved. The concentration of the recombinant keratinase rk27 produced by b. The reaction mixture was centrifuged 2000g10min and read at 280nm in a spectrophotometer.
Screening and characterization of keratinase from bacillus licheniformis isolated from namakkal poultry farm c. Keratinase is an inducible enzyme that is synthesized only when an inducer keratin appears in the environment. The purified enzyme displayed 3 bands in close proximity between 20 to 22 kda in sdspage which were apparent to the zone of hydrolysis in gelatin zymogram. Production and estimation of keratinase by immobilized and. Valkerase is a unique keratinase processing enzyme processing additive that creates better quality, lower cost feather meal naturally discovered by dr. Fifty two keratinase producing bacterial strains were isolated from soils in thailand on a semisolid agar medium.
They produced extracellularly keratinolytic enzymes in enrichment media with 10 %. However, the production of such enzymes is not exclusive to dermatophytes, since geophilic species have demonstrated keratinase production kushwaha and nigam, 1996. Production of a new microbial enzyme starts with screening of microorganisms for. In addition, the enzymes critical for keratin degradation and their. This finding adds to the library of keratinase producing microbial collection for sources of keratinase, a potential replacement agent of the harmful. One type contained rod shaped bacteria and another was a coccus, which appear singly and in chain. In this study thermostable keratinase rk27 of bacillus pumilus ks12 was expressed and secreted in bacillus subtilis wb980 expression system under the control of xylose promoter pxyla. We selected sixteen levels from seven factors which could affect the strains growth, replication, and enzyme production of microbe, set the activity of. Keratinase production was associated with growth at the maximum level of 1.
The enzyme was stable at a ph range of 6585 and up to 65 c. Effective biodegradation of chicken feather waste by co. Tamilmani et al production of an extra cellular feather degrading enzyme by bacillus licheniformis 186 agar plate. Keratinase production was optimized with different ph concentration such as 4, 5, 6,7 and 8. Statistical optimization of keratinase production by bacillus. Keratinase is a particular class of extracellular proteolytic inducible enzyme with the capability of degrading insoluble keratin substrates. Nfh5 might be used for large scale production of keratinase for industrial. The first step was the production of keratinase enzyme from chicken feathers and the second was to check the stability of enzyme. A wide variety of keratinase enzyme options are available to you, such as supply type, grade standard.
He isolated an inducible extracellular homogenous enzyme, which shows a 7. Purification and characterization of keratinase from. Production of keratinase and lmethioninase from aspergillus flavipes utilizing chicken feathers as the substrate under solid state fermentation was reported. Conclusion ubiquitous keratinase enzyme has the capability that replaces most of the conventional proteases in the leather industry and detergent additive application due to their better performance. The isolate produces keratinase and the enzyme showed activity in dehairing of goat skin. Effect of substrate concentration on keratinasemaximum amount of enzyme production was found in production. Secretion of keratinolytic enzymes is associated with dermatophytic fungi, for which keratin is the major substrate. The supernatant was used as the source of extracellular keratinase enzyme. Keratinase production and keratin degradation by a mutant. Enzymes were obtained by centrifugation at 10,000 g for 5 min, and culture supernatants were used as crude enzyme extracts. Poultry feathers consist mainly of the protein keratin, which is rich in. During the batch fermentation by both strains, the ph changed from 7.
Production, partial optimization and characterization of. The smaller keratinase is a monomeric enzyme with a molecular weight of 18 kda. Characterization and overexpression of a novel keratinase. Knowledge of fermentation parameters, purification strategies and properties of the biomolecules is essential to develop efficient enzyme based production process. In brief, 20 mg of chicken feathers were suspended in 3. Microbial degradation of keratinrich porcine byproducts.
Enhancing the ability of keratinase by using immobilised enzymes v. Screening and characterization of keratinase from bacillus. Keratinase is the major enzyme involved in the pathogenesis process howard, 1983 and noteworthy information is available on the keratinase production by different species of dermatophytes takuchi, et al 1984, wawarzkiez, 1991, quin, et al 1992. Jason shih, cofounder of bri, valkerase hydrolyzes keratin peptide bonds in feather waste, resulting in feather meal that is more easily digested by animals. Keratinase production by bacterial isolates from soils in namakkal poultry farm. The purified enzyme showed maximum keratinase activity at temperature 65 oc and ph 10. Fk 28 isolated in thailand dakrong pissuwan and worapot suntornsuk abstract screening for keratinase producing bacteria and their keratinase production were investigated. Three different surfaceactive compounds were added to the fermentation medium in two different concentrations and the growth of microorganism and enzyme activity were monitored. A novel alkaline surfactantstable keratinase with superior.
Pdf optimization of keratinase production for feather. Keratinase enzymes degrade the compact keratin materials, and. Among the dermatophytes, different species of trichophyton were reported as. Raw feather, an important byproduct from the poultry industry, was the selected growth substrate to test the. Keratinase production and biodegradation of some keratinous wastes by alternaria tenuissima and aspergillus nidulans. Optimization of keratinase production by keratinolytic.
Secretion of keratinolytic enzymes is associated with dermatophytic fungi, for which keratin is the major substrate matsumoto, 1996. Development of a keratinase activity assay using recombinant chicken feather keratin substrates article pdf available in plos one 122. The initial optimal ph for keratinase production was 7. Similarly optimum temperature and ph for the enzyme. The keratinase production was accompanied with observable iaa production in the culture media even without tryptophan supplementation which paves way to utilize spent. The molecular weight of most keratinases is concentrated between 30 and 70 kda. Keratinase was purified using chromatographic methods sephadex g75 and q sepharose resulting in 8.
Due to the growing demands for keratinases in industrial application, most studies are focusing on mining of keratinase resources and improving the enzyme production level. Optimization for keratinase enzyme production using. Production of thermostable organic solvent tolerant. Through using the ultraviolet ray produced by ultraviolet light and the compound mutation of sodium nitrite solution, a mutated strain was produced which can yield keratinase with a high activity. Tamilmani et al production of an extra cellular feather degrading enzyme by bacillus licheniformis 185 were plated on nutrient agar medium and incubated t aassayed for keratinase activity. One unit of keratinase activity was defined as the amount of enzyme required to produce an absorbance increase of 0. The purified enzyme showed maximum keratinase activity at temperature 65 o c and ph 10. Therefore, enzyme keratinase expected to be produced from bacillus spp. Response surface methodology was used to optimize the conditions for the. The production of keratinolytic enzymes by chryseobacterium sp. Pdf development of a keratinase activity assay using. Isolation, partial purification and characterization of. The residual hydrolysates decreased gradually with the initial medium alkalinity. Raw feather, an important byproduct from the poultry industry, was the selected growth substrate to test the effect of three variables.
Production, characterization and application of keratinase. They are produced in the keratinous substrates such as feather, hair, wool, nail, horn and hoof by microorganisms. It is important for hydrolyzing hair, feather, and collagen in sewage system during waste water treatment. Keratinase production was optimized by using production media having chicken feathers as sole carbon and nitrogen source which removes the hurdle of huge substrate cost in industrial enzyme production. The enzymeactivity was inhibited by reduced glutathione, pmsf and 2mercaptaethanol. The maximum production was obtained at 4th day of incubation at ph 9 and temperature 55oc. Use of poultry byproduct for production of keratinolytic. Optimization of process parameters for keratinase enzyme. Energetic bacillus subtilis was preliminarily isolated from feather meal selection medium experiment, which could be used to produce keratinase.
Keratinase producing microorganisms are being increasingly utilized for degradation and recycling of poultry feather waste. It was found that keratinase in fungi, streptomyces and bacteria were produced in nearly at alkaline ph and almost thermophilic temperatures. The presence of carbon source in feather medium suppressed the enzyme production, while 0. After five days of incubation, a loss of enzyme activity was observed probably because of enzymatic autolysis and end product inhibition. Higher amount of keratinase enzyme was found to produce by arthrobacter sp. Optimization of parameters for fermentative production of keratinase enzyme in our earlier studies. Screening and characterisation of keratinase enzyme obtained.
Enhancing the keratinase production in the first part of this study the keratinase production from streptomyces sp. Two native strains bf11 bacillus subtilis and bf21 bacillus cereus degrading keratin completely were characterized. Progress in microbial degradation of feather waste. A comparative characterization of indigenous keratinase. Isolation, identification and characterization of a. This was 5 days after cell numbers began to decrease fig. Concerning the use of nitrogen sources different from keratin, the extra addition of nitrogen sources have no or depressive effect on keratinase production as well as solubilization of. Following the application of the enzyme, this waste can be fed to chickens as a highprotein supplement, resulting in the recycling of this product. Determination of keratinolytic activity keratinase activity was assayed by the modified method of yu et al. The outcome recommended the prospective utilization of keratinase in industrial to hydrolyze keratin for the fabrication of bacterial toxin 4, 125, 67 and 27 kda. Partial purification of keratinase from actinomycetes.
Strain improvement resulted in isolation of mbf11 and mbf21 from bf11 and. Optimization for keratinase enzyme production using bacillus. May 18, 2019 five temperatures were chosen between the optimal enzyme production temperatures 23 c and 37 c of the two bacterial strains, and 30 c was found to be the best temperature for feather degradation additional file 1. Use of poultry byproduct for production of keratinolytic enzymes. Enzyme production the organisms were cultivated in feather meal broth 10 g l1 feather meal, 0. Feather waste is a byproduct of the domestic poultry industry and is 90%. Also, the keratinase stand out among proteases in developing costeffective feather byproducts for feed and fertilizers. Maximum keratinase production was peptone specific activity 85. Enzymatic dehairing of goat skin using keratinase from. Keratinases are proteolytic enzymes capable of degrading rigid and insoluble keratinous proteins present in skin and appendages. Keratinase production and biodegradation of some keratinous. Protein precipitate obtained at ammonium sulphate saturation at 60% level and sephacryl s200 column chromatography resulted in 2. Among all the keratincontaining substrates, feather was mostly utilized, followed by hair and wool. Ii, issue 11 february 2015 industry resulted in the generation of an increased quantity of organic solid.
Development of a keratinase activity assay using recombinant. However, the production of such enzymes is not exclusive to dermatophytes, since geophilic species have demonstrated keratinase production. The reaction mixture was centrifuged 2000 g10 min and read at 280 nm in a spectrophotometer. Extracellular keratinase of some dermatophytes, their. Keratinase definition of keratinase by medical dictionary.
The amplified dna was visualized by gel electrophoresis additional file 1. The effect of carbon source on the production of keratinase was tested by adding different concentrations of. Production of an extra cellular feather degrading enzyme. It is also useful in food industry, animal feed preparation etc. Keratinase, an extracellular protease produced by bacillus licheniformis pwd1, can degrade this keratin waste. Optimization of keratinase production by central composite design rsm. Keratinase a feather degrading enzyme useful for mosquito control. The isoelectric point of the keratinase enzyme is known at ph 5. Production of thermostable organic solvent tolerant keratinolytic. Although many keratinases have been identified, the reasons for their substrate specificity towards. Keratinolytic bacteria isolated from feather waste scielo. Optimization of culture conditions for keratinase production.
Screening and selection of fungus for keratinase production. Isolation, identification and characterization of a feather degrading bacterium 691 table 1. The new keratinase enzyme was isolated, purified and characterized from b. The optimum ph and temperature for production of the enzyme were 8 and 55 c, respectively. Keratinase production in various microorganisms was reported on by a number of researchers. The production of organic solvent tolerant keratinolytic protease enzyme by thermoactinomyces sp. The keratinase enzyme production was carried out in the basal medium. On 4 th day enzyme production was highest 140 ku ml1 with 1% feather. C and peptidase production occurred between 22 and 37. They produced extracellularly keratinolytic enzymes in enrichment media with 10%. Production of an extra cellular feather degrading enzyme by. Both the isolates showed lower keratinase production in luria broth and nutrient broth tested with maximum of. Production and preparation of keratinase lysobacter ncimb 9497 was harvested shortly after extracellular protein concentration peaked 31 days of incubation. With optimized conditions, recombinant production of keratinase is possible.