The effect of temperature for keratinase production such as 20c, 30c, 40c, 50c and 60c was studied. Similarly optimum temperature and ph for the enzyme. Supplementationof soyabean meal was observed to enhance the production of the alkaline protease as well as keratinase. Onehundredmilligrams offeather keratinwasincubated at 50c with 50,ug of keratinase in 15 ml of 50 mm phosphatebuffer ph7. In other words these enzymes have wide range of temperature and ph optima. It is, thus, urgently needed to use alternative processes free from pollution. Research article efficient degradation of feather by. Production and estimation of keratinase by immobilized and. The aim of this work was to clone the kera gene in an expression vector followed by expression of keratinase under xylose inducible promoter in bacillus megaterium and investigate the ability of the purified. During the present study, the enzyme production by the bacterial cells in broth has been quantified as units of enzyme activity per milliliter of cell free broth per minute. The strain grew and produced alkaline keratinase using chicken feather and horn.
Mechanism of extracellular keratin degradation by keratinolytic bacteria was proposed by yamamura et al. The effects of additional agricultural wastes to keratinase production were tested. Keratinase production and keratin degradation by a mutant. Further, purified bsker71 could hydrolyze a variety of natural proteins, such as fibrous protein, collagen protein, casein, keratin and bovine serum albumin. A wide variety of keratinase enzyme options are available to you, such as cream. The cell free supernatant was collected and used for the assay of keratinase activity. Biodegradation of wool waste and keratinase production in. To check the keratinase production and optimized with various temperature such as 20c, 30c, 40c, 50c and 60c. Organic solvents such as methanol, ethanol and isopropanol can induce catalytic activity of the enzyme, peek et al. Production of keratinase and lmethioninase from aspergillus flavipes utilizing chicken feathers as the substrate under solid state fermentation was reported. Unit of keratinase activity in the assay is defined as an increase in the a450 of 0. Isolation, partial purification and characterization of.
Gopinath, 1,2,3 periasamyanbu, 4 thangavellakshmipriya, 2 theanhocktang, 2 yengchen, 3 udahashim, 1 a. Production, purification and characterization of an extracellular. The effect of temperature on keratinase activity was determined by the addition of 20 ll keratinase 3 mg ml1 protein to 1. Pdf recently, chicken feathers have been discarded in bulk as waste from poultry processing industries. Optimization of physicochemical parameters for hyper keratinase. Effective production of keratinase by gellan gumimmobilised. Feather was the optimal substrate for keratinase production fig. The medium used for enzyme production was composed of 0.
The enzyme fraction of the first peak showed protease activity only, while the enzyme fraction in. Jul 23, 2009 an extremely alkaliphilic bacterial strain, bacillus sp. The maximum production of keratinase occurred after 48 h in shaking culture at ph 11. The first step was the production of keratinase enzyme from chicken feathers and the second was to check the stability of enzyme. Biodegradation of feather waste by keratinase produced from newly. Efficient degradation of feather by keratinase producing. Bacillus licheniformis showing higher keratinase activity was screened out of the ten different bacterial strains isolated. Purification and characterization of keratinase from feather. Feather degradation by keratinolytic bacteria and biofertilizing. Ppks2, was isolated from rice mill effluents and screened for the production of extracellular keratinase. The ability of gellan gumimmobilised cells of the heavy metaltolerant bacterium alcaligenes sp. Isolation, purification and characterization of keratinase. The 53 kda keratinolytic enzyme is a serralysin optimally active at 4045 c, ph 6. Screening and characterization of keratinase from bacillus.
Effect of substrate concentration on keratinasemaximum amount of enzyme production was found in production. The best activity is seen with freshly prepared solutions. Enhancing the ability of keratinase by using immobilised enzymes. From the previous discussion of leudekingpiret model in 3l fermenter, we could know that 12 h was the maximum point of keratinase increasing rate and 24 h was the stable phase of enzyme production. Optimization of parameters for fermentative production of keratinase enzyme in our earlier studies. Production, characterization and application of keratinase. Medium optimization for enzyme production and its application in. The enzymeactivity was inhibited by reduced glutathione, pmsf and 2mercaptaethanol.
Production of a keratinolytic protease from serratia marcescens p3 was. On 4 th day enzyme production was highest 140 ku ml1 with 1% feather. Optimization of azokeratin hydrolysis by alginateimmobilized. The highest keratinase production was observed at 30c by bacillus spp and aspergillus spp. Cloning and expression of keratinase gene in bacillus. Pdf production of keratinase by free and immobilized cells. Recent developments in keratinase production have attracted. The keratinase gene with and without leader sequence from the chromosomal dna of bacillus licheniformis mku3 was amplified by pcr and cloned into pet30b and transferred into escherichia. Bacillus subtilis atcc 6633 was used for keratinase and protease production by submerged cultivation. Keratinase can be produced by many kinds of microorganisms, and has broad application prospects in the fields of feed, leather, medicine, food and. Partial characterization and its application in feather degradation and dehairing of the goat skin author.
Prolonging the utilization of keratinase by entrapment of. However, the production of such enzymes is not exclusive to dermatophytes, since geophilic species have demonstrated keratinase production. Keratinase production was carried out in the following basal medium nh 4 cl0. Jan 15, 2020 keratinase is one of the proteolytic enzymes, which can hydrolyze insoluble feather keratins into free amino acids and polypeptides gupta et al. These factors including feather concentration, incubation time, ph and temperature were optimized to. Present study showed that the optimum moisture level of 69. Enzyme production was monitored according to the keratinase activity. Recombinant expression and biochemical characterization of. Proteases are ubiquitous enzymes that occur in various biological systems. Review article biotechnological aspects and perspective of. Keratinases have been used because of their ability to separate keratin that cannot be broken down by common proteases in the processing of wool fabrics in the textile industry.
Arshad 1 institute of nano electronic engineering inee, universiti malaysia perlis, kangar, perlis, malaysia. Isolation and identification of a keratinolytic bacillus cereus and. The purified enzyme showed maximum keratinase activity at temperature 65 oc and ph 10. Ammonia production during feather degradation by bacillus sp. Optimisation of the media ph, incubation temperature and immobilisation parameters bead size, bead number, gellan gum concentration was determined for the. Strain improvement resulted in isolation of mbf11 and mbf21 from bf11 and bf21 isolates. Pdf the purpose of the present research was to study the production and.
Ten microbial isolates were screened for their keratinase production. Thus, keratinase enzyme by degrading poultry wastes feather can be an. Carrageenan beads showed higher stability of the enzyme when compared to other substrates from the earlier studies. An additional two to three feeding trials are planned, followed by a largescale, 7week feeding trial once. The strain grew and produced alkaline keratinase using chicken feather and horn meal. Complete degradation was achieved in 4 days with immobilized partial purified keratinase as compared to free cells which took 56 days. The native strains produced more than 10 kuml of enzyme. Currently, various keratinases have been purified or cloned from bacteria, yeasts, and fungi ramnani et al. In this study, to enhance the keratinase production from streptomyces sp.
The optimum ph and ph stability range for the production of. He isolated an inducible extracellular homogenous enzyme, which shows a 7. Nine bacteria, reported to produce extracellular proteases, were screened for production of keratinases. Optimization for keratinase enzyme production using. Purification and characterization of keratinase from. Results and discussion keratinase production was compared between free cells of four mbf strains with immobilized cells in carragenan with tcp. Valkerase is a unique keratinase processing enzyme processing additive that creates better quality, lower cost feather meal naturally discovered by dr. Keratinase can be produced by many kinds of microorganisms, and has broad application prospects in the fields of feed, leather, medicine, food and other industrial, as well as environmental governance. The half lives of the free and immobilized enzyme were 45 and 60. Industrial application of keratinase and soluble proteins. Enhancing the ability of keratinase by using immobilised. Dec 07, 2006 feather was the optimal substrate for keratinase production fig.
Production and characterization of feather degrading. Optimisation of the media ph, incubation temperature and immobilisation parameters bead size, bead number, gellan gum concentration was. Inorganic nitrogensourceintheformofnh 4 clhadnosigni cante ect on the production of keratinase for the mbf isolates, with the yield being either lower than or comparable to controls. The ability of bacillus licheniformis to utilize chicken feather powder as a substrate was tested. After 12 days of incubation of cellfree filtrate with keratin azure, mean of three replicates. Recombinant expression and biochemical characterization of a. Microbial keratinase production and application to improve.
The enzyme is stable 3040oc and active around wide ph range 68. Two native strains bf11 bacillus subtilis and bf21 bacillus cereus degrading keratin completely were characterized. The enzyme production was assayed by bacterium before optimizing the parameter of media components and culture condition. The crude enzyme from the keratinolytic strain stenotrophomonas sp. In our study, the production of enzyme by arthrobacter sp. The maximum production was obtained at 4th day of incubation at ph 9 and temperature 55oc. At the end of the fermentation, the remaining solid material was removed after centrifugation at 5. Keratinase producing microorganisms are being increasingly utilized for degradation and recycling of poultry feather waste. Aq05001 for the semicontinuous production of keratinase enzyme. Aq05001 to utilise both heavy metal free and heavy metalpolluted feathers hmpfs as substrates to produce keratinase enzyme was studied.
Prolonging the utilization of keratinase by entrapment of cells. The results of ph effect on production of keratinase in the range of 5. Screening and characterisation of keratinase enzyme. Of these, lysobacter ncimb 9497 exhibited the highest keratinolytic activity. Maximum keratinase production was peptone specific activity 85. Cloning and expression of keratinase gene in bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain. The crude enzyme solution was able to degrade native feather up to 63% in redox free. Inhibition of the enzyme by edta was verified by letourneau et al.
Production of keratinase by free and immobilized cells of. Jason shih, cofounder of bri, valkerase hydrolyzes keratin peptide bonds in feather waste, resulting in feather meal that is more easily digested by animals. An extremely alkaliphilic bacterial strain, bacillus sp. Water content was varied to achieve optimum keratinase production. However, singleuse aliquots of keratinase solutions can be stored at 20 c. Production of keratinase by free and immobilized cells of bacillus halodurans strain ppks2. Production, partial optimization and characterization of. Pdf production of keratinase by free and immobilized. The study indicates that hmpf can be effectively utilised as a substrate by the immobilisedcell system of alcaligenes sp. Production, partial optimization and characterization of keratinase. The bacterium grew well and highest protease production occurred in 144 h at mesophilic temperature 30 c and alkaline condition ph 810 with enzyme activities of 4 and 168 uml. Keratinase enzyme production data at varying feather analyzed using completely randomized. The aim of this study was to determine the keratinolytic ability of a range of bacteria and subsequently, to characterize the keratinase showing the greatest biotechnological potential. Production, purification and characterization of an.
Solid state fermentation ssf was developed to improve keratinase productivity. Feather meal was the most degraded 97% and induced higher keratinase activity. The ability of a microorganism to degrade keratin and the resulting levels of keratinase produced vary according to the specie, chemical composition, the. In terms of growth, the organisms showed less growth at ph 4. Keratinase is one of the proteolytic enzymes, which can hydrolyze insoluble feather keratins into free amino acids and polypeptides gupta et al. In order to determine the optimal temperature in different stages for efficient temperatureshift process, keratinase activities at 12, 24, and 48. Among all the keratincontaining substrates, feather was mostly utilized, followed by hair and wool. Analytical method for keratinase production keratinase activities were measured by the method described by dozie et al. Microbial bioremediation of feather waste for keratinase production. Optimization of keratinase production by keratinolytic. In addition, this keratinase exhibited high enzyme activity in a wide range of ph and optimal ph of 10. Keratinase is routinely being produced by largescale fermentation, and the application of this enzyme is underway, with two chickenfeeding trials already completed. Optimization for keratinase enzyme production using bacillus.
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