Optimization, characterization and thermodynamic studies on B. licheniformis ALW1 keratinase
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Optimization of B. licheniformis ALW1 keratinase was investigated by using a Plackett – Burman design (PBD) and Central Composite Design (CCD). PBD showed that galactose, inoculum size and corn steep liquorwere the most effectivevariables played a rolein improving the enzyme productivity (87.65U/mL). CCD results recorded an increase in enzyme productivity to about1.4-fold compared to the basal medium (99.1 U/mL). The optimum activity for the partial purified enzyme was obtained at pH 8.5 and 70˚C. The activation and deactivation energy were calculated to be25.37 kJmol-1 and73.38 kJmol-1 respectively. The half-life time was 1380,690,530, and383 min. at 50˚C,55˚C,60˚C and 65˚C respectively. Also, D values were 4600,2300,1769, 1277min. at the same degree respectively. ∆G° (kJmol-1) kept relatively constant between 50-60˚C (191.49 kJmol-1-193.31 kJmol-1) and noticeably increase at 65˚C (212.86 kJmol-1). ∆H° (kJmol-1) recorded minor decrease by the increase of temperature. Approximately, most of the tested metals ions have stimulation effect in enzyme activityand MgSO4.H2O was the best (146%). Among all the tested detergents tween 80 retained 97% of original enzyme activity. DMSO increased the enzyme activity about 11%, while propanol and acetonitrile reduced the enzyme activity to about 14% and 10% respectively. All the reducing agents had a stimulating effect on enzyme activity with variable degrees. The enzyme (980 U) had the ability to hydrolyze 74% of the feather to nutritional valuable protein.Keywords:
Keratinase
Central composite design
Bacillus licheniformis
Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2 U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2 U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65 °C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60 °C for almost 90 min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.
Keratinase
Bacillus licheniformis
Feather meal
Bacillus (shape)
Bacillus sphaericus
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Isolate local Bacillus licheniformis KA-08 known extracellular thermostable keratinase producers. Scale up of thermostable keratinase production can be with cells immobilized. The objective of the research is to thermostable keratinase production of B. licheniformis KA-08 cells immobilization. Thermostable keratinase activities were determined with modification of Brandelli and Riffel method. Protein concentration of enzyme determined with Lowry method. Immobilization of cells by Ca-alginate matrix with Adinarayana method, alginate concentration and amount of alginate bead effects with Beshay method. The result extracellular thermostable keratinase of B. licheniformis KA-08 cells immobilized was maximum produced at 12 times incubation with activity as 9.25 U/mg. Three percent alginate has optimum activity. Three hundred alginate beads has optimum activity. Cells immobilized of B. licheniformis KA-08 has scale up of thermostable keratinase activity at 2 times than free cells. Thermostable keratinase produced by cell immobilized was nine cycles.
Keratinase
Bacillus licheniformis
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Alginate immobilized Bacillus licheniformis (St. 24) were isolated from chicken feather wastes recorded higher keratinase production than free bacterial suspension. 2-3% alginate concentration, medium pellet size, and in addition of keratin powder as adjuvant were optimum for keratinase production by the immobilized bacteria. Encapsulated B. licheniformis st. 24 was successfully produced keratinase for three repeated batch fermentation cycles each 24 hour incubation period. Immobilization of the whole cells proved to be useful for continuous production of keratinase and feather degradation.
Keratinase
Bacillus licheniformis
Pellet
Feather meal
Incubation period
Suspension
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Feathers are composed of beta keratin which is an insoluble protein resistant to degradation. Bacillus licheniformis has a gene that codes for a keratinase enzyme that hydrolyzes feathers. The DNA sequences of the genes coding for keratinase enzymes of various strains of B. licheniformis were compared. Some strains of the bacteria hydrolyze feathers rapidly, some slowly, and some at an intermediate rate. The mature keratinase enzyme is composed of 274 amino acids three of which, aspartic acid, histidine, and serine, are specific for activity at the catalytic site. The keratinase enzyme amino acid sequences from strains of the bacteria that degrade feathers rapidly, slowly and intermediately all contain the three specific amino acids of the catalytic site in their respective positions. Genetic mutations that resulted in changes in the amino acid sequence of the mature keratinase enzymes did not correspond to differences in the ability of the enzymes to degrade feathers. The −10 and −35 sequences of the bacterial promoter site of the keratinase enzyme genes were found to be essentially identical in all three classes of feather degrading strains of bacteria. Subtilisin is a closely related serine protease enzyme produced by B. subtilis. Control of expression of subtilisin is mediated by at least 15 regulator proteins and is still not completely understood. The differences in the feather degrading abilities of various strains of B. licheniformis are probably due to complex patterns of regulation of expression of secreted protease enzymes of the Bacillus genus +. Keywords: Keratinase genes, feathers, Bacillus licheniformis Cite this Article Goldstein Gerald, Celestino-Soper Patricia BS, Morrell Dailey Allison K, et al. Comparison of the keratinase gene sequences of fast, slow and intermediate feather degrading strains of Bacillus licheniformis. Research & Reviews: Journal of Microbiology and Virology. 2015; 5(3): 1–9p. Normal 0 false false false EN-US X-NONE X-NONE
Keratinase
Bacillus licheniformis
Subtilisin
Feather meal
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A novel bacterium, Bacillus licheniformis K-19, which produces a large amount of akeratinase that is extremely thermostable and has a broad resistance to pH, was isolated and characterized. The maximum amount of keratinase activity (about 224 Uml-1) was produced at 37°C when the bacterium was cultured for 72 h in broth containing feather meal with initial pH of 7.5. The keratinase activity was observed over a wide range of temperatures (30 - 90°C) and pH values (pH 6 - 10). It was optimal at 60°C and pH 7.5 - 8 respectively. These results suggest potential biotechnological applications of this bacterium that involve hydrolysis of keratin, including the improvement of the nutritional properties of feathers (and other keratins) used as supplementary feedstuffs.
Key words: Bacillus licheniformis, chicken feather, keratin, keratinolytic protease.
Keratinase
Bacillus licheniformis
Feather meal
Isolation
Bacillus (shape)
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Optimal medium was used to improve the production of keratinase by Bacillus licheniformis ZJUEL31410, which has a promising application in the transformation of feather into soluble protein. The results of single factor design revealed that the concentration of feather at 20 g/l and the initial pH at value 8 was the best for the production of keratinase and the degradation of feather. Ammonia salt and nitrate salt strongly restricted the production of keratinase and the degradation of feather. Result of Box-Behnken design (BBD) experiment which was used to optimize concentrations of glucose, corn steep flour and K 2 HPO 4 for further improvement of keratinase productivity showed that the optimal medium was composed of glucose (20 g/l), corn steep flour (7.5 g/l), K 2 HPO 4 (1 g/l) and feather (20 g/l). The result of submerged batch cultivation of B. licheniformis ZJUEL31410 in the 5 L fermentor indicated that the optimal medium had the highest keratinase and the degree of feather degradation (DFD) at 54.9 U/ml and 72.4%; both were 5 times more than the basal medium. The degradation of feather was verified by the analysis of scanning electron microscopy (SEM). This study provides a foundation for the production of keratinase and the conversion of feather to soluble protein through submerged fermentation process by B. licheniformis ZJUEL31410. Key words: Bacillus licheniformis ZJUEL31410, keratinase, culture medium, optimization, Box-Behnken design, scanning electron microscopy, feather degradation.
Keratinase
Bacillus licheniformis
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B. licheniformis and B. subtilis are capable to produce enzyme, which hydrolyzed feather highly. Keratinase is a group of commercial enzyme, that has many important applications in industries of waste management, leather, food. Hydrolyzed keratin is used to create some rare amino acids, such as, serine, cysteine and proline or used as animal feed, fertilizer, glue that contribute to reducing environmental pollution. We carried out cloning and expression of recombinant keratinase from Bacillus licheniformis and Bacillus subtilis in Escherichia coli BL21(DE3). Ker 1 from the strain B. licheniformis HT10 was integrated into pET22b vector and Ker 2 from B. subtilis Đ.nd1.2 was integrated into the pET32a vector. In the same expression conditions at 37°C, the 1mM IPTG, two obtained proteins showed a differ in size and activity of the enzyme respectively is 28 kDa, 23.5 U/ml and 36 kDa, 18.5 U/ml.
Keratinase
Bacillus licheniformis
Cloning (programming)
Environmental Pollution
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Keratin are insoluble fibrous proteins found in hair, wool, feather, nail, horns and other epithelial covering which is rich in beta helical coil linked through cysteine bridges. Keratinase (EC 3.4.4.25) belongs to the class hydrolase which are able to hydrolyse insoluble keratins more efficient than other proteases. The bacteria Bacillus licheniformis showing higher keratinase activity was screened out of the ten different bacterial strains isolated. The ability of Bacillus licheniformis to utilize chicken feather powder as a substrate was tested. It was found that maximum enzyme activity was 10.76U/ml. Similarly optimum temperature and pH for the enzyme activity was found to be 60 � C and 7.0 respectively. The k m and V max values were 0.22 mg/ml and 0.01 U/ml respectively. The enzyme is stable (30-40 o C) and active around wide pH range (6-8). Among the various metal ions tested zinc, magnesium were found to enhance the enzyme activity where as mercury, copper, cadmium, 1, 10 phenanthroline and EDTA completely inhibit the enzyme activity. It was found from this study, organism such as Bacillus licheniformis isolated from poultry soil can be used as a potential candidate for degradation of feather and for dehairing process in leather industry. (Researcher 2010;2(4):89-96). (ISSN: 1553-9865).
Bacillus licheniformis
Keratinase
Bacillus (shape)
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Isolate local Bacillus licheniformis KA-08 known extracellular thermostable keratinase producers. Scale up of thermostable keratinase production can be with cells immobilized. The objective of the research is to thermostable keratinase production of B. licheniformis KA-08 cells immobilization. Thermostable keratinase activities were determined with modification of Brandelli and Riffel method. Protein concentration of enzyme determined with Lowry method. Immobilization of cells by Ca-alginate matrix with Adinarayana method, alginate concentration and amount of alginate bead effects with Beshay method. The result extracellular thermostable keratinase of B. licheniformis KA-08 cells immobilized was maximum produced at 12 times incubation with activity as 9.25 U/mg. Three percent alginate has optimum activity. Three hundred alginate beads has optimum activity. Cells immobilized of B. licheniformis KA-08 has scale up of thermostable keratinase activity at 2 times than free cells. Thermostable keratinase produced by cell immobilized was nine cycles.
Keratinase
Bacillus licheniformis
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Forty isolates of Bacillus spp. were isolated from fifty samples including different source of soil to detect the ability to produce keratinase enzyme in liquid state fermentation, Bacillus (Bs13)was the highest keratinase producer , it was identified as a strain of Bacillus licheniformis. The optimum conditions for keratinase productions were in a media contains keratin 4% (hooves) as a carbon and nitrogen and energy sources, peptone 1% as a secondary nitrogen source with pH 8 , inculums size 1%, and incubated at 37C o for 24 hrs.
Keratinase
Bacillus licheniformis
Strain (injury)
Bacillus (shape)
Carbon source
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