Bioconversion of chicken feather into amino acids and keratinase production by mesophilic Chryseobacterium proteolyticum and Pseudomonas aeruginosa isolated from municipal waste dumpsites
- Giwu, Nonkonzo https://orcid.org/0000-0001-9416-7896
- Authors: Giwu, Nonkonzo https://orcid.org/0000-0001-9416-7896
- Date: 2021-02
- Subjects: Poultry -- Processing , Proteolytic enzymes
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/22732 , vital:52720
- Description: Chicken feathers are by-products of poultry processing which are generated in large amount because of the global growing demand for poultry meats. They have high contents of crude proteins in the form of keratin which could be valorized into digestible products. Keratinases are classified as a specific collection of proteolytic enzymes that have the ability for the degradation of recalcitrant keratinous substrates. Isolation and characterization of these enzymes from various microbial producers are gaining prominence in recent years due to their industrial and biotechnological application potentials. For this research, the collection of soil samples was done as well as the isolation of bacteria and the screening for keratinolytic activity. 16S rDNA sequencing and phylogenetic analysis were used to identify the isolates with efficient chicken feathers degrading capacity. Optimum conditions for the fermentation prcocess was enhanced for the production of keratinase. The fermentation broth was also analysed for various amino acids of protein, and the biochemical properties of the enzymes were likewise determined. Twenty two (22) bacteria were isolated from the soil samples, and 18 out of the 22 isolates showed proteolytic activity on solid media with diameters of halo zone that ranged from 5 ± 0.71 mm for isolate coded as PSS-03 to 25 ± 1.41 mm for isolate coded as PSS-06. Intact chicken feathers were degraded by proteolytic bacterial isolates in variable degree that ranged from 24percent for PSS-10 and 81percent for DSS-02. Extracellular keratinase production recorded for the isolates ranged from 63.63 ± 4.14 U/mL for PSS-10 to 693.63 ± 62.99 U/mL for DSS-02. Based on 16S rDNA sequence and phylogenetic analysis, the 2 isolates with remarkable keratinolytic activity coded as DSS-02 and PSS-14 were identified as Chryseobacterium proteolyticum FGNn and Pseudomonas aeruginosa GNFx. C. proteolyticum showed the maximum keratinase production of 756.36 U/mL after 72 h of incubation at optimized fermentation conditions which involved initial medium pH (4), incubation temperature (30 oC), inoculum size (2percent; v/v), and chicken feathers (1.5percent; w/v). Similarly, P. aeruginosa optimally produced keratinase (1055.45 U/mL) after 96 h of incubation at optimized fermentation conditions that involved initial medium pH (7-8), incubation temperature (30 oC), inoculum size (5percent; v/v), and chicken feathers (2.5percent; w/v). Furthermore, feather hydrolysate from C. proteolyticum FGNn had relatively higher abundance (>1.5g/100g sample) of arginine (1.85), serine (1.63), glycine (1.9) and lysine (1.62); while P. aeruginosa GNFx feather hydrolysate showed high abundance of arginine, serine, aspartic acid, glutamic acid, glycine, alanine, valine, and leucine with respective concentration of 2.06, 1.67, 2.39, 3.05, 1.87, 1.73, 1.56 and 1.65 (g/100g sample). The results showed that keratinases from the two bacterial isolates were optimally active at pH 8, and temperature of 50 oC for FGNn keratinase and 50-60 oC for GNFx keratinase. The enzymes displayed remarkable pH stability. Keratinase from C. proteolyticum was catalytically inhibited by EDTA and 1,10-phenanthroline but not affected by PMSF; while P. aeruginosa keratinase was not significantly affected by those class of protease inhibitors. Adiitionally, FGNn keratinase demonstrated high residual activity of 90percent, 103percent, 101percent, 110percent, 130, and 105percent in the presence of DTT, hydrogen peroxides, acetonitrile, triton X-100, tween-80 and SDS, respectively. Similarly, catalytic efficiency of GNFx keratinase was promoted in the presence of hydrogen peroxides (119percent), triton X-100 (140percent), tween-80 (150percent) and SDS (147percent) compared to the control. Furthermore, the keratinases from the both bacterial isolates exhibited catalytic efficiency enhancement and remarkable structural stability in the presence of laundry detergents tested. The findings from the study suggest the application potentials of the isolates for the bioconversion of recalcitrant keratinous wastes into digestible and quality protein hydrolysates. The properties of these microbial keratinases indicate that they may be exploited for various biotechnological and industrial processes especially in the formulation of detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Giwu, Nonkonzo https://orcid.org/0000-0001-9416-7896
- Date: 2021-02
- Subjects: Poultry -- Processing , Proteolytic enzymes
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/22732 , vital:52720
- Description: Chicken feathers are by-products of poultry processing which are generated in large amount because of the global growing demand for poultry meats. They have high contents of crude proteins in the form of keratin which could be valorized into digestible products. Keratinases are classified as a specific collection of proteolytic enzymes that have the ability for the degradation of recalcitrant keratinous substrates. Isolation and characterization of these enzymes from various microbial producers are gaining prominence in recent years due to their industrial and biotechnological application potentials. For this research, the collection of soil samples was done as well as the isolation of bacteria and the screening for keratinolytic activity. 16S rDNA sequencing and phylogenetic analysis were used to identify the isolates with efficient chicken feathers degrading capacity. Optimum conditions for the fermentation prcocess was enhanced for the production of keratinase. The fermentation broth was also analysed for various amino acids of protein, and the biochemical properties of the enzymes were likewise determined. Twenty two (22) bacteria were isolated from the soil samples, and 18 out of the 22 isolates showed proteolytic activity on solid media with diameters of halo zone that ranged from 5 ± 0.71 mm for isolate coded as PSS-03 to 25 ± 1.41 mm for isolate coded as PSS-06. Intact chicken feathers were degraded by proteolytic bacterial isolates in variable degree that ranged from 24percent for PSS-10 and 81percent for DSS-02. Extracellular keratinase production recorded for the isolates ranged from 63.63 ± 4.14 U/mL for PSS-10 to 693.63 ± 62.99 U/mL for DSS-02. Based on 16S rDNA sequence and phylogenetic analysis, the 2 isolates with remarkable keratinolytic activity coded as DSS-02 and PSS-14 were identified as Chryseobacterium proteolyticum FGNn and Pseudomonas aeruginosa GNFx. C. proteolyticum showed the maximum keratinase production of 756.36 U/mL after 72 h of incubation at optimized fermentation conditions which involved initial medium pH (4), incubation temperature (30 oC), inoculum size (2percent; v/v), and chicken feathers (1.5percent; w/v). Similarly, P. aeruginosa optimally produced keratinase (1055.45 U/mL) after 96 h of incubation at optimized fermentation conditions that involved initial medium pH (7-8), incubation temperature (30 oC), inoculum size (5percent; v/v), and chicken feathers (2.5percent; w/v). Furthermore, feather hydrolysate from C. proteolyticum FGNn had relatively higher abundance (>1.5g/100g sample) of arginine (1.85), serine (1.63), glycine (1.9) and lysine (1.62); while P. aeruginosa GNFx feather hydrolysate showed high abundance of arginine, serine, aspartic acid, glutamic acid, glycine, alanine, valine, and leucine with respective concentration of 2.06, 1.67, 2.39, 3.05, 1.87, 1.73, 1.56 and 1.65 (g/100g sample). The results showed that keratinases from the two bacterial isolates were optimally active at pH 8, and temperature of 50 oC for FGNn keratinase and 50-60 oC for GNFx keratinase. The enzymes displayed remarkable pH stability. Keratinase from C. proteolyticum was catalytically inhibited by EDTA and 1,10-phenanthroline but not affected by PMSF; while P. aeruginosa keratinase was not significantly affected by those class of protease inhibitors. Adiitionally, FGNn keratinase demonstrated high residual activity of 90percent, 103percent, 101percent, 110percent, 130, and 105percent in the presence of DTT, hydrogen peroxides, acetonitrile, triton X-100, tween-80 and SDS, respectively. Similarly, catalytic efficiency of GNFx keratinase was promoted in the presence of hydrogen peroxides (119percent), triton X-100 (140percent), tween-80 (150percent) and SDS (147percent) compared to the control. Furthermore, the keratinases from the both bacterial isolates exhibited catalytic efficiency enhancement and remarkable structural stability in the presence of laundry detergents tested. The findings from the study suggest the application potentials of the isolates for the bioconversion of recalcitrant keratinous wastes into digestible and quality protein hydrolysates. The properties of these microbial keratinases indicate that they may be exploited for various biotechnological and industrial processes especially in the formulation of detergents. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
Valorization of chicken feather through dekeratinization by keratinolytic Bacillus species to amino acid
- Authors: Matches, Lupho
- Date: 2021-02
- Subjects: Proteolytic enzymes , Poultry -- Processing
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20451 , vital:45667
- Description: The poultry meat processing sector generates chicken feathers as by-products, and they are 90percent keratin in composition. Keratin is an insoluble and structural protein that shows recalcitrance to hydrolysis by classical proteolytic enzymes, including trypsin, pepsin, and papain. Keratinases are a group of proteolytic enzymes endowed with keratin degradation into peptides and amino acids. They are recently gaining traction for their multifaceted potential application in the green industrial space. Hence, keratinolytic bacteria previously isolated from dumpsite were identified using 16S rDNA sequencing. The optimal fermentation conditions were determined for enhanced extracellular keratinase production and chicken feather degradation. Also, the amino acid analysis of the chicken feather hydrolysates was carried out. The biochemical properties of the keratinases were also determined. Based on 16S rDNA sequencing and phylogenetic analysis, the isolates coded as SSN-02 and HSN-03 showed a high percentage of sequence homology with Bacillus spp.; hence, they were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM, respectively. Bacillus sp. NFH5 showed optimal keratinase production of 1149.99 ± 80.99 U/mL after 96 h of incubation time, in optimized fermentation conditions that included pH (4.0), chicken feather (1.5percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). Similarly, Bacillus sp. FHNM demonstrated the maximum keratinase production of 480 ± 41.14 U/mL 144 h post cultivation, in optimized fermentation conditions with pH (7.0), chicken feather (2.0percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). For Bacillus sp. NFH5 chicken feather hydrolysate, the amino acids in relatively higher concentration (>1.0g/100g sample) include arginine (1.8), serine (1.16), aspartic acid (1.95), glutamic acid (2.47), proline (1.16) and glycine (1.45). Bacillus sp. FHNM feather hydrolysates, contained (g/100g of sample): arginine (1.9), serine (1.4), aspartic acid (2.5), glutamic acid (2.51), glycine (1.51), proline (1.13), leucine (1.030, histidine (1.25), and lysine (1.06) (g/100g of sample) in high concentration. The keratinases were optimally active at pH 8.0. Bacillus sp. FHNM showed an optimal temperature of 100 oC; while Bacillus sp. NFH5 keratinase displayed optimal activity at 90 oC. EDTA and 1,10-phenanthroline inhibited the keratinases, and the inhibition pattern indicated that they belong to metalloprotease. Keratinase from Bacillus sp. FHNM showed considerable residual activity in the presence of Co²⁺ (93percent), Fe³⁺ (99percent), and K⁺ (94percent). Bacillus sp. NFH5 keratinase retained 92percent, 92percent, 93percent of the original activity against Ba²⁺, Na⁺ and Fe³⁺ treatment. Bacillus sp. FHNM keratinase was remarkably stable after 60 min of detergents treatment with residual activity of 89percent, 96percent, 81percent, 73percent, 96percent, 88percent, 88percent and 98percent for Omo, Surf, Ariel, Sunlight, Prowash, Freshwave, Sky, and Evaklin, respectively. Maq impacted the enzyme stability negatively, with residual activity of 48percent after 60 min of incubation. Additionally, keratinase Bacillus sp. NFH5 retained 68percent, 78percent, 80percent, 84percent, 57percent, 80percent, 98percent, 106percent and 106percent of the original activity against Omo, Surf, Ariel, Sunlight, Maq, Prowash, Freshwave, Sky and Evaklin, respectively. Therefore, these results suggest that Bacillus spp. could be ideal candidates for sustainable production of active keratinases and valorization of the abundantly generated keratinous biomass. The stability displayed by keratinases from Bacillus sp. FHNM and Bacillus sp. NFH5 suggests their promising candidacy for detergent formulation. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
- Authors: Matches, Lupho
- Date: 2021-02
- Subjects: Proteolytic enzymes , Poultry -- Processing
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/20451 , vital:45667
- Description: The poultry meat processing sector generates chicken feathers as by-products, and they are 90percent keratin in composition. Keratin is an insoluble and structural protein that shows recalcitrance to hydrolysis by classical proteolytic enzymes, including trypsin, pepsin, and papain. Keratinases are a group of proteolytic enzymes endowed with keratin degradation into peptides and amino acids. They are recently gaining traction for their multifaceted potential application in the green industrial space. Hence, keratinolytic bacteria previously isolated from dumpsite were identified using 16S rDNA sequencing. The optimal fermentation conditions were determined for enhanced extracellular keratinase production and chicken feather degradation. Also, the amino acid analysis of the chicken feather hydrolysates was carried out. The biochemical properties of the keratinases were also determined. Based on 16S rDNA sequencing and phylogenetic analysis, the isolates coded as SSN-02 and HSN-03 showed a high percentage of sequence homology with Bacillus spp.; hence, they were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM, respectively. Bacillus sp. NFH5 showed optimal keratinase production of 1149.99 ± 80.99 U/mL after 96 h of incubation time, in optimized fermentation conditions that included pH (4.0), chicken feather (1.5percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). Similarly, Bacillus sp. FHNM demonstrated the maximum keratinase production of 480 ± 41.14 U/mL 144 h post cultivation, in optimized fermentation conditions with pH (7.0), chicken feather (2.0percent, w/v), inoculum size (3percent, v/v) and temperature (30 oC). For Bacillus sp. NFH5 chicken feather hydrolysate, the amino acids in relatively higher concentration (>1.0g/100g sample) include arginine (1.8), serine (1.16), aspartic acid (1.95), glutamic acid (2.47), proline (1.16) and glycine (1.45). Bacillus sp. FHNM feather hydrolysates, contained (g/100g of sample): arginine (1.9), serine (1.4), aspartic acid (2.5), glutamic acid (2.51), glycine (1.51), proline (1.13), leucine (1.030, histidine (1.25), and lysine (1.06) (g/100g of sample) in high concentration. The keratinases were optimally active at pH 8.0. Bacillus sp. FHNM showed an optimal temperature of 100 oC; while Bacillus sp. NFH5 keratinase displayed optimal activity at 90 oC. EDTA and 1,10-phenanthroline inhibited the keratinases, and the inhibition pattern indicated that they belong to metalloprotease. Keratinase from Bacillus sp. FHNM showed considerable residual activity in the presence of Co²⁺ (93percent), Fe³⁺ (99percent), and K⁺ (94percent). Bacillus sp. NFH5 keratinase retained 92percent, 92percent, 93percent of the original activity against Ba²⁺, Na⁺ and Fe³⁺ treatment. Bacillus sp. FHNM keratinase was remarkably stable after 60 min of detergents treatment with residual activity of 89percent, 96percent, 81percent, 73percent, 96percent, 88percent, 88percent and 98percent for Omo, Surf, Ariel, Sunlight, Prowash, Freshwave, Sky, and Evaklin, respectively. Maq impacted the enzyme stability negatively, with residual activity of 48percent after 60 min of incubation. Additionally, keratinase Bacillus sp. NFH5 retained 68percent, 78percent, 80percent, 84percent, 57percent, 80percent, 98percent, 106percent and 106percent of the original activity against Omo, Surf, Ariel, Sunlight, Maq, Prowash, Freshwave, Sky and Evaklin, respectively. Therefore, these results suggest that Bacillus spp. could be ideal candidates for sustainable production of active keratinases and valorization of the abundantly generated keratinous biomass. The stability displayed by keratinases from Bacillus sp. FHNM and Bacillus sp. NFH5 suggests their promising candidacy for detergent formulation. , Thesis (MSc) -- Faculty of Science and Agriculture, 2021
- Full Text:
- Date Issued: 2021-02
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