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Chryseobacterium aquifrigidense FANN1 Produced Detergent-Stable Metallokeratinase and Amino Acids Through the Abasement of Chicken Feathers.

ABSTRACT: Microbial keratinases' versatility in the beneficiation of keratinous waste biomass into high-value products prompts their application in diverse spheres hence, advancing green technology and the bioeconomy. Consequently, a feather-degrading Chryseobacterium aquifrigidense FANN1 (NCBI: MW169027) was used to produce keratinase, and its biochemical properties were determined. The optimization of physicochemical parameters and analysis of the free amino acid constituents of the feather hydrolysate were also carried out. FANN1 showed a maximum keratinase yield of 1,664.55 ± 42.43 U/mL after 72 h, at optimal process conditions that included initial medium pH, incubation temperature, inoculum size, and chicken feather concentration of 8, 30°C, 4% (v/v), and 15 (g/L), respectively. Analysis of degradation product showed 50.32% and 23.25% as the protein value and total free amino acids, respectively, with a relatively high abundance of arginine (2.25%) and serine (2.03%). FANN1 keratinase was optimally active at pH 8.0 and relatively moderate to high temperature (40-50°C). EDTA and 1,10-phenanthroline inhibited the keratinase activity, and that suggests a metallo-keratinase. The enzyme showed remarkable stability in the presence of chemical agents, with residual activity 141 ± 10.38%, 98 ± 0.43%, 111 ± 1.73%, 124 ± 0.87%, 104 ± 3.89%, 107 ± 7.79%, and 112 ± 0.86% against DTT, H₂O₂, DMSO, acetonitrile, triton X-100, tween-80, and SDS, respectively. The residual activity of FANN1 keratinase was enhanced by Sunlight (129%), Ariel (116%), MAQ (151%), and Surf (143%) compared to the control after 60 min preincubation. Likewise, the enzyme was remarkably stable in the presence Fe³⁺ (120 ± 5.06%), Ca²⁺ (100 ± 10.33%), Na⁺ (122 ± 2.95%), Al³⁺ (106 ± 10.33%); while Co²⁺ (68 ± 8.22%) and Fe²⁺ (51 ± 8.43%) elicited the most repressive effect on keratinase activity. The findings suggest that C. aquifrigidense FANN1 is a potential candidate for keratinous wastes bio-recycling, and the associated keratinase has a good prospect for application in detergent formulation.

SUBMITTER: Bokveld A

PROVIDER: S-EPMC8377754 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Beta-keratin in poultry feathers is a structural protein that is resistant to degradation due to disulfide and hydrogen bonds. Feather meal can be a valuable feed compound if the digestibility can be increased. The objective of the present study was to analyze the effects of chemical, enzymatic, and pressure-thermic treatments for chicken feathers on solubility, in vitro protein digestibility (IVPD), and amino acid composition of solubilized and residual fractions. Two experiments were conducted. In experiment 1, models for solubility and IVPD were developed including the above factors applying a central composite face-centered design. Addition of sodium hydroxide (NaOH) and sodium sulfite (Na2SO3), and autoclaving time affected solubility and IVPD of the feather hydrolysates, but not addition of keratinolytic enzyme. In experiment 2, 7 combinations of the hydrolysis factors NaOH, Na2SO3, and autoclaving time with a predicted IVPD of 900 g/kg of DM, calculated for the sum of solubilized and residual feather fractions, were included to measure effects on IVPD and amino acid composition in each fraction. The IVPD values were higher for solubilized than residual fractions when treated with NaOH and autoclaving, but no differences were found when treated with Na2SO3 and autoclaving. Losses of cystine were substantial for all treatments, but lower for Na2SO3 than for NaOH. Furthermore, use of lower Na2SO3 concentration and longer autoclaving time reduced losses of cystine. Compared with NaOH treatments, Na2SO3 gave lower losses of threonine, arginine, serine, and tyrosine. With reference to the ideal protein profile for Atlantic salmon (Salmo salar L.), the treatments with 60 or 90 min autoclaving and 0.36 or 0.21% Na2SO3 had the highest chemical scores. The scores were generally higher for amino acids in residual than solubilized fractions, but with 90 min autoclaving and 0.21% Na2SO3 differences were small. In conclusion, hydrolysis of chicken feathers with low concentrations of Na2SO3 combined with autoclaving results in feather meal with high nutritional value for Atlantic salmon; separation of solubilized and residual fractions is not necessary.

Project description:Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) → ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell's resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

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Chryseobacterium aquifrigidense FANN1 Produced Detergent-Stable Metallokeratinase and Amino Acids Through the Abasement of Chicken Feathers.

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