Prediction of Bioactive Peptides From Chicken Feather and Pig Hair Keratins Using In Silico Analysis Based on Fragmentomic Approach

Background: Keratin is among the most abundant structural proteins of animal origin, however it remains broadly underutilized. Objective: Bioinformatic investigation was performed to evaluate selected keratins originating from mass-produced waste products, i.e., chicken feathers and pig hair, as potential sources of bioactive peptides. Methods: Pepsin, trypsin, chymotrypsin, papain, and subtilisin were used for in silico keratinolysis with the use of “Enzyme(s) action” and fragmentomic analysis of theoretical products was performed using “Profiles of potential biological activity” in BIOPEP-UWM database of bioactive peptides. Bioactivity probability calculation and toxicity prediction of the peptides obtained were estimated using PeptideRanker and ToxinPred tools, respectively. Results: Our results showed that the keratins are a potential source of a variety of biopeptides, including dipeptidyl peptidase IV, angiotensin converting enzyme, prolyl endopeptidase inhibitory and antioxidative. Papain and subtilisin were found to be the most appropriate enzymes for keratin hydrolysis. This study presents possible structures of keratin-derived bioactive peptides that have not been previously described. Conclusion: Our data suggest additional in vitro and in vivo studies to verify theoretical predictions and further investigate the possibility of using keratin-rich waste as a source of peptide nutraceuticals.

Bio-plastic Films Production from Feather Waste Degradation by Keratinolytic Bacteria Bacillus cereus

Plastic materials have become a necessity of human life especially in the packaging of food commodities and biomedical procedures. Bioplastic is emerging as an effective alternative to fossil oil-based materials to avoid the environmental hazards of the plastic industry. During this study, chicken feathers were used as a substrate to isolate keratin degrading bacteria. Among 14 identified isolates, Bacillus sp BAM3 was found to be the most promising isolate. Partial 16S rDNA analysis-based molecular characterization revealed it is a strain of Bacillus cereus. Bacillus sp BAM3 can grow and produce keratinase in feathers containing basal medium as the sole carbon and energy source. The maximum keratinase production (730U/ml) was achieved within 24 h under optimum reaction conditions. The optimized reaction pH and temperature were noted as 9.0 and 50 °C for crude keratinase activity, respectively. The chicken feathers were used as a substrate in 2, 5, and 10 wt% glycerol to synthesize keratin-based bioplastic with keratinolytic bacterium Bacillus cereus BAM3. Bioplastic prepared from keratin with 2% of glycerol was found to possess good mechanical properties. Therefore, the results present a novel keratinolytic isolate of Bacillus cereus BAM3, which may have potential biotechnological applications in keratin hydrolysis processes. The development of keratin-based bioplastics possessing superior crystalline morphology requires further investigations to substitute fossil oil-based materials.

BIOSTIMULATOR MADE OF WASTE OF KERATIN-CONTAINING RAW MATERIALS

Ecosystem degradation forces us to introduce highly productive, environmentally friendly biotechnologies in agricultural production. In this regard, interest in organic farming is growing. Demand for organic products is ahead of supply. This, in its turn, causes interest in substances that, on the one hand, are safe for the environment, and on the other hand, increase yield and quality of crops. These substances include biostimulants. They stimulate the natural processes of assimilation of nutrients, increase stress resistance of plants. In this regard, keratin-containing raw materials deserve special attention, which, in terms of chemical composition, is a protein concentrate (90-95%) . But its use is limited due to the low functionality of the keratin protein of this raw material. Enzymatic keratin hydrolysis will provide a biostimulant for plants with a high content of amino acids. The aim of the study was to develop an enzymatic method for keratin hydrolysis with a high content of amino acids in the final product. Fermentation was carried out by Str. chromogeness.g. 0832 proteinase. The content of soluble protein, peptides and amino acids was carried out photocolorimetrically. Temperature (4000C) and pH (8.0) conditions of Str. chromogeness.g. 0832 enzyme complex have been determined, which allowed further hydrolysis of keratin-containing raw materials with a maximum yield of final products - amino acids (99.57 g/100g). Treatment of corn seeds with the obtained amino acid solution enabled to increase growth-promoting activity by 48%.

Feather Keratin Hydrolysis by an Aquatic Bacterium Meiothermus I40 from Hot Spring Water

After undergoing keratinase digestion, feather wastes could have a great potential as a source of proteins and amino acids for many applications. In this study, the fermentation conditions of feather-degrading Meiothermus sp. strain I40 (I40) were optimized to enhance the biodegradation of chicken feather to hydrolysates. Initially, the factors essential for I40 keratinase production in submerged fermentation were screened, whereas response surface methodology (RSM) was then employed to evaluate the interactions among the effective factors. At first stage, eight fermentation parameters were screened using a Plackett-Burman (PB) design. Four effective factors identified by PB screening, namely feather concentration, tryptone concentration, yeast extract concentration, and incubation temperature, were further investigated their effects on keratinase production by RSM using central composite design (CCD). The I40 fermentation conditions for maximal keratinase activity were as follows: tryptone concentration 0.16 percent (w/v), yeast extract concentration 0.27 percent (w/v), feather concentration 0.08 percent (w/v), and incubation temperature at 51.7°C for 72 hr under 120 rev/min shaking. Compared to the initial stage, a 13.3-fold increase in keratinase activity was achieved when I40 incubated in the optimized conditions.

Subtilisins ofBacillusspp. hydrolyze keratin and allow growth on feathers

Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. Studies presented herein demonstrate that B. licheniformis Carlsberg strain NCIMB 6816, which produces the well-characterized serine protease subtilisin Carlsberg, also degrades and grows on feathers. The PWD-1 and Carlsberg strains showed a similar time-course of enzyme production, and the purified serine proteases have similar enzymatic properties on insoluble azokeratin and soluble FITC-casein. Kinetic analysis of both enzymes demonstrated that they have high specificity for aromatic and hydrophobic amino acids in the P1 substrate position, although keratinase discriminates more than subtilisin Carlsberg against charged residues at this site. Nucleotide sequence analysis of the serine protease genes from B. licheniformis strains PWD-1, Carlsberg NCIMB 6816, ATCC 12759, and NCIMB 10689 showed that the kerA-encoded protease of PWD-1 differs from the others only by having V222, rather than A222, near the active site serine S220. Further, high-level expression of subE-encoded subtilisin from B. subtilis (78% similar to subtilisin Carlsberg) also confers growth on feathers on a protease-deficient B. subtilis strain. While strain PWD-1 and the kerA protease efficiently degrade keratin, keratin hydrolysis and growth on feathers is a property that can be conferred by appropriate expression of the major subtilisins, including the industrially produced enzymes.Key words: keratin hydrolysis, Bacillus, subtilisin, keratinase.