Keratinase Production by Endophytic Bacteria Aneurinibacillus aneurinilyticus VRCS-4 Isolated from Xerophytic Plant Opuntia ficus - indica (Prickly pear)

Bacterial endophytes colonize an ecological niche which is unexplored site makes them suitable to produce pharmacologically active substances with vast biotechnological potential therefore, xerophytes were chosen to isolate the endophytes. In the present study forty endophytic bacterial isolates were isolated from xerophytic plants grown near poultry farms and feather dumping sites. Of them eight isolates showed zone of hydrolysis and the maximum zone of hydrolyisis of 36mm was with VRCS-4 on skimmed milk agar. This isolate exhibited efficient feather degradation and was identified as Aneurinibacillus aneurinilyticus based on its morphological, biochemical test and molecular sequencing method. The isolate was deposited in NCBI with an accession number MW227423.The isolate showed maximum en-zyme activity of 140.24U/ml at 72h, pH 7.5 and 40º C at 140 rpm. Chicken feather 1% (w/v) used as a sole source of carbon and nitrogen. Feather deg-radation by A.aneurinilyticus VRCS-4 showed 90% degradation in feather meal broth. Ours appears to be the first report on keratinase production by endophytic bacteria from xerophytic plant (Opuntia ficus -indica).

Molecular Identification of Keratinase DgokerA from Deinococcus gobiensis for Feather Degradation

Keratin is a tough fibrous structural protein that is difficult to digest with pepsin and trypsin because of the presence of a large number of disulfide bonds. Keratin is widely found in agricultural waste. In recent years, especially, the development of the poultry industry has resulted in a large accumulation of feather keratin resources, which seriously pollute the environment. Keratinase can specifically attack disulfide bridges in keratin, converting them from complex to simplified forms. The keratinase thermal stability has drawn attention to various biotechnological industries. It is significant to identify keratinases and improve their thermostability from microorganism in extreme environments. In this study, the keratinases DgoKerA was identified in Deinococcus gobiensis I-0 from the Gobi desert. The amino acid sequence analysis revealed that DgoKerA was 58.68% identical to the keratinase MtaKerA from M. thermophila WR-220 and 40.94% identical to the classical BliKerA sequence from B. licheniformis PWD-1. In vitro enzyme activity analysis showed that DgoKerA exhibited an optimum temperature of 60 °C, an optimum pH of 7 and a specific enzyme activity of 51147 U/mg. DgoKerA can degrade intact feathers at 60 °C and has good potential for industrial applications. The molecular modification of DgoKerA was also carried out using site-directed mutagenesis, in which the mutant A350S enzyme activity was increased by nearly 30%, and the results provide a theoretical basis for the development and optimization of keratinase applications.

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51–3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants’ effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5–100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D137N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5–19.3 and aliphatic index between 74.7–75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D137N of KerS13uv+ems (affinity: −7.17; S score: −6.54 kcal/mol) and seven mutants of KerS26uv (affinity: −7.43; S score: −7.17 kcal/mol) compared to the wild predicted structure (affinity: −6.57; S score: −6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry.

Study of proteolytic and keratinolytic enzymes of bacillus sp. A5.3

Feathers, as a major part of the waste from the industrial poultry industry, require a special approach for disposal and, at the same time, are of interest as a source of feed protein. Feather biomass consists of 90% β-keratin, hydrolysates of which can be a valuable source of pepton, but feather keratin is highly resistant to most proteolytic enzymes. For hydrolysis, therefore, keratin must be subjected to special treatment, the purpose of which is to break down the compact structure of the keratin molecule to produce polypeptides, peptides and single amino acids. For enzymatic hydrolysis of keratin, proteases with keratinase activity are used, capable of cleaving keratin disulfide bonds. A strain of Bacillus sp. A5.3 was isolated from feather waste sites and showed high proteolytic and keratinolytic activity. The strain is able to grow on minimal feather medium and has caseinolytic, collagenase and β-keratinolytic activity. The secretory proteome of the strain was studied using nano HPLC/Q-TOF-MS. As a result, 154 proteins were identified, 13 of which are proteases and peptidases. The genes for 3 proteases and peptidases clpY, clpX and ytjP were amplified from the genomic DNA of Bacillus sp. A5.3, sequenced, and the nucleotide sequence of the genes was deposited in the GenBank database. A study of a strain of Bacillus sp. A5.3 showed that the strain is capable of effective feather degradation and promising as a producer of proteolytic and keratinolytic enzymes.

A Novel Thermostable Keratinase from Deinococcus geothermalis with Potential Application in Feather Degradation

Keratinase can specifically attack disulfide bridges in keratin to convert them from complex to simplified forms. Keratinase thermal stability has drawn attention to various biotechnological industries. In this study, a keratinase DgeKer was identified from a slightly thermophilic species, D. geothermalis. The in silico analysis showed that DgeKer is composed of signal peptide, N-terminal propeptide, mature domain, and C-terminal extension. DgeKer and its C-terminal extension-truncated enzyme (DgeKer-C) were cloned and expressed in E. coli. The purified DgeKer and DgeKer-C showed maximum activity at 70 °C and pH 9–The thermal stability assay (60 °C) showed that the half-life value of DgeKer and DgeKer-C were 103.45 min and 169.10 min, respectively. DgeKer and DgeKer-C were stable at the range of pH from 9 to 11 and showed good tolerance to some metal ions, surfactants and organic solvent. Furthermore, DgeKer could degrade feathers at 70 °C for 60 min. However, the medium became turbid with obvious softening of barbules after being treated with DgeKer-C, which might be due to C-terminal extension. In summary, a thermostable keratinase DgeKer with high efficiency degradation of feathers may have great potential in industry.

Bioutilization of Chicken Feather Wastes by Newly Isolated Keratinolytic Bacteria Into Protein Hydrolysates With Improved Functionalities

In this study, a novel feather-degrading bacteria B. amyloliquefaciens KB1 was isolated from chicken farm bed (CFB), identified by morphological, physico-biochemical tests followed by 16s rDNA analysis. Among observed isolates, bacterial isolate (KB1) showed the highest degree of feather degradation (74.78 ± 2.94 %) and total soluble protein (205 ± 0.03 mg/ g). Using the same species of bacteria, the optimum fermentation condition was found at 40 oC, pH 9, and 1 % (w/v) feather concentration that produced 260 mg/ g of soluble protein and 86.16 % feather degradation using response surface methodology in a Box-Behnken design space. The obtained hydrolysates exhibited bioactive properties. The amino acid profile showed the increase in concentration of essential amino acid compared with feather meal broth. The selection of safe screening source of this new bacteria in CFB produced hydrolysates with enhanced bioactivity applicable for food, feed, and cosmetic applications along with environmental remediation.

Optimization of Keratinase Production and Utilization of Bacillus pumilus for Feather Degradation

Soil samples were collected from the feather dumped area where Bacillus pumilus was isolated and used for keratinase production and keratinolytic activity. In the optimization study, optimal condition for enzyme production was observed at 144 h, pH 7, temperature 37°C. The organism was utilized for feather degradation study. The maximum degradation of 57% was obtained at 37°C, pH 7 and 6 days incubation. The size of keratinase was determined by SDS- PAGE and was observed as 52 KDa.