Thermoactive metallo-keratinase from Bacillus sp. NFH5: Characterization, structural elucidation, and potential application as detergent additive.

In recent times, robust green technological developments have advanced the goal of a circular economy by minimizing waste generation. The study was undertaken to explore the keratinolytic activity of chicken feather-degrading bacteria from South African soil. Isolates coded as SSN-01 and HSN-01 were identified as Bacillus sp. NFH5 and Bacillus sp. FHNM and their sequences were deposited in GenBank, with accession numbers MW165830.1 and MW165831.1, respectively. Extracellular enzyme production and thiol group generation by Bacillus sp. NFH5 peaked at 120 h with 1879.09 ± 88.70 U/mL and 9.49 ± 0.78 mM, respectively. Glutamic acid (4.44%), aspartic acid (3.50%), arginine (3.23%), glycine (2.61%), serine (2.08%), and proline (2.08%) were relatively higher in concentration. Keratinase (KerBAN) activity was highest at pH 8.0 and 90 °C but was inhibited by both EDTA and 1,10-phenanthroline. In addition, the keratinase-encoding gene (kerBAN) accessioned OK033360 had 362 amino acid residues, with molecular weight and theoretical isoelectric point of 39 kDa and 8.81, respectively. Findings from this study highlight the significance of Bacillus sp. NFH5 in the bio-recycling of recalcitrant keratinous wastes to protein hydrolysates - potential dietary supplements for livestock feeds. The properties of KerBAN underscore its application potential in green biotechnological processes.

Chryseobacterium aquifrigidense keratinase liberated essential and nonessential amino acids from chicken feather degradation.

Keratinous biomass valorization for value-added products presents a high prospect in ecological management and the advancement of the bio-economy. Consequently, soil samples from the poultry dumpsite were collected. The bacteria isolated on the basal salt medium were screened for keratinolytic activity. The potent chicken feathers degrading bacteria were identified through 16S rRNA gene sequencing and phylogenetic analysis. Fermentation process conditions were optimized, and the amino acid compositions of the feather hydrolysate were likewise quantified. Ten (10) proteolytic bacteria evaluated on skimmed milk agar showed intact chicken feather degradation ranging from 33% (WDS-03) to 88% (FPS-09). The extracellular keratinase activity ranged from 224.52 ± 42.46 U/mL (WDS-03) to 834.55 ± 66.86 U/mL (FPS-07). Based on 16S rRNA gene sequencing and phylogenetic analysis, the most potent keratinolytic isolates coded as FPS-07, FPS-09, FPS-01, and WDS-06 were identified as Chryseobacterium aquifrigidense FANN1, Chryseobacterium aquifrigidense FANN2, Stenotrophomonas maltophilia ANNb, and Bacillus sp. ANNa, respectively. C aquifrigidense FANN2 maximally produced keratinase (1460.90 ± 26.99 U/mL) at 72 h of incubation under optimal process conditions of pH (6), inoculum side (5%; v/v), temperature (30°C), and chicken feather (25 g/L). The feather hydrolysate showed a protein value of 67.54%, with a relative abundance of arginine (2.84%), serine (3.14%), aspartic acid (3.33%), glutamic acid (3.73%), and glycine (2.81%). C. aquifrigidense FANN2 yielded high keratinase titre and dismembered chicken feathers into amino acids-rich hydrolysate, highlighting its significance in the beneficiation of recalcitrant keratinous wastes into dietary proteins as potential livestock feed supplements.

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

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.

Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO.

OBJECTIVES: Decaying wood samples were collected, and actinomycetes were isolated and screened for laccase production. The identity of the efficient laccase-producing isolate was confirmed by using a molecular approach. Fermentation conditions for laccase production were optimized, and laccase biochemical properties were studied. RESULTS: Based on the 16S rRNA gene sequencing and phylogenetic analysis, the isolate coded as HWP3 was identified as Streptomyces sp. LAO. The time-course study showed that the isolate optimally produced laccase at 84 h with 40.58 ± 2.35 U/mL activity. The optimized physicochemical conditions consisted of pH 5.0, ferulic acid (0.04%; v/v), pine back (0.2 g/L), urea (1.0 g/L), and lactose (1 g/L). Streptomyces sp. LAO laccase was optimally active at pH and temperature of 8.0 and 90 °C, respectively, with remarkable pH and thermal stability. Furthermore, the enzyme had a sufficient tolerance for organic solvents after 16 h of preincubation, with laccase activity > 70%. Additionally, the laccase maintained considerable residual activity after pretreatment with 100 mM of chemical agents, including sodium dodecyl sulphate (69.93 ± 0.89%), ethylenediaminetetraacetic acid (93.1 ± 7.85%), NaN3 (96.28 ± 3.34%) and urea (106.03 ± 10.72%). CONCLUSION: The laccase's pH and thermal stability; and robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry.

Microbial keratinase and the bio-economy: a three-decade meta-analysis of research exploit.

Microbial keratinase research has been on an upward trajectory due to the robustness and efficiency of the enzyme toward various green technological processes that promote economic development and environmental sustainability. A compendium of research progression and advancement within the domain was achieved through a bibliometric study to understand the trend of research productivity, scientific impacts, authors' involvement, collaboration networks, and the advancement of knowledge gaps for future research endeavours. A three-decade (1990 to 2019) scholarly published articles were retrieved from the web of science database using a combination of terms "keratinas* or keratinolytic proteas* or keratinolytic enzym*", and subsequently analyzed for bibliometric indicators. A collection of 330 peer-reviewed, research, articles were retrieved for the survey period and authored by 1063 researchers with collaboration index of 3.27. Research productivity was most in 2013 with total research output of 28 articles. The top three authors' keywords were keratinase, keratin and protease with a respective frequency of 188, 26 and 22. India, China and Brazil ranked top in terms of keratinase research outputs and total citation with respective article productivity (total citations) of 85 (1533), 57 (826), and 36 (764). This study evaluated the trend of keratinase research outputs, scientific impact, collaboration networks and biotechnology innovations. It has the potentials to influence positively decision making on future research direction, collaborations and development of products for the bio-economy.

Bacillus sp. CSK2 produced thermostable alkaline keratinase using agro-wastes: keratinolytic enzyme characterization.

BACKGROUND: Chicken feathers are the most abundant agro-wastes emanating from the poultry processing farms and present major concerns to environmentalists. Bioutilization of intractable feather wastes for the production of critical proteolytic enzymes is highly attractive from both ecological and biotechnological perspectives. Consequently, physicochemical conditions influencing keratinase production by Bacillus sp. CSK2 on chicken feathers formulation was optimized, and the keratinase was characterized. RESULTS: The highest enzyme activity of 1539.09 ± 68.14 U/mL was obtained after 48 h of incubation with optimized conditions consisting of chicken feathers (7.5 g/L), maltose (2.0 g/L), initial fermentation pH (5.0), incubation temperature (30 °C), and agitation speed (200 rpm). The keratinase showed optimal catalytic efficiency at pH 8.0 and a temperature range of 60 °C - 80 °C. The keratinase thermostability was remarkable with a half-life of above 120 min at 70 °C. Keratinase catalytic efficiency was halted by ethylenediaminetetraacetic acid and 1,10-phenanthroline. However, keratinase activity was enhanced by 2-mercaptoethanol, dimethyl sulfoxide, tween-80, but was strongly inhibited by Al3+ and Fe3+. Upon treatment with laundry detergents, the following keratinase residual activities were achieved: 85.19 ± 1.33% (Sunlight), 90.33 ± 5.95% (Surf), 80.16 ± 2.99% (Omo), 99.49 ± 3.11% (Ariel), and 87.19 ± 0.26% (Maq). CONCLUSION: The remarkable stability of the keratinase with an admixture of organic solvents or laundry detergents portends the industrial and biotechnological significance of the biocatalyst.

Biochemical and Molecular Characterization of a Thermostable Alkaline Metallo-Keratinase from Bacillus sp. Nnolim-K1.

Keratinases are considerably gaining momentum in green technology because of their endowed robustness and multifaceted application potentials, such as keratinous agro-wastes valorization. Therefore, the production of novel keratinases from relatively nonpathogenic bacteria grown in agro-wastes formulated medium is cost-effective, and also imperative for the sustainability of thriving bioeconomy. In this study, we optimized keratinase production by Bacillus sp. Nnolim-K1 grown in chicken feather formulated medium. The produced keratinase (KerBNK1) was biochemically characterized and also, the keratinase-encoding gene (kerBNK1) was amplified and sequenced. The optimal physicochemical conditions for extracellular keratinase production determined were 0.8% (w/v) xylose, 1.0% (w/v) feather, and 3.0% (v/v) inoculum size, pH 5.0, temperature (25 °C) and agitation speed (150 rpm). The maximum keratinase activity of 1943.43 ± 0.0 U/mL was achieved after 120 h of fermentation. KerBNK1 was optimally active at pH and temperature of 8.0 and 60 °C, respectively; with remarkable pH and thermal stability. KerBNK1 activity was inhibited by ethylenediamine tetra-acetic acid and 1,10-phenanthroline, suggesting a metallo-keratinase. The amplified kerBNK1 showed a band size of 1104 bp and the nucleotide sequence was submitted to the GenBank with accession number MT268133. Bacillus sp. Nnolim-K1 and the keratinase displayed potentials that demand industrial and biotechnological exploitations.

Exoproduction and characterization of a detergent-stable alkaline keratinase from Arthrobacter sp. KFS-1.

Arthrobacter sp. KFS-1 previously isolated from a dump site was used to produce keratinase in basal medium. The physico-chemical conditions were optimized to enhance the keratinase production, and biochemical properties of the enzyme were also evaluated. Arthrobacter sp. KFS-1 optimally produced keratinase in a basal medium that contained 1.0 g/L xylose, 2.5-5.0 g/L chicken feather; with initial pH, incubation temperature and agitation speed of 6.0, 30 °C and 200 rpm, respectively. Maximum keratinase activity of 1559.09 ± 29.57 U/mL was achieved at 96 h of fermentation; while optimal thiol concentration of 665.13 ± 38.73 µM was obtained at 144 h. Furthermore, the enzyme was optimally active at pH 8.0 and 60 °C. The enzyme activity was inhibited by ethylene diamine tetraacetic acid and 1,10-phenanthroline, but not affected by phenylmethylsulfonyl floride. In addition, the crude enzyme retained 55%, 63%, 80%, 81% and 90% of the original activity after respective pretreatment with some commercial detergents (Maq, Omo, Surf, Sunlight and Ariel). Moreso, the enzyme showed remarkable stability in the presence of reducing agents, surfactants, and organic solvents. Arthrobacter sp. KFS-1 significantly produced keratinase which exhibited excellent stability in presence of chemical agents and commercial laundry detergents; hence, suggesting its industrial application potentials especially in detergent formulation.

Proteolytic bacteria isolated from agro-waste dumpsites produced keratinolytic enzymes.

Microbial bioconversion of carbonoclastic materials is an efficient tool for the exploitation and valorization of underutilized agro-industrial wastes. The agro-industrial sector accumulates tones of keratinous wastes biomass which may be valorized into high value products. Consequently, the keratinolytic potentials of some bacteria isolated from terrestrial milieu was evaluated. Soil samples were collected from dumpsites, keratinase producing bacteria were isolated. Bacterial species were identified through 16S rRNA gene sequences. The keratinase activity was assessed in relation to thiol formation, percentage feather degradation and quantitation of keratinase produced. Keratinolytic bacteria were identified as Bacillus spp. (accession numbers: MG214989 - MG214992, MG214997, MG214998, MG215000, MG215002-MG215005) and Arthrobacter sp. (accession numbers; MG215001). The degree of chicken feather degradation ranged from 61.5 ± 0.71 % to 85.0 ± 1.41 %. Similarly, the activity of keratinase, total protein and thiol group ranged from 198.18 ± 15.43-731.83 ± 14.14 U/mL; 0.09 ± 0.01-0.87 ± 0.05 mg/mL; and 0.69 ± 0.12-2.89 ± 0.11 mM respectively. Notably, Bacillus sp. Nnolim-K1 displayed the best keratinolytic potential with extracellular keratinase activity and feather degradation of 731.83 ± 14.14 U/mL and 85.0 ± 1.41 % respectively, and that is an indication of a potential relevance biotechnologically.

Bacillus sp. FPF-1 Produced Keratinase with High Potential for Chicken Feather Degradation.

Chicken feathers are predominantly composed of keratin; hence, valorizing the wastes becomes an imperative. In view of this, we isolated keratinase-producing bacteria and identified them through the 16S rDNA sequence. The process condition for keratinase activity was optimized, and electron micrography of the degradation timelines was determined. Keratinolytic bacteria were isolated and identified as Bacillus sp. FPF-1, Chryseobacterium sp. FPF-8, Brevibacillus sp. Nnolim-K2, Brevibacillus sp. FPF-12 and Brevibacillus sp. FSS-1; and their respective nucleotide sequences were deposited in GenBank, with the accession numbers MG214993, MG214994, MG214995, MG214996 and MG214999. The degree of feather degradation and keratinase concentration among the isolates ranged from 62.5 ± 2.12 to 86.0 ± 1.41(%) and 214.55 ± 5.14 to 440.01 ± 20.57 (U/mL), respectively. In the same vein, 0.1% (w/v) xylose, 0.5% (w/v) chicken feather, an initial fermentation pH of 5.0, fermentation temperature of 25 °C and an agitation speed of 150 rpm, respectively, served as the optimal physicochemical conditions for keratinase activity by Bacillus sp. FPF-1. The time course showed that Bacillus sp. FPF-1 yielded a keratinase concentration of 1698.18 ± 53.99(U/mL) at 120 h. The electron microscopic imaging showed completely structural dismemberment of intact chicken feather. Bacillus sp. FPF-1 holds great potential in the valorization of recalcitrant keratinous biomass from the agro sector into useful products.

Microbial Keratinase: Next Generation Green Catalyst and Prospective Applications.

The search for novel renewable products over synthetics hallmarked this decade and those of the recent past. Most economies that are prospecting on biodiversity for improved bio-economy favor renewable resources over synthetics for the potential opportunity they hold. However, this field is still nascent as the bulk of the available resources are non-renewable based. Microbial metabolites, emphasis on secondary metabolites, are viable alternatives; nonetheless, vast microbial resources remain under-exploited; thus, the need for a continuum in the search for new products or bio-modifying existing products for novel functions through an efficient approach. Environmental distress syndrome has been identified as a factor that influences the emergence of genetic diversity in prokaryotes. Still, the process of how the change comes about is poorly understood. The emergence of new traits may present a high prospect for the industrially viable organism. Microbial enzymes have prominence in the bio-economic space, and proteases account for about sixty percent of all enzyme market. Microbial keratinases are versatile proteases which are continuously gaining momentum in biotechnology owing to their effective bio-conversion of recalcitrant keratin-rich wastes and sustainable implementation of cleaner production. Keratinase-assisted biodegradation of keratinous materials has revitalized the prospects for the utilization of cost-effective agro-industrial wastes, as readily available substrates, for the production of high-value products including amino acids and bioactive peptides. This review presented an overview of keratin structural complexity, the potential mechanism of keratin biodegradation, and the environmental impact of keratinous wastes. Equally, it discussed microbial keratinase; vis-à-vis sources, production, and functional properties with considerable emphasis on the ecological implication of microbial producers and catalytic tendency improvement strategies. Keratinase applications and prospective high-end use, including animal hide processing, detergent formulation, cosmetics, livestock feed, and organic fertilizer production, were also articulated.