Characterization of a Novel Keratinase With Surfactant Stability in a Wide Range of pH Activity From a Native Isolate, Bacillus Mojavensis FUM125

Keratinases are enzymes with the most diverse sources and applications. Different forms of keratinase have been applied in environment and variety of industries, highlighting the necessity for novel potential keratinases, which could be applicable in variety of industries. Accordingly, the present study aimed to identify and characterize a novel keratinase producing bacterium with high potential in variety of industries. In the present study, the native isolate of Bacillus sp. FUM125 was isolated, identified and optimized for the keratinolytic activity. The keratinase was purified and characterized using biochemical assays. The Bacillus sp. FUM125 isolate was identified as Bacillus mojavensis R-OH-1 with 99.8% similarity. The isolate showed the maximum keratinolytic activity at pH of 8.5 after 24-hour incubation at 37°C (2.1-fold enzyme production). According to the biochemical analysis, the keratinase belonged to a serine protease family, whit 33.5 kDa molecular weight and was stable in a wide range of pH and temperature with maximum keratinolytic activity at 60°C and pH 8. Among the metal ions, K+, Ca2+, Na2+ and Mg2+ increased the enzyme activity. The activity was increased by the reducing agents of DTT and beta-mercaptoethanol. Based on the substrate profile findings, the enzyme was active in various soluble and insoluble substrates. The enzyme showed a half-life of 98 min in the optimal temperature and the ratio of keratinolytic:caseinolytic to be 0.95. Our enzyme with higher temperature and pH stability compared to existing commercial enzymes can be considered as a potential candidate for use in various industries.

Production of microbial keratinase using a newly isolated strain of Stenotrophomonas maltophilia and its characterization and applications.

Biodegradation of keratin containing wastes viz. bird feathers, nails, hairs, animalwool is a low-cost, nutrient-rich biotechnological process that turns this plentiful waste into a low-cost, nutrient-rich substance. It is a sustainable green approach towards the solution of environmental threats created by these wastes. This study aimed to screen for potential keratinase-producing microorganisms and to optimize physicochemical parameters to produce keratinase and its characterization. Samples were collected from various poultry farms of Surat (India). Screening for keratinase-production was carried using a feather basal medium. Among these samples, 16 isolates showed keratinolytic activity in primary screening using skim milk agar medium among which, nine isolates showed keratinolytic activity using keratin agar medium. Isolate KA1a gave the highest yield of keratinase and was identified as a strain of Stenotrophomonas maltophilia (NCBI Accession number MT478451), based on 16S rRNA sequencing. It delivered highest enzyme production (108 U/ml) in the medium with feathers (2.2%), NaCl (0.5%), K2HPO4(0.3%), KH2PO4(0.4%), pH7, with 10% inoculum of young cell mass, at 37°C for 24 h under shaking conditions. Optimum temperature for keratinase activity was 40°C and pH 7.0. Enzyme showed stability over different temperature and pH for up to 90 min. It showed potential applications as a detergent additive, animal feed, and organic fertilizer.

The keratinolytic bacteria Bacillus cytotoxicus as a source of novel proteases and feather protein hydrolysates with antioxidant activities

Background Argentina’s geothermal areas are niches of a rich microbial diversity. In 2020, species of Bacillus cytotoxicus were isolated for the first time from these types of pristine natural areas. Bacillus cytotoxicus strains demonstrated the capability to grow and degrade chicken feathers with the concomitant production of proteases with keratinolytic activity, enzymes that have multitude of industrial applications. The aim of this research was to study the production of the proteolytic enzymes and its characterization. Also, feather protein hydrolysates produced during fermentation were characterized. Results Among the thermotolerant strains isolated from the Domuyo geothermal area (Neuquén province, Argentina), Bacillus cytotoxicus LT-1 and Oll-15 were selected and put through submerged cultures using feather wastes as sole carbon, nitrogen, and energy source in order to obtain proteolytic enzymes and protein hydrolysates. Complete degradation of feathers was achieved after 48 h. Zymograms demonstrated the presence of several proteolytic enzymes with an estimated molecular weight between 50 and > 120 kDa. Optimum pH and temperatures of Bacillus cytotoxicus LT-1 crude extract were 7.0 and 40 °C, meanwhile for Oll-15 were 7.0 and 50 °C. Crude extracts were inhibited by EDTA and 1,10 phenanthroline indicating the presence of metalloproteases. Feather protein hydrolysates showed an interesting antioxidant potential measured through radical-scavenging and Fe3+-reducing activities. Conclusion This work represents an initial approach on the study of the biotechnological potential of proteases produced by Bacillus cytotoxicus. The results demonstrated the importance of continuous search for new biocatalysts with new characteristics and enzymes to be able to cope with the demands in the market.

Feather protein lysate optimization and feather meal formation using YNDH protease with keratinolytic activity afterward enzyme partial purification and characterization

Incubation parameters used for the creation of a protein lysate from enzymatically degraded waste feathers using crude keratinase produced by the Laceyella sacchari strain YNDH were optimized using the Response Surface Methodology (RSM); amino acids quantification was also estimated. The optimization elevated the total protein to 2089.5 µg/ml through the application of the following optimal conditions: a time of 20.2 h, a feather concentration (conc.) of 3 g%, a keratinase activity of 24.5 U/100 ml, a pH of 10, and a cultivation temperature of 50 °C. The produced Feather Protein Lysate (FPL) was found to be enriched with essential and rare amino acids. Additionally, this YNDH enzyme group was partially purified, and some of its characteristics were studied. Crude enzymes were first concentrated with an Amicon Ultra 10-k centrifugal filter, and then concentrated proteins were applied to a "Q FF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranging from 6 to 10 kDa. The maximum enzyme activity was observed at 70 °C and for a pH of 10.4. Most characteristics of this protease/keratinase group were found to be nearly the same when the activity was measured with both casein and keratin-azure as substrates, suggesting that these three protein bands work together in order to degrade the keratin macromolecule. Interestingly, the keratinolytic activity of this group was not inhibited by ethylenediamine tetraacetic acid (EDTA), phenylmethanesulfonyl fluoride (PMSF), or iron-caused activation, indicating the presence of a mixed serine–metallo enzyme type.

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.

Proteases in the diet of monogastric animals

There are many proteases, and about 2% of the human genome is involved in the regulation of their formation. The share of proteases involved in digestion accounts for only a small part. Despite this, the mechanisms of action of digestive proteases are less studied than carbohydrases and lipases. The incorporation of exogenous proteases into young animal feeds is often accompanied by improved utilization of protein and other nutrients. Exogenous proteases degrade inhibitors of the endogenous protease and lectins in feed. Alkaline proteases are of interest due to their broader substrate specificity and activity throughout the entire gastrointestinal tract. This group includes keratinases, which digest proteins inaccessible for cleavage by proteases and peptidases of animals. Keratinases digest agglutinins, glycinin and b-conglycinin and connective tissue proteins, which are resistant to the action of gastrointestinal enzymes and a number of exogenous proteases. The alleged reasons for the inconsistent results when using feed proteases are described. Their mediated positive effects not associated with proteolysis are indicated. It is advisable to use proteases with keratinolytic activity as fodder proteases.

Feather Biodegradation For Feather Protein Lysate/ Feather Meal Mutual Formations: Optimization Using Crude YNDH Protease/Keratinase; Enzyme Partial Purification and Characterization

Incubation parameters used for the production of a protein lysate from enzymatically degraded waste feather using crude keratinase produced by laceyella sacchari YNDH was optimized using RSM, amino acids quantification was estimated as well. The optimization resulted in total soluble protein 2089.5µg/ml. The optimal conditions were, time 20.2h, feather concentration 3g% and keratinase activity 24.5U/100ml at pH, 10; and cultivation temperature 50oC. The FPL was found enriched with essential and rare amino acids.In parallel, this YNDH enzyme group had been partially purified and some characteristics of it were studied. Crude enzymes were first concentrated with Amicon Ultra 10k centrifugal filter, and then concentrated proteins were applied to "QFF" strong anion column chromatography. The partially purified enzyme has an estimated molecular masses ranged 6 to 10 kDa. The maximum enzyme activity was observed at 70°C and pH 10.4 when measured by both casein and keratin azure as substrates. Interestingly, keratinolytic activity of this group was not affect by EDTA, PMSF and DTT. Generally the overall characters of this group protease/keratinase nearly the same when its activity was measured with both substrates suggesting that all these 3 protein bands working together as a group of keratinases.

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.

Isolation of Bacillus Strains with Keratinolitic Activity

Environmental safety and economic feasibility determine the search for new ways of processing waste in poultry farms. Most of this waste is down and feathers, which are 90% β-keratin. Feathers can be a valuable source of amino acids and peptones when properly processed. The most effective is enzymatic treatment of feather keratin. The search for new strains producing keratinolytic enzymes seems to be a promising direction. On the territory of the poultry farm, 4 strains were isolated from the places of accumulation of feathers. They are able to use chicken feathers as their sole source of organic matter. Based on morphological, genomic, and proteomic analyzes, the isolated strains were identified as Bacillus sp. It was found that the strains secrete proteolytic enzymes that hydrolyze collagen, casein, β-keratin and do not hydrolyze bovine serum albumin. Feather hydrolysis experiments showed that the Bacillus sp. A5.3 possesses maximum keratinolytic activity, and on the second day, the destruction of the second order barbs is observed. The keratinase activity of the strain on azokeratin after an hour of incubation on feather medium was 27.4 U/ml. The optimal conditions for the complex of secreted proteolytic enzymes are pH 7.0-8.0 and temperature 35-40 °C. The isolated Bacillus sp. A5.3 strain is a promising source of proteases and keratinases.