Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 µg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Utilizing immobilized recombinant serine alkaline protease from Bacillus safensis lab418 in wound healing: Gene cloning, heterologous expression, optimization, and characterization.

Microbial proteases have proven their efficiency in various industrial applications; however, their application in accelerating the wound healing process has been inconsistent in previous studies. In this study, heterologous expression was used to obtain an over-yielding of the serine alkaline protease. The serine protease-encoding gene aprE was isolated from Bacillus safensis lab 418 and expressed in E. coli BL21 (DE3) using the pET28a (+) expression vector. The gene sequence was assigned the accession number OP610065 in the NCBI GenBank. The open reading frame of the recombinant protease (aprEsaf) was 383 amino acids, with a molecular weight of 35 kDa. The yield of aprEsaf increased to 300 U/mL compared with the native serine protease (SAFWD), with a maximum yield of 77.43 U/mL after optimization conditions. aprEsaf was immobilized on modified amine-functionalized films (MAFs). By comparing the biochemical characteristics of immobilized and free recombinant enzymes, the former exhibited distinctive biochemical characteristics: improved thermostability, alkaline stability over a wider pH range, and efficient reusability. The immobilized serine protease was effectively utilized to expedite wound healing. In conclusion, our study demonstrates the suitability of the immobilized recombinant serine protease for wound healing, suggesting that it is a viable alternative therapeutic agent for wound management.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen.

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Isolation and Functional Characterization of Erythrofibrase: An Alfa-Fibrinogenase Enzyme from Trimeresurus erythrurus Venom of North-East India.

Green pit viper bites induce mild toxicity with painful local swelling, blistering, cellulitis, necrosis, ecchymosis and consumptive coagulopathy. Several bite cases of green pit vipers have been reported in several south-east Asian countries including the north-eastern region of India. The present study describes isolation and characterization of a haemostatically active protein from Trimeresurus erythrurus venom responsible for coagulopathy. Using a two-step chromatographic method, a snake venom serine protease erythrofibrase was purified to homogeneity. SDS-PAGE of erythrofibrase showed a single band of ~30 kDa in both reducing and non-reducing conditions. The primary structure of erythrofibrase was determined by ESI LC-MS/MS, and the partial sequence obtained showed 77% sequence similarity with other snake venom thrombin-like enzymes (SVTLEs). The partial sequence obtained had the typical 12 conserved cysteine residues, as well as the active site residues (His57, Asp102 and Ser195). Functionally, erythrofibrase showed direct fibrinogenolytic activity by degrading the Aα chain of bovine fibrinogen at a slow rate, which might be responsible for causing hypofibrinogenemia and incoagulable blood for several days in envenomated patients. Moreover, the inability of Indian polyvalent antivenom (manufactured by Premium Serum Pvt. Ltd., Maharashtra, India) to neutralize the thrombin-like and plasmin-like activity of erythrofibrase can be correlated with the clinical inefficacy of antivenom therapy. This is the first study reporting an α-fibrinogenase enzyme erythrofibrase from T. erythrurus venom, which is crucial for the pathophysiological manifestations observed in envenomated victims.

Purification, and characterization of detergent-compatible serine protease from Bacillussafensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry.

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopteruscuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz.Bacillus,Priestia,Aeromonas,Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillussafensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B.safensis strain PRN1 includes 72 h (OD600 = 0.56,1303 U/mL), pH 8 (OD600 = 0.83, 403.29 U/mL), 40 °C (OD600 = 1.75, 1849.11 U/mL), fructose (OD600 = 1.22, 1502 U/mL), and gelatin (OD600 = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

SP-1, a Serine Protease from the Gut Microbiota, Influences Colitis and Drives Intestinal Dysbiosis in Mice.

Increased protease activity has been linked to the pathogenesis of IBD. While most studies have been focusing on host proteases in gut inflammation, it remains unclear how to address the potential contribution of their bacterial counterparts. In the present study, we report a functional characterization of a newly identified serine protease, SP-1, from the human gut microbiota. The serine protease repertoire of gut Clostridium was first explored, and the specificity of SP-1 was analyzed using a combinatorial chemistry method. Combining in vitro analyses and a mouse model of colitis, we show that oral administration of recombinant bacteria secreting SP-1 (i) compromises the epithelial barrier, (ii) alters the microbial community, and (ii) exacerbates colitis. These findings suggest that gut microbial protease activity may constitute a valuable contributor to IBD and could, therefore, represent a promising target for the treatment of the disease.

Proteome Analysis and In Vitro Antiviral, Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms.

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Novel recombinant keratin degrading subtilisin like serine alkaline protease from Bacillus cereus isolated from marine hydrothermal vent crabs.

Microbial secondary metabolites from extreme environments like hydrothermal vents are a promising source for industrial applications. In our study the protease gene from Bacillus cereus obtained from shallow marine hydrothermal vents in the East China Sea was cloned, expressed and purified. The protein sequence of 38 kDa protease SLSP-k was retrieved from mass spectrometry and identified as a subtilisin serine proteinase. The novel SLSP-k is a monomeric protein with 38 amino acid signal peptides being active over wide pH (7-11) and temperature (40-80 °C) ranges, with maximal hydrolytic activities at pH 10 and at 50 °C temperature. The hydrolytic activity is stimulated by Ca2+, Co2+, Mn2+, and DTT. It is inhibited by Fe2+, Cd2+, Cu2+, EDTA, and PMSF. The SLSP-k is stable in anionic, non-anionic detergents, and solvents. The ability to degrade keratin in chicken feather and hair indicates that this enzyme is suitable for the degradation of poultry waste without the loss of nutritionally essential amino acids which otherwise are lost in hydrothermal processing. Therefore, the proteinase is efficient in environmental friendly bioconversion of animal waste into fertilizers or value added products such as secondary animal feedstuffs.

Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system.

Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E.coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.

In vitro biochemical characterization and genotoxicity assessment of Sapindus saponaria seed extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. AIM OF THE STUDY: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. MATERIALS AND METHODS: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. RESULTS: Seed proteins extract of S. saponaria was evaluated until 100 µg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. CONCLUSION: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.

Insight into the collagen-degrading activity of a serine protease in the latex of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease. Earlier study on a serine protease from F. carica cultivar Brown Turkey showed that it specifically degraded collagen. In this study, we characterized the collagenolytic activity of a serine protease in the latex of F. carica cultivar Masui Dauphine. The serine protease degraded denatured, but not undenatured, acid-solubilized type I collagen. It also degraded bovine serum albumin, while the collagenase from Clostridium histolyticum did not. These results indicated that the serine protease in Masui Dauphine is not collagen-specific. The protease was purified to homogeneity by two-dimensional gel electrophoresis, and its partial amino acid sequence was determined by liquid chromatography-tandem mass spectrometry. BLAST searches against the Viridiplantae (green plants) genome database revealed that the serine protease was a subtilisin-like protease. Our results contrast with the results of the earlier study stating that the serine protease from F. carica is collagen-specific.

Insights into substrate specificity of proteases for screening efficient dehairing enzymes.

Though numerous proteases have been isolated and screened for the dehairing purpose, their use in the leather industry is limited mainly due to high cost, the need for expertise, and control during unit operation and alterations in the quality of leather due to lack of the right kind of substrate specificity of the enzymes used. This paper deals with the comparative specificity and dehairing efficiency of proteases isolated from Bacillus cereus VITSP01 (PE2) and Brevibacterium luteolum VITSP02 (PE). PE2 and PE were found to be trypsin-like and elastase-like serine proteases respectively. The protease of VITSP02 degraded the proteoglycans efficiently in comparison to that of VITSP01. The results suggest that the possible targets of the studied proteases might be skin proteoglycans, including those cementing the hair root bulb. Hence, an in-depth study on the substrate specificity of the dehairing proteases would help in designing an improved screening method for isolating potent dehairing enzymes.

Two steps purification, biochemical characterization, thermodynamics and structure elucidation of thermostable alkaline serine protease from Nocardiopsis alba strain OM-5.

The Nocardiopsis alba strain OM-5 showed maximum protease production in submerged culture. The OM-5 protease was purified by hydrophobic interaction chromatography. The purified protease of 68 kDa showed maximum activity (3312 ± 1.64 U/mL) at 70 °C and was quite stable at 80 °C up to 4 M NaCl (w/v) at pH 9. The purified protease showed significant activity and stability in different cations, denaturing agents, metal ions, and osmolytes. The thermodynamic parameters including deactivation rate constant (Kd) and half lives (t1/2) at 50-80 °C were in the range of 2.50 × 10-3 to 5.50 × 10-3 and 277.25-111.25 min respectively at 0-4 M NaCl. The structural stability of the OM-5 protease under various harsh conditions was elucidated by circular dichroism (CD) spectroscopy followed by K2D3 analysis revealed that the native structure of OM-5 protease was stable even in sodium dodecyl sulfate and Tween 20 indicated by increased α-helices content assisted with decreased ß-sheets content.

A novel serine protease with anticoagulant and fibrinolytic activities from the fruiting bodies of mushroom Agrocybe aegerita.

A novel fibrinolytic enzyme, ACase was isolated from fruiting bodies of a mushroom, Agrocybe aegerita. ACase was purified by using ammonium sulfate precipitation, gel filtration, ion exchange and hydrophobic chromatographies to 237.12 fold with a specific activity of 1716.77 U/mg. ACase was found to be a heterodimer with molecular mass of 31.4 and 21.2 kDa by SDS-PAGE and appeared as a single band on Native-PAGE and fibrin-zymogram. The N-terminal sequence of the two subunits of ACase was AIVTQTNAPWGL (subunit 1) and SNADGNGHGTHV (subunit 2). ACase had maximal activity at 47 °C and pH 7.6. It's activity was improved by Cu2+, Na+, Fe3+, Zn2+, Ba2+, K+ and Mn2+, but inhibited by Fe2+, Mg2+ and Ca2+. PMSF, SBTI, aprotinine and Lys inhibited the enzyme activity, which suggested that ACase was a serine protease. ACase could degrade all three chains (α, ß and γ) of fibrinogen. Moreover, the enzyme acted as both, a plasmin-like fibrinolytic enzyme and a plasminogen activator. It could hydrolyze human thrombin slightly, which indicated that the ACase could inhibit the activity of thrombin and acted as an anticoagulant to prevent thrombosis. Based on these results, ACase might act as a therapeutic agent for treating thrombosis, or as a functional food. Further investigation of the enzyme is underway.

Biochemical characterization of a partially purified protease from Aspergillus terreus 7461 and its application as an environmentally friendly dehairing agent for leather industry.

Proteases can be used in several biotechnological processes including detergent, food and leather industries. In the leather industry, dehairing is carried out by chemicals, which pollute the environment. Therefore, to make the hair removal process environmentally friendly, a protease produced by Aspergillus terreus has been purified, biochemically characterized and had an efficient ability to remove hair from bovine leather. The protease was produced using 1% wheat bran and was purified 2.3-fold using two chromatographic steps showing a molecular weight of 90 kDa. Optimal temperature and pH were 50 °C and 6.5, respectively. Thermal stability was up to 1 h at 50 °C. Protease was stable to detergents like Tween 80 and to organic solvents. The activity was activated by Ca2+ and inhibited by Hg2+ and Cu2+. The enzyme was classified as serine protease, by the inhibition by PMSF and was stable to reducing agents. It hydrolyzed casein, azocasein, BSA, egg albumin and BTpNA. The Km and Vmax values were 0.65 ± 0.03 mg/mL and 3.66 ± 0.18 µmol/min, respectively. Remarkable properties about temperature, pH, stability to detergents and reducing agents ensure that the protease from A. terreus can be an excellent candidate for industrial applications, particularly in the leather industry.

A Thermostable Aluminum-Tolerant Protease Produced by Feather-Degrading Bacillus thuringiensis Isolated from Tea Plantation.

BACKGROUND: Proteases with keratinolytic activity are widely used in biotechnologies. The feather-degrading Bacillus thuringensis isolated from soil sample of a tea plantation produced high level of extracellular keratinase. OBJECTIVE: This study aimed to analyze the properties by biochemical and enzymological methods to gain information for better utilization of the enzyme. METHODS: The enzyme was purified with ion exchange and size exclusion chromatography. The substrate preference, optimal pH and temperature, and the effects of organic solvents and ions were checked. Circular dichroism was performed to compare the secondary structures of the native and apo-enzyme. RESULTS: The enzyme worked best at 50°C, and it was an acidic serine protease with an optimal pH of 6.2. Ions Ca2+ and Mg2+ were essential for its activity. Organic solvents and other metal ions generally deactivated the enzyme in a concentration-dependent manner. However, Mn2+ and DMSO, which were frequently reported as inhibitors of protease, could activate the enzyme at low concentration (0.01 to 2 mmol/L of Mn2+; DMSO <2%, v/v). The enzyme exhibited high resistance to Al3+, which might be explained by the soil properties of its host's residence. Circular dichroism confirmed the contribution of ions to the structure and activity. CONCLUSION: The enzyme was a thermostable aluminum-tolerant serine protease with unique biochemical properties.

Biochemical Study of Fibrinolytic Protease from Euphausia superba Possessing Multifunctional Serine Protease Activity.

BACKGROUND: Fibrinolytic protease from Euphausia superba (EFP) was isolated. OBJECTIVE: Biochemical distinctions, regulation of the catalytic function, and the key residues of EFP were investigated. METHODS: The serial inhibition kinetic evaluations coupled with measurements of fluorescence spectra in the presence of 4-(2-aminoethyl) benzene sulfonyl fluoride hydrochloride (AEBSF) was conducted. The computational molecular dynamics (MD) simulations were also applied for a comparative study. RESULTS: The enzyme behaved as a monomeric protein with a molecular mass of about 28.6 kD with Km BApNA = 0.629 ± 0.02 mM and kcat/Km BApNA = 7.08 s-1/mM. The real-time interval measurements revealed that the inactivation was a first-order reaction, with the kinetic processes shifting from a monophase to a biphase. Measurements of fluorescence spectra showed that serine residue modification by AEBSF directly caused conspicuous changes of the tertiary structures and exposed hydrophobic surfaces. Some osmolytes were applied to find protective roles. These results confirmed that the active region of EFP is more flexible than the overall enzyme molecule and serine, as the key residue, is associated with the regional unfolding of EFP in addition to its catalytic role. The MD simulations were supportive to the kinetics data. CONCLUSION: Our study indicated that EFP has an essential serine residue for its catalyst function and associated folding behaviors. Also, the functional role of osmolytes such as proline and glycine that may play a role in defense mechanisms from environmental adaptation in a krill's body was suggested.

Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management.

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.

Extraction, partial purification and characterization of proteases from the red seaweed Gracilaria edulis with similar cleavage sites on κ-casein as calf rennet.

Enzymes currently used in cheesemaking have various drawbacks, and there is a continual need to find new coagulants. This study describes the extraction and biochemical characterization of two proteases from the red alga Gracilaria edulis. The proteases were extracted with phosphate buffer and partially purified by ammonium sulphate precipitation and dialysis. The enzymes exhibited optimum caseinolytic activity at 60 °C and a pH range of 6-8. They showed a high ratio of milk-clotting over caseinolytic activity, indicating they had an excellent milk-clotting ability. The proteases were confirmed to be serine protease and metalloprotease with molecular weight (MW) of 44 and 108 kDa. They exhibited high hydrolytic activity on κ-caseins, cleaving κ-casein at four main sites, one of which being the same as that of calf rennet, which is the first reported for an algal protease. The findings demonstrated that the proteases could potentially be used as a milk coagulant in cheesemaking.