Enzymatic properties of a bee venom serine protease from the bumblebee Bombus ignitus

We previously reported that bumblebee (Bombus ignitus) venom serine protease (Bi-VSP) acts as a prophenoloxidase-activating factor in arthropods and a fibrin(ogen)olytic enzyme in mammals. In the present study, we characterized the enzymatic properties of Bi-VSP purified from B. ignitus venom. The 34-kDa active form of Bi-VSP was purified from the venom of B. ignitus worker bees. Glycoprotein staining showed that approximately 20% of the total molecular mass of Bi-VSP is due to carbohydrate moieties. Bi-VSP had an optimal pH and temperature of pH 9.0 and 40 °C, respectively, and was stable at 50 °C for at least 10 min. Bi-VSP activity decreased abruptly below pH 6.0, indicating that Bi-VSP activity is almost completely inhibited at pH 5.4 of B. ignitus venom. The protease activity of Bi-VSP was strongly inhibited by typical serine protease inhibitors such as phenylmethanesulfonyl fluoride, leupeptin, and soybean trypsin inhibitor.     

Triacontyl p-coumarate: An inhibitor of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A₂). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH – minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments.     

Effects of the bumblebee (Bombus ignitus) venom serine protease inhibitor on serine protease and phospholipase A2 of B. ignitus venom

Bumblebee venom contains serine proteases and serine protease inhibitors. In this study, we characterized whether the bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibits B. ignitus venom serine protease (Bi-VSP) or phospholipase A₂ (Bi-PLA₂). Bi-KTI did not inhibit Bi-VSP activity at pH 5.4 or 7.4, whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 after a 30 min preincubation. The Bi-VSP activity that converts prothrombin into thrombin and fibrin into fibrin degradation products was not significantly affected by Bi-KTI. Additionally, Bi-KTI or Bi-VSP did not inhibit Bi-PLA₂ activity. These findings indicate that each bee venom component appears to a play a toxic role via a unique function.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

A novel fibrinolytic metalloproteinase,barnettlysin-I from Bothrops barnetti (barnett´s pitviper) snake venom with anti-platelet properties

BackgroundViperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential.MethodsBar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function.ResultsBar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC₅₀ = 1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation.ConclusionA non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function.General significanceThis study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.     

Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.     

Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom,Chaetoceros gracilis

We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.     

Effects of pore characters of mesoporous resorcinol–formaldehyde carbon gels on enzyme immobilization

This paper demonstrates, for the first time, the use of resorcinol–formaldehyde carbon gels (RFCs) as enzyme carriers. The immobilization behavior of Bacillus licheniformis serine protease in RFCs of different pore characters was investigated. RFCs derived with (RF1) and without (RF2) cationic surfactant (trimethylstearylammonium chloride; C18) resulted in predominantly microporous, and mesoporous characters, respectively. It was found that support pore size and volume were key parameters in determining immobilized enzyme loading, specific activity, and stability. RF2, with higher mesopore volume (V_mes: RF1 = 0.21 cm³/g; RF2 = 0.81 cm³/g) and mesopore size radius (RF1 = 1.7–3.8 nm; RF2 = 7.01 nm), accommodated approximately fourfold more enzyme than RF1. Serine protease loading in RF2 could reach as high as 21.05 unit/g support. In addition, RF2 was found to be a better support in terms of serine protease operation and storage stability. Suitable mesopore size likely helped preventing immobilized enzyme from structural denaturation due to external forces and heat. However, immobilized enzyme in RF1 gave 12.8-fold higher specific activity than in RF2, and 2.1-fold higher than soluble enzyme. Enzyme leaching was found to be problematic in both supports, nonetheless, higher desorption was observed in RF2. Enhancement of interaction between serine protease and RFCs as well as pore size adjustment will be necessary for repeated use of the enzyme and further process development.     

A centrifugal ultrafiltration strategy for isolating the low-molecular weight (≤ 25 K) component of human plasma proteome

The low-molecular weight fraction (LMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. In this study, a separation and enrichment strategy based on centrifugal ultrafiltration was developed for the LMF (i.e., ≤ 25 K) of plasma routinely prepared from normal, healthy volunteers. Four commercially-available filter membranes of similar nominal molecular weight cut-off (NMWC), but differing membrane chemistries and filter orientations (Microcon®, Millipore; Centrisart®, Sartorius; Amicon Ultra®, Millipore; Vivaspin®, Sartorius), were evaluated. Of these filtration devices, only the Sartorius Vivaspin® tangential membrane, NMWC 20 K was effective in the non-retention of M_r > 50 K, and recovery and enrichment of low-M_r components from human plasma. This filter membrane device was further optimized with respect to plasma buffer composition, centrifugal force, duration and temperature. Optimal ultrafiltration conditions were obtained using 100 µL of normal plasma in 10% acetonitrile, and a centrifugation force of 4000 × g for 35 min at 20 °C. In this LMF, 44 proteins (from 266 unique peptides) were identified using a combination of 1D-SDS-PAGE / nano-LC-MS/MS and a stringent level of identification (FDR < 1%). We report the identification of several proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization (HUPO) Plasma Proteome Project datasets. When compared with the low-M_r human plasma/serum proteome datasets of Zhou et al. (Electrophoresis, 2004. 25, 1289–98), Gundry et al. (Proteomics Clin. Appl., 2007. 1, 73–88) and Villanueva et al. (Anal Chem, 2004. 76, 1560–70), 64% of our identifications (28 proteins) were novel; these include cofilin-1, PPIase A, and the SH3 domain-binding glutamic acid-rich-like protein 3. In addition to intact proteins, many peptide fragments from high-abundance proteins (e.g., fibrinogen, clusterin, Factor XIIIa, transferrin, kinogen-1, and inter-alpha-trypsin inhibitor), presumably derived by ex vivo proteolysis, were observed.     

Direct acting anti-thrombotic serine protease from brown seaweed Costaria costata

A direct acting antithrombotic serine protease (CCP) was purified from brown seaweed Costaria costata. CCP was a monomeric protease with molecular mass of 60,547.598 daltons as determined by mass spectrometry. The N-terminal sequence of CCP was SCNSCLDKVDADGLN. Proteolytic activity was inhibited by PMSF and APMSF. CCP exhibited high amidolytic activity toward substrate S-2251 with apparent K_m and V_max values were 14.5 μM and 183.5 U/ml respectively. Fibrin plate and fibrin zymography results revealed that CCP was able to degrade fibrin clots directly. It specifically hydrolyzed Aα and α and Bβ and β chains followed by γ and γ–γ chains of human fibrinogen and fibrin respectively. Cleavage of fibrin clot and fibrinogen was emphasized by observing the alteration of secondary structure using FTIR spectroscopy. Morphological alteration of fibrin clot was also evidenced by fluorescent microscopic observation. CCP reduced thrombus effectively in vitro. In vivo observation showed that it prevented/decreased thrombus formation in carrageenan-induced mice tail model. CCP prolonged activated partial thromboplastin time (APTT) and had little effect on prothrombin time (PT). Platelet function analyzer (PFA-100) tests showed that CCP prolonged closure time (CT). These data suggest that CCP could have therapeutic potential for the treatment of thrombosis.     

A novel surfactant-stable alkaline serine-protease from a newly isolated Bacillus mojavensis A21. Purification and characterization

An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21 on the basis of the 16S rRNA gene sequencing and biochemical properties. The A21 alkaline protease was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43-fold increase in specific activity and 16.56% recovery. The molecular weight of the purified enzyme was estimated to be 20 kDa by SDS-PAGE and gel filtration. The enzyme was highly active over a wide range of pH from 7.0 to 13.0, with an optimum at pH 8.5. The relative activities at pH 11.0 and 12.0 were about 80 and 71.7% of that obtained at pH 8.5. The enzyme was extremely stable in the pH range of 7.0–12.0. It exhibited maximal activity at 60 °C. The thermostability of the enzyme was significantly increased by the addition of CaCl₂. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease.The N-terminal amino acid sequence of the first 20 amino acids of the purified protease was DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the enzyme is a new protease.The alkaline protease showed high stability towards anionic (0.1% SDS) and non-ionic (1 and 5% Tween 80 and 1% Triton X-100) surfactants. In addition, the enzyme was relatively stable towards oxidizing agents, retaining more than 79 and 70% of its initial activity after 1 h incubation in the presence of 1% H₂O₂ and 0.1% sodium perborate, respectively. The enzyme showed excellent stability with a wide range of commercial solid and liquid detergents at 30 and 40 °C. Considering its promising properties, B. mojavensis A21 may find potential application in laundry detergents.     

Purification,characterization and secondary structure elucidation of a detergent stable,halotolerant, thermoalkaline protease from Bacillus cereus SIU1

A thermoalkaline protease with a molecular weight of 22 kDa was purified from the Bacillus cereus SIU1 strain using a combination of Q-Sepharose and Sephadex G-75 chromatography. The kinetic analyses revealed the K_m, V_max and k_cat to be 1.09 mg ml^?1, 0.909 mg ml^?1 min^?1 and 3.11 s^?1, respectively, towards a casein substrate. The protease was most active and stable at pH 9.0 and between a temperature range of 45–55 °C. It was fully stable at 0.0–2.0% and moderately stable at 2.5–10.0% (w/v) sodium chloride. Phenyl methyl sulfonyl fluoride, ethylene diamine tetra acetic acid and ascorbic acid were inhibitory with regard to enzyme activity, whereas cysteine, β-mercaptoethanol, calcium, magnesium, manganese and copper at concentration of 1.0 mM increased enzyme activity. Sodium dodecyl sulfate, Triton X-100, Tween 80, hydrogen peroxide and sodium perborate significantly enhanced protease activity at 0.1 and 1.0% concentrations. In the presence of 0.1 and 1.0% (w/v) detergents, the protease was fairly stable and retained 50–76% activity. Therefore, it may have a possible application in laundry formulations. An initial analysis of the circular dichroism (CD) spectrum in the ultraviolet range revealed that the protease is predominantly a β-pleated structure and a detailed structural composition showed ～50% β-sheets. The CD-based conformational evaluation of the protease after incubation with modulators, metal ions, detergents and at different pH values, revealed that the change in the β-content directly corresponded to the altered enzyme activity. The protease combined with detergent was able to destain blood stained cloth within 30 min.     

A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

Purification and characterization of three thermostable alkaline fibrinolytic serine proteases from the polychaete Cirriformia tentaculata

Three distinct alkaline serine proteases (named CTSP-1, -2, and -3) were purified from the polychaete Cirriformia tentaculata and characterized in terms of their enzymatic properties and kinetics. The estimated molecular masses of CTSP-1, -2, and -3 enzymes were found to be 28.8, 30.9, and 28.4 kDa, respectively. The enzymes were active at the temperature range of 50–60 °C under pH 8.5–9.0 and completely inactivated by phenylmethanesulfonyl fluoride and diisopropyl fluorophosphates, but not by 1,10-phenanthroline and bestatin, suggesting that they are all typical serine proteases and not metalloproteases or cysteine proteases. CTSP-1 and -2 cleaved arginine, whereas CTSP-3 digested tyrosine residue at the carboxyl sides in their peptide substrates. A typical hepta-sequence (I-X-X-G-X-X-A) conserved in serine proteases from annelid species was found in N-termini of all CTSPs. CTSP-2 was the most active enzyme among the proteases purified as shown by kinetic values. The enzymes cleaved all chains of fibrinogen within 20 min and also hydrolyzed actively fibrin polymer as well as cross-linked fibrin. In addition, the enzymes could actively digest the fibrin clot in blood plasma milieu. Taken together, the results obtained demonstrate that CTSP enzymes have a potential of becoming therapeutic agents for thrombus dissolution.     

Purification and characterization of a serine protease extracellularly produced by Aspergillus fumigatus in a shrimp and crab shell powder medium

A fungus with protease and chitinase activities was isolated from the soil. It has been identified as Aspergillus fumigatus Fresenius TKU003. A. fumigatus TKU003 produced proteases and chitinases when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine waste. An extracellular protease was purified from the culture supernatant of A. fumigatus TKU003. The molecular weight of TKU003 protease was 124 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pI for TKU003 protease was 8.3. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU003 protease was pH 8, 40 °C, 6–10, and 50 °C, respectively. The activity of the enzyme was strongly inhibited by PMSF. TKU003 serine protease, same as most other serine proteases of A. fumigatus, belongs to protease with alkaline pI. The unique characteristics of TKU003 protease is its high molecular weight.     

Cathepsin G activity lowers plasma LDL and reduces atherosclerosis

Cathepsin G (CatG), a serine protease present in mast cells and neutrophils, can produce angiotensin-II (Ang-II) and degrade elastin. Here we demonstrate increased CatG expression in smooth muscle cells (SMCs), endothelial cells (ECs), macrophages, and T cells from human atherosclerotic lesions. In low-density lipoprotein (LDL) receptor-deficient (Ldlr^–/–) mice, the absence of CatG reduces arterial wall elastin degradation and attenuates early atherosclerosis when mice consume a Western diet for 3 months. When mice consume this diet for 6 months, however, CatG deficiency exacerbates atherosclerosis in aortic arch without affecting lesion inflammatory cell content or extracellular matrix accumulation, but raises plasma total cholesterol and LDL levels without affecting high-density lipoprotein (HDL) or triglyceride levels. Patients with atherosclerosis also have significantly reduced plasma CatG levels that correlate inversely with total cholesterol (r = –0.535, P < 0.0001) and LDL cholesterol (r = –0.559, P < 0.0001), but not with HDL cholesterol (P = 0.901) or triglycerides (P = 0.186). Such inverse correlations with total cholesterol (r = –0.504, P < 0.0001) and LDL cholesterol (r = –0.502, P < 0.0001) remain significant after adjusting for lipid lowering treatments among this patient population. Human CatG degrades purified human LDL, but not HDL. This study suggests that CatG promotes early atherogenesis through its elastinolytic activity, but suppresses late progression of atherosclerosis by degrading LDL without affecting HDL or triglycerides.     

Hydrolytic and chromatographic studies on the PEGylation of dextranase from Penicillium sp.

Dextranases catalyze the hydrolysis of the α-l,6-glucosidic bond of the polysaccharide dextran. Dextranases have been isolated from bacteria, yeast and fungi. Purified dextranase enzyme from Penicillium sp. was PEGylated (polyethylene glycol modification) with mPEG (5000 Da) and showed an increase in the dextranase protein molecular weight as estimated by Superose 12 (23 ml) column and this increment in the molecular weight is directly proportional to mPEG (5000 Da) concentration until a complete dextranase enzyme PEGylation (disappearance of dextranase peak). The residual activity of partially PEGylated dextranase (mPEG 5000 of 5.8 mg/ml) was 33.8% and for the completely PEGylated dextranase (mPEG 5000 of 29 mg/ml) it was 25.75%. Dextranase PEGylated with mPEG (30,000 Da) showed a little PEGylation at mPEG concentration of 5.8 mg/ml but at a concentration of 29 mg/ml several PEGylated peaks were produced with a difference in dextranase activity toward dextran T500, retardation in the activity with the increasing in the molecular weight was clearly appeared with Sephadex G75 but for Sephadex G200 a little retardation than Sephadex G75 has been appeared.     

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.