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.

Nondestructive Single-Atom-Thick Crystallographic Scanner via Sticky-Note-Like van der Waals Assembling-Disassembling.

Crystallographic characteristics, including grain boundaries and crystallographic orientation of each grain, are crucial in defining the properties of two-dimensional materials (2DMs). To date, local microstructure analysis of 2DMs, which requires destructive and complex processes, is primarily used to identify unknown 2DM specimens, hindering the subsequent use of characterized samples. Here, a nondestructive large-area 2D crystallographic analytical method through sticky-note-like van der Waals (vdW) assembling-disassembling is presented. By the vdW assembling of veiled polycrystalline graphene (PCG) with a single-atom-thick single-crystalline graphene filter (SCG-filter), detailed crystallographic information of each grain in PCGs is visualized through a 2D Raman signal scan, which relies on the interlayer twist angle. The scanned PCGs are seamlessly separated from the SCG-filter using vdW disassembling, preserving their original condition. The remaining SCG-filter is then reused for additional crystallographic scans of other PCGs. It is believed that the methods can pave the way for advances in the crystallographic analysis of single-atom-thick materials, offering huge implications for the applications of 2DMs.

Float-stacked graphene-PMMA laminate.

Semi-infinite single-atom-thick graphene is an ideal reinforcing material that can simultaneously improve the mechanical, electrical, and thermal properties of matrix. Here, we present a float-stacking strategy to accurately align the monolayer graphene reinforcement in polymer matrix. We float graphene-poly(methylmethacrylate) (PMMA) membrane (GPM) at the water-air interface, and wind-up layer-by-layer by roller. During the stacking process, the inherent water meniscus continuously induces web tension of the GPM, suppressing wrinkle and folding generation. Moreover, rolling-up and hot-rolling mill process above the glass transition temperature of PMMA induces conformal contact between each layer. This allows for pre-tension of the composite, maximizing its reinforcing efficiency. The number and spacing of the embedded graphene fillers are precisely controlled. Notably, we accurately align 100 layers of monolayer graphene in a PMMA matrix with the same intervals to achieve a specific strength of about 118.5 MPa g-1 cm3, which is higher than that of lightweight Al alloy, and a thermal conductivity of about 4.00 W m-1 K-1, which is increased by about 2,000 %, compared to the PMMA film.

Uniform Synthesis of Bilayer Hydrogen Substituted Graphdiyne for Flexible Piezoresistive Applications.

Graphdiyne (GDY) has garnered significant attention as a cutting-edge 2D material owing to its distinctive electronic, optoelectronic, and mechanical properties, including high mobility, direct bandgap, and remarkable flexibility. One of the key challenges hindering the implementation of this material in flexible applications is its large area and uniform synthesis. The facile growth of centimeter-scale bilayer hydrogen substituted graphdiyne (Bi-HsGDY) on germanium (Ge) substrate is achieved using a low-temperature chemical vapor deposition (CVD) method. This material's field effect transistors (FET) showcase a high carrier mobility of 52.6 cm2 V-1 s-1 and an exceptionally low contact resistance of 10 Ω µm. By transferring the as-grown Bi-HsGDY onto a flexible substrate, a long-distance piezoresistive strain sensor is demonstrated, which exhibits a remarkable gauge factor of 43.34 with a fast response time of ≈275 ms. As a proof of concept, communication by means of Morse code is implemented using a Bi-HsGDY strain sensor. It is believed that these results are anticipated to open new horizons in realizing Bi-HsGDY for innovative flexible device applications.

Investigation of Potential cGMP-Specific PDE V and Aminopeptidase N Inhibitors of Allium ampeloprasum L. and Its Bioactive Components: Kinetic and Molecular Docking Studies.

The primary objectives of this study were to assess the inhibitory effects of Allium ampeloprasum L. extract (AAE) and its derived organosulfur and polyphenolic compounds on the enzymatic activities of cGMP-specific PDE V (PDE5) and aminopeptidase N (APN). Additionally, the study aimed to investigate their potential as inhibitors against these two target enzymes through kinetic analyses and molecular docking studies. The in vitro enzyme assays demonstrated that both AAE and its derived compounds significantly decreased the activity of PDE5 and APN. Further analyses involving kinetics and molecular docking provided insights into the specific inhibitor types of AAE and its derived compounds along with the proposed molecular docking models illustrating the interactions between the ligands (the compounds) and the enzymes (PDE5 and APN). In particular, AAE-derived polyphenolic compounds showed relatively stable binding affinity (-7.2 to -8.3 kcal/mol) on PDE5 and APN. Our findings proved the potential as an inhibitor against PDE5 and APN of AAE and AAE-derived organosulfur and polyphenolic compounds as well as a functional material for erectile dysfunction improvement.

Partial Purification and Biochemical Evaluation of Protease Fraction (MA-1) from Mycoleptodonoides aitchisonii and Its Fibrinolytic Effect.

The antioxidative proteolytic fraction, MA-1, was partially purified from Mycoleptodonoides aitchisonii. MA-1 was purified to homogeneity using a two-step procedure, which resulted in an 89-fold increase in specific activity and 42.5% recovery. SDS-PAGE revealed two proteins with a molecular weight of 48 kDa. The zymography results revealed proteolytic activity based on the MA-1 band. MA-1 was found to be stable in the presence of Na+, Ca2+, Fe3+, K+, and Mg2+. MA-1 was also stable in methanol, ethanol, and acetone, and its enzyme activity increased by 15% in SDS. MA-1 was inhibited by ethylenediaminetetra-acetic acid or ethylene glycol tetraacetic acid and exerted the highest specificity for the substrate, MeO-Suc-Arg-Pro-Tyr-pNA, for chymotrypsin. Accordingly, MA-1 belongs to the family of chymotrypsin-like metalloproteins. The optimum temperature was 40 °C and stability was stable in the range of 20 to 35 °C. The optimum pH and stability were pH 5.5 and pH 4-11. MA-1 exhibited stronger fibrinolytic activity than plasmin. MA-1 hydrolyzed the Aα, Bß, and γ chains of fibrinogen within 2 h. MA-1 exhibited an antithrombotic effect in animal models. MA-1 was devoid of hemorrhagic activity at a dose of 80,000 U/kg. Overall, our results show that M. aitchisonii produces an acid-tolerant and antioxidative chymotrypsin-like fibrinolytic enzyme, and M. aitchisonii containing MA-1 could be a beneficial functional material for the prevention of cardiovascular diseases and possible complications.

Short-Term Effects of PJE Administration on Metabolic Parameters in Diet-Induced Obesity Mice.

The study investigated the effects of Petasites japonicus (Siebold & Zucc.) Maxim. extract (PJE) and fenofibrate on diet-induced obesity (DIO) in mice. PJE was found to contain various bio-active polyphenolic compounds, including kaempferol, p-hydroxybenzoic acid, ferulic acid, gallic acid, chlorogenic acid, 3,4-dicaffeoylquinic acid, caffeic acid, quercetin, rutin, protocatechuic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, p-coumaric acid, apigenin, and 1,3-dicaffeoylquinic acid. The results showed that PJE treatment up to 1000 µg/mL did not affect the viability of 3T3-L1 cell line, and it reduced the feed efficiency ratio in DIO mice. PJE administration also resulted in a significant reduction in body weight gain and fat accumulation in the liver compared to the DIO control group. Additionally, PJE administration improved the levels of lipid and related parameters, including total cholesterol, triacylglycerol, low-density lipoprotein, very low-density lipoprotein, glucose, insulin, insulin resistance, leptin, and atherogenic or cardiac indexes compared to the DIO control group. The study suggested that PJE may have a beneficial effect on insulin resistance, lipid profiles, atherogenesis, adipokines, and cardiac risk associated with diet-induced obesity.

Low-Temperature Direct Growth of Amorphous Boron Nitride Films for High-Performance Nanoelectronic Device Applications.

We successfully demonstrated the improvement and stabilization of the electrical properties of a graphene field effect transistor by fabricating a sandwiched amorphous boron nitride (a-BN)/graphene (Gr)/a-BN using a directly grown a-BN film. The a-BN film was grown via low-pressure chemical vapor deposition (LPCVD) at a low growth temperature of 250 °C and applied as a protection layer in the sandwiched structure. Both structural and chemical states of the as-grown a-BN were verified by various spectroscopic and microscopic analyses. We analyzed the Raman spectra of Gr/SiO2 and a-BN/Gr/a-BN structures to determine the stability of the device under exposure to ambient air. Following exposure, the intensity of the 2D/G-peak ratio of Gr/SiO2 decreased and the position of the G and 2D peaks red-shifted due to the degradation of graphene. In contrast, the peak position of encapsulated graphene is almost unchanged. We also confirmed that the mobility of a-BN/Gr/a-BN structure is 17,941 cm2/Vs. This synthetic strategy could provide a facile way to synthesize uniform a-BN film for encapsulating various van der Waals materials, which is beneficial for future applications in nanoelectronics.

In Vitro Antithrombotic, Hematological Toxicity, and Inhibitor Studies of Protocatechuic, Isovanillic, and p-Hydroxybenzoic Acids from Maclura tricuspidata (Carr.) Bur.

In blood coagulation, circulating platelets and coagulation factors are crucial for the primary process because thrombi are generated by fibrin clotting with fibrinogen, thrombin, FXIIIa, and platelet activation. Therefore, strategies to reduce the activity of key coagulation factors, or interfere with their functions and delay the activation of platelets can be used as important tools to suppress excessive blood clot formation and platelet hyperactivation. This study examined the antithrombotic activity and hematological toxicity of PA, IVA, and 4-HA isolated from M. tricuspidata (Carr.) Bur in several in vitro experiments and inhibitor assays. We found that PA, IVA, and 4-HA attenuated the formation of fibrin polymers/clots and degraded the blood clots. These compounds inhibited the activities of procoagulant proteases and fibrinoligase, and prolonged the coagulation time. There was a significant reduction in platelet function and ATP or serotonin levels in thrombin-activated platelets. An inhibitor study showed that PA exhibited a mixed inhibition type for thrombin, an uncompetitive inhibition type for FXa, and a non-competitive inhibition type for FXIIIa and IVA, while 4-HA exhibited an uncompetitive inhibition type for thrombin and non-competitive inhibition type for FXa and FXIIIa. These three compounds (up to 50 µg/mL) were not toxic to blood cells.

Cytotoxicity, metabolic enzyme inhibitory, and anti-inflammatory effect of Lentinula edodes fermented using probiotic lactobacteria.

We found that the fermented Lentinula edodes (FLE) products exhibited various differences in terms of proximate composition, free sugar, and amino acid. In particular, there were higher levels of ergosterol, and ergothioneine in FLE-Pediococcus pentosaceus (PP) and -Lactobacillus acidophilus (LA) than in the L. edodes (LE) products. The survival rates of lactic acid bacteria (LAB) strains on artificial gastric juice, artificial bile, or heat (50-60°C) were observed to vary from 60%-66%, 60%-66%, to 42%-79%, respectively. The FLE products up to 300 µg/ml had no cytotoxicity on RAW264.7, AGS, and RBL-2H3 cells, but inhibited the activities of α-amylase, α-glucosidase, and pancreatic lipase, as well as the production of nitrite, IL-1ß, IL-4, TNF-α, and prostaglandin E2 (PGE2) from lipopolysaccharide (LPS)-induced inflammatory response. Our findings suggest that FLE products have metabolic enzyme inhibitory and anti-inflammatory effects. PRACTICAL APPLICATIONS: Fermentation plays a critical role in improving the functional and nutritional properties of food. In addition, lactobacteria are the main microorganisms involved in the fermentation of food known to have a variety of biological activities. Therefore, the utilization of lactobacteria for research and development of mushroom food materials can be used as a key strategy to improve the biological activity characteristics of mushroom food materials and to increase their active ingredient content. The present results show that FLE products had promising inhibitory efficacies against the activities of obesity-related metabolic enzymes and LPS-induced inflammatory response. These suggest that FLE products have the potential to be developed as functional probiotic dietary supplements or food products.

Fibrinolytic and Thrombolytic Effects of an Enzyme Purified from the Fruiting Bodies of Boletus pseudocalopus (Agaricomycetes) from Korea.

A fibrinolytic enzyme with thrombolytic, anticoagulant activities was purified from fruiting bodies of wild-growing mushroom Boletus pseudocalopus Hongo and homogenized with a two-step procedure with a 6.11-fold increase in specific activity and 3.2% recovery. The molecular weight of the enzyme was estimated to be 63.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The enzyme was active at 40°C and pH 7, and activity was inhibited by zinc metal ion and by serine protease and trypsin inhibitors phenylmethylsulfonyl fluoride and N-α-tosyl-l-lysinyl-chloromethylketone. The enzyme displayed high specificity for Pyro-Glu-Gly-Arg-pNA. In vitro assays showed that the enzyme was able to degrade fibrin and blood clots, inhibit thrombin and activated factor X, and alter the density or structural change of fibrin clots. It could also delay activated partial thromboplastin time and prothrombin time. These results suggest that the enzyme may have characteristics of a trypsin or serine-like enzyme with fibrinolytic and thrombolytic activities and may have potential as an antithrombotic agent for blood clotting disorders.

Short-term Cudrania tricuspidata fruit vinegar administration attenuates obesity in high-fat diet-fed mice by improving fat accumulation and metabolic parameters.

Previous studies have suggested that vinegar intake can help to reduce body fat and hyperglycemia. Therefore, this study aimed to evaluate the anti-obesity efficacy of vinegar fermented using Cudrania tricuspidata fruits (CTFV) and its main phenolic constituents and to analyze its molecular mechanism and changes in obesity-related metabolizing enzymatic activities. We found that HFD significantly caused hepatic steatosis; increases in body fats, feed efficiency, liver mass, lipids, insulin, oxidative parameters, cardiovascular-associated risk indices, lipase and α-amylase activities, whereas CTFV efficaciously attenuated HFD-induced oxidant stress, fat accumulation, obesity-related enzymatic activity, and the activation or reduction of obesity-related molecular reactions via improving metabolic parameters including phosphorylated insulin receptor substrate 1, protein tyrosine phosphatase 1B, phosphorylated phosphoinositide 3-kinase/protein kinase B, phosphorylated mitogen-activated protein kinases, sterol regulatory element-binding protein 1c, CCAAT/enhancer-binding protein, and fatty acid synthase; and decreases in adiponectin receptor 1, leptin receptor, adenosine monophosphate-activated protein kinase, acetyl-CoA carboxylase, and peroxisome proliferator-activated receptor, subsequently ameliorating HFD-induced obesity. Therefore, CTFV might provide a functional food resource or nutraceutical product for reducing body fat accumulation.

Layer-engineered large-area exfoliation of graphene.

The competition between quality and productivity has been a major issue for large-scale applications of two-dimensional materials (2DMs). Until now, the top-down mechanical cleavage method has guaranteed pure perfect 2DMs, but it has been considered a poor option in terms of manufacturing. Here, we present a layer-engineered exfoliation technique for graphene that not only allows us to obtain large-size graphene, up to a millimeter size, but also allows selective thickness control. A thin metal film evaporated on graphite induces tensile stress such that spalling occurs, resulting in exfoliation of graphene, where the number of exfoliated layers is adjusted by using different metal films. Detailed spectroscopy and electron transport measurement analysis greatly support our proposed spalling mechanism and fine quality of exfoliated graphene. Our layer-engineered exfoliation technique can pave the way for the development of a manufacturing-scale process for graphene and other 2DMs in electronics and optoelectronics.

Anti-Inflammatory and Cytotoxicity Effects of Cudrania tricuspidata Fruits Vinegar in a Co-Culture System with RAW264.7 Macrophages and 3T3-L1 Adipocytes.

Vinegar has been found to have in vitro improvement effect on inflammatory biomarkers, and clinically used to improve inflammation and obesity-related diseases. This study was designed to analyze in vitro anti-inflammatory effects of Cudrania tricuspidata fruits vinegar (CTFV) in a co-culture system with macrophages and adipocytes. We analyzed the physicochemical properties and polyphenolic ingredients of CTFV, and investigated in vitro anti-inflammatory effects of CTFV in a co-culture system with macrophages and adipocytes. The cells were cultured in the presence of CTFV for 24 h in contact with each other, then, harvested. The levels of monocyte chemoattractant protein (MCP)-1, tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), nitric oxide (NO), and interleukin (IL)-6 were evaluated by using the Griess reagent, western blot, or enzyme-linked immunosorbent assay assays. We found that increasing levels for NO, iNOS, TNF-α, IL-6 and MCP-1 were caused by LPS treatment and co-culture using the contact method, whereas CTFV efficaciously attenuated inflammatory response by improving inflammatory parameters including NO, iNOS, TNF-α, IL-6 and MCP-1. The present study indicates that CTFV might provide a nutraceutical product or functional food resource for improving inflammation processed via the interaction of adipocytes and macrophages.

Effects of Maclura tricuspidata (Carr.) Bur fruits and its phytophenolics on obesity-related enzymes.

The purpose of the present study was to investigate whether several phytophenolic ingredients isolated from Maclura tricuspidata (Carr.) Bur fruits inhibit the activity of obesity-related enzymes including pancreatic lipase, α-amylase, ß-glucosidase, phosphodiesterase IV, alkaline phosphatase, and citrate synthase, and the compounds play as an inhibitor against the target enzymes in kinetic studies. The enzyme assays indicated that the fruit extract and its phytophenolic compounds inhibited significantly the enzymatic activity of the five target enzymes. The kinetic studies demonstrated that the inhibitory properties of p-hydroxybenzoic acid (4-HA), protocatechuic acid (PA), and isovanillic acid (IA) against pancreatic lipase, ß-glucosidase, citrate synthase, or alkaline phosphatase. Our results suggested that the compounds detected from Maclura tricuspidata (Carr.) Bur fruit extract may regulate carbohydrate/lipid/energy metabolism by obesity-related enzymes' inhibition. PRACTICAL APPLICATIONS: The obesity-related metabolizing enzymes affect (in)directly the metabolites absorption on carbohydrate/lipid/energy metabolism. Accordingly, it is an important strategy to treat obesity through target pathways and enzymes which include the reduction in energy intake and consumption. In our results, Maclura tricuspidata (Carr.) Bur fruit extract and its phytophenolic compounds inhibited significantly the enzymatic activity of the five target enzymes, in particular, 4-HA, PA, and IA have each specific inhibition type on pancreatic lipase, ß-glucosidase, citrate synthase, and alkaline phosphatase. Therefore, M. tricuspidata (Carr.) Bur fruit may be a strong candidate as a food material or therapeutic agent for obesity improvement.

Antioxidant and antithrombotic properties of Dendropanax morbifera Léveille (Araliaceae) and its ferments produced by fermentation processing.

The present study investigates and evaluates the antioxidant and antithrombotic effects of Dendropanax morbifera Léveille (Araliaceae) extracts (DME) and its ferment (FDME). Antioxidant activities were improved by FDME treatment, especially 66.74% of hydroxyl radical scavenging activity was observed at 100 µg of FDME treatment. In vitro antithrombotic activity was also improved by accelerated fibrinolysis and blood clot degradation as well as prolonged prothrombin time. Similar to those results, in vivo experiment also showed that the formation of thrombus was alleviated and paralysis or death due to thromboembolism was also prevented to some extent. PRACTICAL APPLICATIONS: A bioprocessing technology, fermentation is known to enhance nutritional qualities which has beneficial effects on health by improving antioxidant activity, lipid levels, and antithrombotic activities. DME, healthy food source has been known to have a potential for improving blood circulation improvement. The present study provides beneficial information on the potential and excellence of the antithrombotic efficacy of DME and FDME. The present results elucidate the antithrombotic activities of DME and FDME, which is useful in the development of new food materials for cardiovascular health and new antithrombotic agents for clinical application.

p-Hydroxybenzyl alcohol inhibits four obesity-related enzymes in vitro.

Recently, antiobesity studies using the method of inhibiting enzymatic activity of obesity-related enzymes as targets have received considerable attention. The aims of the current study were to investigate whether p-hydroxybenzyl alcohol (HBA), identified from Cudrania tricuspidata fruits with antiobesity effects, inhibits the activity of digestive and obesity-related enzymes and acts as an inhibitor against four target enzymes in kinetic studies. In vitro enzyme assays showed HBA at the highest concentration significantly reduced the enzymatic activity of four targets: pancreatic lipase (IC50 = 2.34-3.70 µM), α-glycosidase (IC50 = 9.08 µM), phosphodiesterase IV (IC50 = 4.99 µM), and citrate synthase (IC50 = 2.07 µM) enzymes. Based on the results of kinetic assays, the types of inhibition were investigated. Our findings indicate that HBA could have antiobesity efficacy, and it deserves further study.

Purification and Antithrombotic Potential of a Fibrinolytic Enzyme from Shiitake Culinary- Medicinal Mushroom, Lentinus edodes GNA01 (Agaricomycetes).

We purified Lentinus edodes GNA01 fibrinolytic enzyme (LEFE) and identified it as a novel metalloprotease. LEFE was purified to homogeneity through a 2-step procedure, with an 8.28-fold increase in specific activity and 5.3% recovery. The molecular mass of LEFE was approximately 38 kDa, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its optimal pH, optimal temperature, pH stability, and thermal stability were 5, 30°C, 6-7, and 40°C, respectively. LEFE was inhibited by zinc and magnesium ions, and by EDTA and EGTA, indicating that LEFE is a metalloprotease. The protease exhibited fibrinolytic activity and a degradative effect on clot formation and blood clots. The protease prolonged activated partial thromboplastin time, prothrombin time, and coagulation time as induced by platelet aggregators (collagen and epinephrine). Taken together, our results indicate that L. edodes GNA01 produces a metalloprotease/fibrinolytic enzyme and that this enzyme might be applied as a new thrombolytic and antithrombotic agent for thrombosis-related cardiovascular disorders.

In vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid.

We discovered recently in vitro and in vivo antithrombotic and cytotoxicity effects of ferulic acid. The cytotoxicity assays showed that ferulic acid (∼300 µg/mL) did not cause any significant toxicity on three cell lines, platelets, leukocytes, and erythrocytes. In vitro assays showed inhibitory effects of ferulic acid on thrombin (THR)- or collagen/epinephrine-stimulated platelet activation by inhibiting platelet aggregation, and decreasing clot retraction activity. The in vitro effect of ferulic acid on THR-stimulated platelet activation was proved by the decrease in the secretion of serotonin from the platelets. The anticoagulant effects of ferulic acid were confirmed by the prolongation of the intrinsic or/and extrinsic pathways and the delay of recalcification time in plasma coagulation. Ferulic acid had antithrombotic effect in acute thromboembolism model in vivo, and decreased the expression of αIIb ß3 /FIB and phosphorylation of AKT in THR-stimulated platelet activation in vivo, and their antithrombotic efficacies hold promise for therapeutic targeting in our ongoing studies.

Purification and partial characterization of a low molecular fibrinolytic serine metalloprotease C142 from the culture supernatant of Bacillus subtilis C142.

Novel serine metalloprotease-like enzyme, C142 was purified from the culture supernatant of Bacillus subtilis C142. The C142 was purified to homogeneity by a two-step procedure with a 20.7-fold increase in specific activity and 0.9% recovery. The molecular mass of C142 was approximately 23.5kDa based on SDS-PAGE. The N-terminal amino acid sequence of the first 21 amino acids of C142 was AQSVPYGISQIKAPALHSQGY. Its optimum pH, optimum temperature, pH stability, and thermal stability were pH 6, 40°C, pH 6-8, and 20-35°C, respectively. C142 was strongly inhibited by PMSF and EGTA, suggesting that C142 was a serine metalloprotease-like enzyme. C142 showed the highest specificity toward the substrate for t-PA. The apparent Km, Vmax, and Kcat values of C142 toward H-d-Ile-Pro-Arg-pNA were determined as 0.34mM, 0.25mmolmg-1min-1, and 46.83s-1. C142 exhibited fibrinolytic activity, which is stronger than that of plasmin. C142 hydrolyzed Aα, and Bß-chains of fibrinogen, but did not cleave γ-chains. C142 had antithrombotic effect in three animal models. C142 was devoid of hemorrhagic activity at a dose of 20,000FU/kg. Taken together, our results indicate that B. subtilis C142 produces a serine metalloprotease-like enzyme/fibrinolytic enzyme and this enzyme might be used as a new thrombolytic agent.