scholarly journals Switchable deep eutectic solvent driven micellar extractive fermentation of ultrapure fibrin digesting enzyme from Bacillus subtilis

2021 ◽  
Author(s):  
Ramya Muniasamy ◽  
Senthilkumar Rathnasamy
Keyword(s):  
Hydrogen Bond ◽  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Deep Eutectic Solvent ◽  
Temperature Response ◽  
Extractive Fermentation ◽  
Two Phase ◽  
Anion Exchange Column

Abstract Fibrinolytic protease (FLP) is a therapeutic enzyme used in the treatment of thrombolytic diseases. The present study proposed the concept of pH-driven swappable micellar two-phase extraction for the concurrent production and purification of FLP from Bacillus subtilis at cloud point extraction. Extractive fermentation was carried out with a pH swap mechanism, and FLP was extracted to the top phase by surfactant deep eutectic solvents (SADES). Shrimp waste was chosen as a sustainable low-cost substrate that yielded a maximum protease of 185 U/mg. Six SDESs were synthesized with nonionic surfactants as hydrogen bond donors and quaternary ammonium salts as hydrogen bond acceptors, and their association was confirmed by H1 NMR. Thermophysical investigation of the synthetic SADES was accomplished as a function of temperature. Response surface methodology for extractive fermentation was performed with the concentration of SADES (35% w/v), Na2SO4 (15% w/v) and pH (6.3) as variables and the enzyme activity (248 IU/mg) as a response. Furthermore, purification using gel filtration chromatography was used to quantify the amount of enzyme obtained in the extraction phase (849 IU/ml). After final purification with an anion exchange column, the maximum purity fold (22.32) with enzyme activity (1172 IU/ml) was achieved. The in vitro fibrinolytic activity was confirmed using a fibrin plate assay.

scholarly journals Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

2007 ◽  
Vol 189 (19) ◽  
pp. 7069-7076 ◽  
Author(s):  
Sumarin Soonsanga ◽  
Mayuree Fuangthong ◽  
John D. Helmann
Keyword(s):  
Hydrogen Bond ◽  
Bacillus Subtilis ◽  
Conformational Changes ◽  
Active Site ◽  
Mutational Analysis ◽  
High Sensitivity ◽  
Oxidative Modification ◽  
Active Site Residues

ABSTRACT Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29′-Y40′ hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

scholarly journals Potential of some bacteria for biological control of postharvest citrus green mould caused by Penicillium digitatum

2017 ◽  
Vol 53 (No. 3) ◽  
pp. 134-143 ◽  
Author(s):  
Mohammadi Parisa ◽  
Tozlu Elif ◽  
Kotan Recep ◽  
Kotan Merve Şenol
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Antifungal Activity ◽  
Mycelial Growth ◽  
Spore Production ◽  
Penicillium Digitatum ◽  
Broth Culture ◽  
Agrobacterium Radiobacter ◽  
Green Mould

Ten bacteria isolate (4 Bacillus subtilis, 2 Bacillus pumilus, 2 Bacillus cereus, 1 Bacillus megaterium, and 1 Agrobacterium radiobacter) were tested in vitro for antagonistic properties against Penicillium digitatum, the causal agent of citrus green mould. The effect of these bacteria was also observed on mycelial growth, spore germination, and spore production of the pathogenic fungus in broth culture. Extracellular enzyme activities of the bacteria were determined. According to the results of in vitro antagonistic tests and enzymes activities, the most promising bacteria were Bacillus subtilis and Agrobacterium radiobacter. These bacteria were tested for disease suppression on lemon fruits. In addition, these bacterial isolates also showed remarkable antifungal activity against the pathogen on lemon fruits. The results of this study showed that Bacillus subtilis and Agrobacterium radiobacter showed remarkable antifungal activity against the pathogen. Chitinase and glucanase enzyme activity of all the tested bacteria was positive. Protease enzyme activity was positive in all tested bacteria with the exception of Agrobacterium radiobacter. In addition, all bacteria inhibited mycelial growth and spore germination (except Agrobacterium radiobacter) of the fungus. Bacillus subtilis, Bacillus cereus, and Agrobacterium radiobacter inhibited spore production in broth culture. Bacillus subtilis and Agrobacterium radiobacter were tested on lemon fruits significantly reduced disease severity. Consequently, these isolates can be used as new biocontrol agents in controlling the post-harvest decay of citrus fruits caused by Penicillium digitatum.

scholarly journals Purification of phosphatidylinositol kinase from bovine brain myelin

1987 ◽  
Vol 241 (3) ◽  
pp. 759-763 ◽  
Author(s):  
A R Saltiel ◽  
J A Fox ◽  
P Sherline ◽  
N Sahyoun ◽  
P Cuatrecasas
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Bovine Brain ◽  
Anion Exchange Column ◽  
Ionic Detergent ◽  
Membrane Bound ◽  
Phosphatidylinositol 4 ◽  
Pi Kinase

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

scholarly journals THE IDENTIFICATION, PRODUCTION AND CHARACTERIZATION OF NATTOKINASE, BACTERIOCIN FROM BACTERIAL SPECIES

2019 ◽  
Vol 2 (4) ◽  
pp. 91
Author(s):  
Lal Krishna
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Blood Clot ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Bacterial Strains ◽  
Wide Range

The study was aimed at identification, production and characterization of nattokinase, bacteriocin from bacterial species. Nattokinase and bacteriocins finds a wide range of applications in Pharmaceutical industry, health care and medicine. Nattokinase is a highly active fibrinolytic enzyme secreted by Bacillus subtilis and bacteriocins are proteinaceous toxins produced by Lactobacillus to inhibit the growth of closely related bacterial strains. Bacillus subtilis and Lactobacillus isolates shown positive results to microscopic, biochemical analysis.  The nattokinase and bacteriocins were produced by optimizing the media. The enzymes were purified by ammonium sulfate precipitation and HPLC. The enzyme activity for nattokinase was found at 7 mg/ml, pH 8.0 and temperature 48 ºC and the enzyme activity for bacteriocin was found at 3.9 mg/ml, pH 6.5 and temperature 30 °C. Bacteriocins from Lactobacillus showed good antagonistic activity against pathogenic bacteria. Nattokinase from Bacillus subtilis played a significant role in thrombolytic and anti-coagulation at in vitro. The results indicated that the pure enzyme has a potential in dissolving blood clot.

scholarly journals Silver Nanoparticles and Polyphenol Inclusion Compounds Composites for Phytophthora cinnamomi Mycelial Growth Inhibition

Antibiotics ◽  
2018 ◽  
Vol 7 (3) ◽  
pp. 76 ◽  
Author(s):  
Petruta Matei ◽  
Jesús Martín-Gil ◽  
Beatrice Michaela Iacomi ◽  
Eduardo Pérez-Lebeña ◽  
María Barrio-Arredondo ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Growth Inhibition ◽  
Inclusion Compounds ◽  
Mycelial Growth ◽  
Water Solubility ◽  
Phytophthora Cinnamomi ◽  
Deep Eutectic Solvent ◽  
Phase System ◽  
Two Phase

Phytophthora cinnamomi, responsible for “root rot” or “dieback” plant disease, causes a significant amount of economic and environmental impact. In this work, the fungicide action of nanocomposites based on silver nanoparticles and polyphenol inclusion compounds, which feature enhanced bioavailability and water solubility, was assayed for the control of this soil-borne water mold. Inclusion compounds were prepared by an aqueous two-phase system separation method through extraction, either in an hydroalcoholic solution with chitosan oligomers (COS) or in a choline chloride:urea:glycerol deep eutectic solvent (DES). The new inclusion compounds were synthesized from stevioside and various polyphenols (gallic acid, silymarin, ferulic acid and curcumin), in a [6:1] ratio in the COS medium and in a [3:1] ratio in the DES medium, respectively. Their in vitro response against Phytophthora cinnamomi isolate MYC43 (at concentrations of 125, 250 and 500 µg·mL−1) was tested, which found a significant mycelial growth inhibition, particularly high for the composites prepared using DES. Therefore, these nanocomposites hold promise as an alternative to fosetyl-Al and metalaxyl conventional systemic fungicides.

Production and partial characterization of elastase of Bacillus subtilis isolated from the cervices of human females

1988 ◽  
Vol 34 (7) ◽  
pp. 855-859 ◽  
Author(s):  
Mohinder Kaur ◽  
K. K. Tripathi ◽  
Meenakshi Gupta ◽  
P. K. Jain ◽  
M. R. Bansal ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Ammonium Sulphate ◽  
Phosphate Buffer ◽  
Gel Filtration ◽  
Ammonium Sulphate Precipitation ◽  
Tris Maleate ◽  
Culture Supernatants

Conditions are described for the production of extracellular elastase by Bacillus subtilis. The yield of enzyme was maximum in shake–cultures grown in Syncase medium at 37 °C and was stable in culture supernatants. The enzyme, purified by ammonium sulphate precipitation and Sephadex G-75 gel filtration, showed a molecular weight of 25 000 and activity between pH 6.0 and 9.5, with an optimum of 9.0 in Tris–maleate buffer. Elastinolytic activity was maximum in glycine–NaOH buffer and minimum in phosphate buffer. Enzyme activity was adversely affected by temperature ≥ 40 °C.

scholarly journals Conditions for an in vitro culture of murine mixed hematopoietic colonies and their putative cellular origin

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 99-107 ◽  
Author(s):  
BA Miller ◽  
DE Siedler ◽  
CD Dunn ◽  
AT Huang
Keyword(s):  
Gel Filtration ◽  
Colony Growth ◽  
Antigenic Determinants ◽  
Anion Exchange Column ◽  
Erythroid Progenitor ◽  
Cellular Origin ◽  
Mouse Liver Cell ◽  
Fetal Mouse Liver

Abstract The supernatant fluid of stimulated spleen cells (PHA-SCM) supported in vitro colony growth of murine marrow. In the absence of exogenous erythropoietin, it stimulated the growth of (1) myeloid colonies and (2) distinct mixed colonies containing erythroid cells, granulocytes, macrophages, and infrequently megakaryocytes in a setting structurally resembling biopsied marrow. The cells that form mixed colonies reside in a density range of 1.058–1.068 g/ml in a discontinuous albumin gradient. Active supernatant was produced by T cells in combination with a macrophage factor. DNA synthesis correlated with activity. PHA- SCM differed from erythropoietin (EPO) when chromatographed on lectin columns and did not contain EPO activity as demonstrated by the fetal mouse liver cell (FMLC) assay. The activity for mixed colony growth could be eluted from an anion exchange column with 0.07 M NaCl and eluted in a gel filtration column at a distance corresponding to a molecular weight of 39,000. Mixed colony-forming cells responsive to PHA-SCM were found to be Ia-H-2+. BFU-Es, CFU-Cs, and progenitors for myeloid colonies responsive to PHA-SCM were also H-2+ but showed significant sensitivity to anti-Ia antisera reflecting variable antigenic density. The mixed colony-forming cell appeared less differentiated than myeloid or erythroid progenitor cells examined, and its antigenic determinants are consistent with those observed for the pluripotent stem cell assayed in vivo (CFU-S).

scholarly journals Conditions for an in vitro culture of murine mixed hematopoietic colonies and their putative cellular origin

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 99-107
Author(s):  
BA Miller ◽  
DE Siedler ◽  
CD Dunn ◽  
AT Huang
Keyword(s):  
Gel Filtration ◽  
Colony Growth ◽  
Antigenic Determinants ◽  
Anion Exchange Column ◽  
Erythroid Progenitor ◽  
Cellular Origin ◽  
Mouse Liver Cell ◽  
Fetal Mouse Liver

The supernatant fluid of stimulated spleen cells (PHA-SCM) supported in vitro colony growth of murine marrow. In the absence of exogenous erythropoietin, it stimulated the growth of (1) myeloid colonies and (2) distinct mixed colonies containing erythroid cells, granulocytes, macrophages, and infrequently megakaryocytes in a setting structurally resembling biopsied marrow. The cells that form mixed colonies reside in a density range of 1.058–1.068 g/ml in a discontinuous albumin gradient. Active supernatant was produced by T cells in combination with a macrophage factor. DNA synthesis correlated with activity. PHA- SCM differed from erythropoietin (EPO) when chromatographed on lectin columns and did not contain EPO activity as demonstrated by the fetal mouse liver cell (FMLC) assay. The activity for mixed colony growth could be eluted from an anion exchange column with 0.07 M NaCl and eluted in a gel filtration column at a distance corresponding to a molecular weight of 39,000. Mixed colony-forming cells responsive to PHA-SCM were found to be Ia-H-2+. BFU-Es, CFU-Cs, and progenitors for myeloid colonies responsive to PHA-SCM were also H-2+ but showed significant sensitivity to anti-Ia antisera reflecting variable antigenic density. The mixed colony-forming cell appeared less differentiated than myeloid or erythroid progenitor cells examined, and its antigenic determinants are consistent with those observed for the pluripotent stem cell assayed in vivo (CFU-S).

Macro creatine kinase BB in serum, and some data on its prevalence.

1979 ◽  
Vol 25 (3) ◽  
pp. 461-465 ◽  
Author(s):  
P Urdal ◽  
S Landaas
Keyword(s):  
Creatine Kinase ◽  
Enzyme Assay ◽  
Clinical Laboratory ◽  
Gel Filtration ◽  
Normal Activity ◽  
Anion Exchange Column ◽  
B Subunit ◽  
Inhibitory Antibodies ◽  
In Vitro Stability

Abstract We report the case of a patient with persistently above-normal activity of creatine kinase (CK) in serum, a major fraction of which on electrophoresis moved as a band between the MM and MB isoenzymes and on anion-exchange column chromatography eluted in the MB fraction. Measurements in the presence of specific M or B subunit-inhibitory antibodies indicated that 93% of the activity consisted of B-isomers. From these experiments we conclude that the abnormal CK is of BB nature. Gel filtration and immunoglobulin precipitation showed that the CK-BB was complexed with IgG. Normal CK-BB, when mixed with the patient's serum, was converted to macro CK-BB. In vitro stability of 37 degrees C of the abnormal enzyme was much greater than that of normal BB and MM isoenzymes. Following this finding, we then assessed 310 sera, received for enzyme assay by the clinical laboratory, for electrophoretically abnormally migrating CK isoenzymes. Of these, five (1.6%) contained such enzymes, all being of BB nature. They were of increased molecular mass, and at least three of them were complexed with IgG.

scholarly journals Effects of Arsenic on Laccase Enzyme Activity in Strains of Bacillus Subtilis in Vitro

2019 ◽  
Vol 26 (6) ◽  
pp. 79-85
Author(s):  
Amir Hoseyn Momen ◽  
Saied Shalbaf ◽  
Safora Salahi ◽  
◽  
◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Laccase Enzyme
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