scholarly journals Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

Catalysts ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 254 ◽  
Author(s):  
David Aregger ◽  
Christin Peters ◽  
Rebecca M. Buller
Keyword(s):  
Enantiomeric Excess ◽  
Asymmetric Hydrogenation ◽  
Paenibacillus Polymyxa ◽  
Solvent Tolerance ◽  
Old Yellow Enzyme ◽  
Conversion Rates ◽  
Specificity Constant ◽  
Km Value ◽  
Ph Range

Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (Topt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a kcat/KM value of 287 mM−1 s−1. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone.

scholarly journals Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina

1996 ◽  
Vol 318 (2) ◽  
pp. 711-716 ◽  
Author(s):  
Marco PICCININI ◽  
Adalberto MERIGHI ◽  
Renato BRUNO ◽  
Paolo CASCIO ◽  
Magda CURTO ◽  
...  
Keyword(s):  
Gene Product ◽  
Primary Structure ◽  
Protein Gene ◽  
Affinity Purification ◽  
Hydrolytic Activity ◽  
Protein Gene Product 9.5 ◽  
Protein Gene Product ◽  
Km Value ◽  
Ph Range

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin–Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.

scholarly journals Purification and characterization of rat intestinal peroxidase. Its activity towards 2-t-butyl-4-methoxyphenol (BHA)

1988 ◽  
Vol 250 (2) ◽  
pp. 501-507 ◽  
Author(s):  
M Valoti ◽  
L Della Corte ◽  
K F Tipton ◽  
G Sgaragli
Keyword(s):  
Specific Activity ◽  
Maximum Velocity ◽  
Purification And Characterization ◽  
Reaction Progress ◽  
Reversible Aggregation ◽  
Aggregation Behaviour ◽  
Apparent Homogeneity ◽  
Specificity Constant ◽  
Km Value

Impure preparations of rat intestinal peroxidase were shown to aggregate at low ionic strengths and to disaggregate at higher values. This aggregation was accompanied by a decrease in specific activity, which could lead to hysteretic behaviour of reaction progress curves. Advantage was taken of this reversible aggregation to obtain a relatively pure extract, which was subsequently purified to apparent homogeneity by affinity chromatography on concanavalin A-Sepharose followed by hydrophobic chromatography. The purified enzyme did not show the ionic-strength-dependent aggregation behaviour, behaving as a monomer of Mr 50,000. The purified enzyme was shown to catalyse the peroxidatic conversion of the commonly used antioxidant 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA) to form 3,3′-di-t-butyl-2,2′-dihydroxy-5,5′-dimethoxybiphenyl, with a Km value of 176 microM and a maximum velocity of 8 mumol/min per mg. The specificity constant, kcat./Km, for this substrate was similar to that shown towards the substrate guaiacol.

scholarly journals Gluconeogenic fructose-1,6-bisphosphatase from the mature sporocarps of common aquatic ferns: partial purification and basic characterization of this enzyme from Marsilea minuta (Polypodiopsida)

2020 ◽  
Vol 77 (5) ◽  
pp. 386-397
Author(s):  
S.S. Ghosh ◽  
◽  
M. Das ◽  
S. Basu ◽  
J. Adhikari ◽  
...  
Keyword(s):  
Partial Purification ◽  
Light Illumination ◽  
Azolla Pinnata ◽  
Fructose 1,6 Bisphosphatase ◽  
Salvinia Natans ◽  
Km Value ◽  
Gluconeogenic Enzyme ◽  
Ph Range ◽  
Fructose 1,6 Bisphosphate

The present communication reports substantial activity of gluconeogenic fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) in three common heterosporous aquatic ferns (Marsilea minuta, Salvinia natans, and Azolla pinnata) and also describes a protocol for its partial purification from mature sporocarps of Marsilea minuta. The cytosolic FBPase, obtained from Marsilea minuta, Salvinia natans, and Azolla pinnata was recognized as gluconeogenic enzyme due to its drastic catabolic inactivation in presence of externally administered glucose and its insensitivity towards photosynthetic light illumination. Cytosolic gluconeogenic FBPase was partially purified from mature sporocarps of Marsilea minuta to about 22-fold over homogenate following low-speed centrifugation (11, 400 × g), 30–80% ammonium sulfate fractionation followed by subsequent chromatography using matrices like CM-Cellulose, Sephadex G-200, and Ultrogel AcA 34. The profile of partially purified FBPase in PAGE under non-denaturing condition was recorded. The enzyme activity increased linearly with respect to protein concentration to about 100 µg and with respect to time up to 75 minutes. Temperature optimum was found at 35 °C. The effect of substrate concentration and kinetic analyses for FBPase were carried out using D-fructose-1,6-bisphosphate (D-FBP, the substrate) in the range of 0.0 to 1.0 mM at an interval of 0.1 mM concentration. The Km value for D-FBP of FBPase was 0.06129 mM and Vmax was 4525 nmole Pi released (mg)-1 protein h-1 as determined by nonlinear regression kinetics using Prism 8 software (Graph Pad). The enzyme was functional in a constricted pH range of 7.0 to 8.0, giving maxima at pH 7.5. This cytosolic enzyme was significantly stimulated by Mg2+ and strongly inhibited by Hg2+, Cu2+ and Zn2+.

scholarly journals Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey

1985 ◽  
Vol 230 (3) ◽  
pp. 715-721 ◽  
Author(s):  
Y Takuwa ◽  
E Ogata
Keyword(s):  
Phosphate Uptake ◽  
Phosphate Transport ◽  
Hill Coefficient ◽  
Epithelial Cell Line ◽  
Carbonyl Cyanide ◽  
Dependent Component ◽  
Km Value ◽  
Ph Range ◽  
Energy Dependent

Phosphate uptake was studied in confluent monolayers of an epithelial-cell line (JTC-12) derived from monkey kidney. Phosphate uptake consisted of a saturable, Na+-dependent, component, which accounted for about 80% of the uptake, and a nonsaturable, Na+-independent, component. The saturable component was specifically dependent on the presence of extracellular Na+ and has an apparent Km value for phosphate of 0.12 mM at 137-mM-Na+, which is close to those reported in the brush-border membranes in mammalian kidneys. The presence of Na+ in the uptake solution decreased the Km for phosphate without affecting the Vmax. Phosphate uptake was inhibited by carbonyl cyanide p-trifluoromethoxyphenylhydrazone and ouabain, suggesting that phosphate transport is an active, energy-dependent, process and is dependent on an Na+ gradient across cell membranes. With respect to the effect of external Na+ concentration, a sigmoid relation was seen between the initial velocity of phosphate uptake and Na+ concentrations, and Hill analysis gave a Hill coefficient of 1.8. In the pH range 6.6-7.4, phosphate uptake declined with increasing pH. Phosphate uptake was stimulated when cells were cultured in the presence of insulin, and was also affected by changes in phosphate concentrations in cultured medium. These results indicate that JTC-12 cells have an Na+-dependent phosphate-transport system with many of the features of phosphate transport in the proximal tubule.

scholarly journals Xylanase SMXL1 from Stenocarpella maydis: Purification and biochemical characterization

BioResources ◽  
2020 ◽  
Vol 15 (2) ◽  
pp. 2947-2960
Author(s):  
Edna M. Hernández-Domínguez ◽  
Jorge Álvarez-Cervantes ◽  
Pedro Gersain Lucio-Ávila ◽  
Gerardo Díaz-Godínez ◽  
Yuridia Mercado-Flores
Keyword(s):  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Birchwood Xylan ◽  
Michaelis Constant ◽  
Sds Page ◽  
Km Value ◽  
Ph Range ◽  
Stenocarpella Maydis

This study aimed to develop a method for the purification of a xylanase called SMXL1 produced by Stenocarpella maydis and its biochemical characterization. The enzyme was purified using a Rotofor preparative chamber and one chromatographic step in an ion exchange column coupled to equipment FPLC. Posteriorly the protein was characterized, and its effect on the birchwood xylan degradation was determine by HPLC. The purified enzyme showed a molecular weight of 55 kDa calculated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purification process obtained a yield of 6.5  0.3 %. The activity was stable at a pH range of 4 to 10 and temperatures of 45 to 60 °C. The optimum values of temperature and pH were 55 °C and 4, respectively. The Michaelis constant (Km) value was 2.61 mg/mL and the Vmax was 3.02 µmol/mL/min using birchwood xylan as substrate and the Michaelis-Menten equation. The enzyme is inhibited by the cations Mn2+ and by Fe3+ and degrades the birchwood xylan being the principal products the xylobiose and the xylose. This work is the first report of the purification and biochemical characterization of a xylanase called SMXL1 produced by S. maydis.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals Identification and characterization of a novel glucomannanase from Paenibacillus polymyxa

3 Biotech ◽  
2021 ◽  
Vol 11 (3) ◽  
Author(s):  
Kuikui Li ◽  
Chaofeng Jiang ◽  
Haidong Tan ◽  
Junyan Li ◽  
Yali Xu ◽  
...  
Keyword(s):  
Paenibacillus Polymyxa ◽  
Identification And Characterization

scholarly journals Incidence and characterisation of Bacillus cereus bacteriophages from Thua Nao, a Thai fermented soybean product

2021 ◽  
Vol 12 (1) ◽  
pp. 85-93
Author(s):  
Wallapat Phongtang ◽  
Ekachai Chukeatirote
Keyword(s):  
Bacillus Cereus ◽  
Food Industry ◽  
Pathogenic Bacteria ◽  
Morphological Analysis ◽  
Food Poisoning ◽  
Fermented Soybean ◽  
Foodborne Pathogenic Bacteria ◽  
Ph Range ◽  
Potential Use

Abstract Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus. Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae. Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis. The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

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