AN ISOPHENOXAZINE SYNTHASE FROM PYGNOPORUS COCCINEUS (FR.) BOND. AND SING.

1964 ◽  
Vol 42 (11) ◽  
pp. 1515-1526 ◽  
Author(s):  
P. M. Nair ◽  
L. C. Vining
Keyword(s):  
Electron Acceptor ◽  
Specific Activity ◽  
Fold Increase ◽  
Maximum Activity ◽  
Flavin Mononucleotide ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Hydroxyanthranilic Acid ◽  
Pycnoporus Coccineus ◽  
Citrate Phosphate

Mycelium from cultures of the red polypore Pycnoporus coccineus (Fr.) Bond. and Sing, contains an enzyme which catalyzes the oxidative condensation of 2 molecules of 2-aminophenol to yield 2-amino-3H-isophenoxazin-3-one. Fractionation of the crude extract has given a preparation with an 893-fold increase in specific activity. The purified enzyme has a pH optimum in citrate-phosphate buffer of 5.0, and a temperature optimum of 55°. The Km value is 4.35 × 10−4 M. FMN and Mn++ ions were required for maximum activity. FAD also served as an electron acceptor. Of the metal ions tested only Mn++ activated the reaction. Hg++ and Fe++ inhibited strongly. The course of the reaction when cofactors were added separately suggested that flavin mononucleotide is the initial electron acceptor and that Mn++ ions are required for reoxidation of the flavin. The enzyme has narrow specificity, and does not catalyze the oxidation of 3-hydroxyanthranilic acid, 3-hydroxykynurenine, or pyrocatechol.

A Dpnh-Specific Nitrate Reductase From Germinating Wheat

1959 ◽  
Vol 12 (2) ◽  
pp. 181 ◽  
Author(s):  
D Spencer
Keyword(s):  
Nitrate Reductase ◽  
Flavin Adenine Dinucleotide ◽  
Potassium Cyanide ◽  
Fold Increase ◽  
Maximum Activity ◽  
Flavin Mononucleotide ◽  
Enzyme Complex ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Isolation And Purification

The isolation and purification of a nitrate reductase from the embryos of germinating wheat is described. This is a soluble enzyme, which is coupled specifically to reduced diphosphopyridine nucleotide (DPNH). Addition of flavin adenine dinucleotide to the purified enzyme results in a three-fold increase in activity. Flavin mononucleotide is without effect. Potassium cyanide and sodium azide cause 50 per cent. inhibition of enzyme activity at 1� 6 X IO-6M and 2 X IO-5M respectively. The effect of a range of other inhibitors is reported. The presence of inorganic phosphate is required for maximum activity. The dissociation constant (Ks ) of the nitrate-enzyme complex is 3�8 X IO-4M, and that of the DPNH-enzyme complex is 8 X IO-6M. The pH optimum for enzymatic activity is 7 �4.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

Solubilization of microsomal-associated phosphatidylserine synthase and phosphatidylinositol synthase from Saccharomyces cerevisiae

1981 ◽  
Vol 27 (11) ◽  
pp. 1140-1149 ◽  
Author(s):  
George M. Carman ◽  
Jonathan Matas
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Activity ◽  
Phosphatidyl Inositol ◽  
Maximum Activity ◽  
Enzymatic Activities ◽  
Ph Optimum ◽  
Phosphatidylserine Synthase ◽  
Phosphatidylinositol Synthase ◽  
Microsome Fraction ◽  
Triton X

Membrane-associated cytidine 5′-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):L-serine O-phosphatidyltransferase (phosphatidylserine synthase, EC 2.7.8.8.) and CDP-diacylglycerol: myo-inositol phosphatidyltransferase (phosphatidyl-inositol synthase, EC 2.7.8.11) were solubilized from the microsomal fraction of Saccharomyces cerevisiae. A variety of detergents were examined for their ability to release phosphatidylserine synthase and phosphatidylinositol synthase activities from the microsome fraction. Both enzymes were solubilized from the microsome fraction with Renex 690 in yields over 80% with increases in specific activity of 1.6-fold. Both solubilized enzymatic activities were dependent on manganese ions and Triton X-100 for maximum activity. The pH optimum for each reaction was 8.0. The apparent Km values for CDP-diacylglycerol and serine for the phosphatidylserine synthase reaction were 0.1 and 0.25 mM, respectively. The apparent Km values for CDP-diacylglycerol and inositol for the phosphatidylinositol synthase reaction were 70 μM and 0.1 mM, respectively. Thiore-active agents inhibited both enzymatic activities. Both solubilized enzymatic activities were thermally inactivated at temperatures above 30 °C.

scholarly journals Solubilization, partial purification and properties of N-methylglutamate dehydrogenase from Pseudomonas aminovorans

1977 ◽  
Vol 161 (2) ◽  
pp. 357-370 ◽  
Author(s):  
C W Bamforth ◽  
P J Large
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Specific Activity ◽  
Partial Purification ◽  
Ph Optimum ◽  
Transport Chain ◽  
Purification And Properties ◽  
Solubilized Enzyme ◽  
Triton X ◽  
Sepharose 4B

1. Extracts of amine-grown Pseudomonas aminovorans contained a particle-bound N-methylglutamate dehydrogenase (EC 1.5.99.5). The enzyme was not present in succinate-grown cells, and activity appeared before growth began in succinate-grown cells which had been transferred to methylamine growth medium. 2. Membrane-containing preparations from methylamine-grown cells catalysed an N-methylglutamate-dependent uptake of O2 or reduction of cytochrome c, which was sensitive to inhibitors of the electron-transport chain. 3. N-Methylglutamate dehydrogenase activity with phenazine methosulphate or 2,6-dichlorophenol-indophenol as electron acceptor could be solubilized with 1% (w/v) Triton X-100. The solubilized enzyme was much less active with cytochrome c as electron acceptor and did not sediment in 1 h at 150000g. Solubilization was accompanied by a change in the pH optimum for activity. 4. The solubilized enzyme was partially purified by Sepharose 4B and hydroxyapatite chromatograpy to yield a preparation 22-fold increased in specific activity over the crude extract. 5. The partially-purified enzyme was active with sarcosine, N-methylalanine and N-methylaspartate as well as with N-methylglutamate. Evidence suggesting activity with N-methyl D-amino acids as well as with the L-forms was obtained. 6. The enzyme was inhibited by p-chloromercuribenzoate, iodoacetamide and by both ionic and non-ionic detergents. 2-Oxoglutarate and formaldehyde were also inhibitors. 7. Kinetic analysis confirmed previous workers' observations of a group transfer (Ping Pong) mechanism. 8. Spectral observations suggested that the partially purified preparation contained flavoprotein and a b-type cytochrome. 9. The role of the enzyme in the oxidation of methylamine is discussed.

Properties of formate dehydrogenase from Desulfovibrio gigas

1986 ◽  
Vol 32 (5) ◽  
pp. 430-435 ◽  
Author(s):  
Mary Ann Riederer-Henderson ◽  
Harry D. Peck Jr.
Keyword(s):  
Cytochrome C ◽  
Electron Acceptor ◽  
Formate Dehydrogenase ◽  
Specific Activity ◽  
Ph Optimum ◽  
Benzyl Viologen ◽  
Desulfovibrio Gigas ◽  
Diaphorase Activity ◽  
Lag Period ◽  
Molecular Radius

The formate dehydrogenase from extracts of Desulfovibrio gigas was partially purified to a specific activity of 5600 nmol CO2 ∙ min−1 ∙ mg protein−1. Uniquely for a formate dehydrogenase from anaerobes, the enzyme was stable when stored aerobically. Nevertheless, thiols were required in the assay mixture for enzymatic activity. If the enzyme first catalyzed the transfer of electrons from thiols to benzyl viologen (a diaphorase activity), then formate was oxidized rapidly without a lag period. The enzyme had a molecular weight of approximately 240 000, a pH optimum of 7.5–8.0, and a temperature optimum of 56 °C. Activity with cytochrome c3 (molecular radius (Mr) = 13 000) was about twice that with ferredoxin or flavodoxin as the electron acceptor. These results suggest that the formate dehydrogenase from D. gigas can be activated by transferring electrons from thiols to an electron acceptor and uses cytochrome c3 as the natural electron carrier for the oxidation of formate.

scholarly journals Purification and properties of a 1,3-1,4-β-d-glucanase (lichenase, 1,3-1,4-β-d-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli

1988 ◽  
Vol 255 (3) ◽  
pp. 833-841 ◽  
Author(s):  
J D Erfle ◽  
R M Teather ◽  
P J Wood ◽  
J E Irvin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Glucanase Activity ◽  
Ph Optimum ◽  
Beta Glucanase ◽  
Purification And Properties ◽  
Hydrolysis Of

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

Purification of a third distinct xylanase from the xylanolytic system of Trichoderma harzianum

1986 ◽  
Vol 32 (7) ◽  
pp. 570-576 ◽  
Author(s):  
Ken K. Y. Wong ◽  
Larry U. L. Tan ◽  
John N. Saddler ◽  
Makoto Yaguchi
Keyword(s):  
Molecular Mass ◽  
Trichoderma Harzianum ◽  
Thermal Tolerance ◽  
Specific Activity ◽  
Gene Products ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Polysulfone Membrane ◽  
Xylan Hydrolysis ◽  
Hydrolysis Of

Three of the xylanases produced by Trichoderma harzianum E58 passed through a polysulfone membrane with molecular mass cut-off of 10 000 daltons, even though their molecular mass had been estimated to be 20 000, 22 000, and 29 000 daltons. The 22 000 dalton xylanase was purified to homogeneity from a preparation containing a mixture of 22 000 and 20 000 dalton xylanase using a combination of hydrophobic column chromatography and chromatofocusing. This enzyme has a pI of 8.5, a specific activity of 0.28 U/mg, a temperature optimum between 45 and 50 °C, a pH optimum between 4.5 and 5.0, and the ability to cleave xylotriose. It differs from the other two xylanases by having a lower pI, a lower specific activity, and a lower thermal tolerance. All three xylanases are highly specific for xylan hydrolysis and they do not cleave xylobiose or release arabinose substituents from arabinoxylan. Their amino acid compositions suggest that they are three distinct gene products. The three enzymes are major components of the xylanolytic system of T. harzianum, which consists of at least two other xylanases and two β-xylosidases which are responsible for the release of arabinose substituents and the hydrolysis of xylobiose.

scholarly journals Characterization of a chelator-resistant proteinase from Thermus strain Rt4A2

1993 ◽  
Vol 295 (2) ◽  
pp. 463-469 ◽  
Author(s):  
S A Freeman ◽  
K Peek ◽  
M Prescott ◽  
R Daniel
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Substrate Inhibition ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
High Sequence Similarity

The Thermus isolate Rt4A2 was found to produce an extracellular chelator-resistant proteinase. The proteinase was purified to homogeneity by (NH4)2SO4 precipitation, cation-exchange chromatography, gel-filtration chromatography, and weak anion-exchange chromatography. The Rt4A2 proteinase was found to have properties typical of an alkaline serine proteinase. It had a pH optimum of 9.0 and was specifically inhibited by phenylmethanesulphonyl fluoride. Its isoelectric point was greater than 10.25. Its molecular-mass was 31.6 kDa as determined by SDS/PAGE. N-terminal sequencing has shown it to have high sequence similarity with other serine proteinases from Thermus species. The proteinase hydrolysed a number of substrates including fibrin, casein, haemoglobin, collagen, albumin and the synthetic chromogenic peptide substrate Suc-Ala-Ala-Pro-Phe-NH-Np. The specific activity of the purified proteinase using azocasein as substrate was 313 units/mg. Substrate inhibition was observed above an azocasein concentration of 0.05% (w/v). Esterase activity was directed mainly towards those substrates containing the aliphatic or aromatic residues of alanine, glycine, tryptophan, tyrosine and phenylalanine. Thermostability half-lives of greater than 7 days at 70 degrees C, 43 h at 80 degrees C and 90 min at 90 degrees C were found in the presence of 5 mM CaCl2. At 90 degrees C increasing the CaCl2 concentration 100-fold (0.5 mM to 50 mM) caused a 4.3-fold increase in the half-life of the enzyme from 30 to 130 min. Half-lives of 19.4 min at 100 degrees C and 4.4 min at 105 degrees C were found in the presence of 50 mM CaCl2. The metal chelators EGTA and EDTA reduced the stability at higher temperatures but had no effect on the activity of the proteinase. Activity was not stimulated by common metal activators such as Ca2+, Mg2+ and Zn2+.

scholarly journals Preparation and Characterization of 5′-Phosphodiesterase from Barley Malt Rootlets

2010 ◽  
Vol 5 (2) ◽  
pp. 1934578X1000500
Author(s):  
Jie Hua ◽  
Ke-long Huang
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Barley Malt

Two 5′- phosphodiesterases (5′-PDE-a and 5′-PDE-b) were isolated from barley malt rootlets, and further purified by gel filtration on Sephadex G-25 and Sephadex G-75. 5′-PDE-a had a pH optimum of 5.0, temperature optimum of 70oC, and specific activity of 0.0143 mM ·mg−1-min−1. 5′-PDE –b had a pH optimum of 6.0, temperature optimum of 65°C and specific activity of 0.0125 mM ·mg−1·min−1. Both enzymes can be used to hydrolyze RNA to form 5′-nucleotides. The enzymes were quite stable at 70oC for 420 minutes. The Km was 0.24 mM for 5′-PDE-a and 0.16 mM for 5′-PDE-b with t-RNA (yeast) as substrate.

Pyruvate carboxylase from Thiobacillus novellus: properties and possible function

1984 ◽  
Vol 30 (5) ◽  
pp. 532-539 ◽  
Author(s):  
A. M. Charles ◽  
D. W. Willer
Keyword(s):  
Initial Velocity ◽  
Pyruvate Carboxylase ◽  
Maximum Activity ◽  
Temperature Optimum ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Citrate Cycle ◽  
End Products ◽  
Sigmoidal Curve ◽  
Hill Coefficients

Pyruvate carboxylase (EC 6.4.1.1) from Thiobacillus novellus (ATCC 8093) was highly purified and found to have a pH optimum of 7.6, a temperature optimum of 25–35 °C, and a requirement for a monovalent and a divalent cation, as well as a CoA derivative, for maximum activity. These were best served by K+, Mg2+, and acetyl-CoA. Km values for pyruvate, ATP, HCO3− and Mg2+ were 0.25, 0.04, 0.27, and 0.44 mM, respectively. Initial velocity plots of increasing acetyl-CoA concentrations gave a sigmoidal curve with Ka of 4.2 μM, and Hill coefficients of 2.2. Plots of fixed acetyl-CoA concentrations against varying concentrations of pyruvate, ATP, or CO2 all gave rectangular hyperbolae. Apart from end products, only hydroxypyruvic acid was found to be inhibitory. The enzyme was very sensitive to mercurials. This enzyme is not believed to serve an anaplerotic function, because of the simultaneous presence of the highly regulated phosphoenolpyruvate carboxylase in the organism. Instead, it may function either to supply oxaloacetate to the citrate cycle or as part of the system that provides reduced NADP+.

Sign in / Sign up

Export Citation Format

Share Document