scholarly journals Purification and properties of malonyl-CoA synthetase from Rhizobium japonicum

1991 ◽  
Vol 273 (3) ◽  
pp. 511-516 ◽  
Author(s):  
Y S Kim ◽  
H Z Chae
Keyword(s):  
Carbon Sources ◽  
Gel Filtration ◽  
Rhizobium Japonicum ◽  
Purification And Properties ◽  
Malonyl Coa ◽  
Sds Page ◽  
Optimum Ph ◽  
Soybean Nodule ◽  
Terminal Amino ◽  
Single Polypeptide

A novel malonyl-CoA synthetase was found in Rhizobium japonicum bacteroid of the soybean nodule. The levels of the enzyme in the free-living cells grown on a variety of carbon sources including glucose were similar, indicating that this enzyme is not inducible. The malonyl-CoA synthetase from glucose-grown Rhizobium japonicum was purified to homogeneity. The Mr of the enzyme was determined to be 58,000 by gel filtration on a Sephacryl S-300 and by SDS/PAGE respectively, indicating a single polypeptide enzyme. N-Terminal amino acid of the enzyme was methionine but the enzyme preparation contained about 40% de-methionylated protein. The enzyme catalyses the formation of malonyl-CoA, AMP and PPi directly from malonate, CoA and ATP in the presence of Mg2+. High substrate specificity on malonate and ATP was revealed, but Mn2+ could be substituted for Mg2+ without any difference in activity. Optimum pH was 7.9. Kinetic constants, Km and Vmax, for malonate, CoA and ATP were 200 microM and 21.3 mumol/min per mg, 87 microM and 41.7 mumol/min per mg, and 33.3 microM and 29.4 mumol/min per mg respectively. Succinate inhibited the enzyme noncompetitively, whereas AMP and ADP inhibited competitively. Diethylpyrocarbonate and pyridoxal-5′-phosphate severely inhibited the enzyme, but iodoacetamide, p-chloromercuriphenylsulphonate, N-acetylimidazole and phenylglyoxal did not.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Purification and properties of an exocellular β-glucosidase of Candida molischiana (Zikes) Meyer and Yarrow capable of hydrolyzing soluble cellodextrins

1985 ◽  
Vol 63 (11) ◽  
pp. 1160-1166 ◽  
Author(s):  
Pierre Gondé ◽  
Robert Ratomahenina ◽  
Alain Arnaud ◽  
Pierre Galzy
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Purification And Properties ◽  
Optimum Ph

The exocellular enzyme β-glucosidase of Candida molischiana was studied. This strain is able to ferment soluble cellodextrins. The enzyme was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 120 000; its optimum pH was between 4 and 4.5 and its optimum temperature was 60 °C. This enzyme was active against different soluble glucosides and was inhibited by p-chloromercuribenzoate, gluconolactone, and glucose. A "glucosyltransferase" activity appeared in the presence of ethanol. The biosynthesis of the enzyme was constitutive but repressed by glucose.

scholarly journals Purification and properties of the enzyme arylamine N-acetyltransferase from the housefly Musca domestica

1993 ◽  
Vol 295 (1) ◽  
pp. 149-154 ◽  
Author(s):  
D P Whitaker ◽  
M W Goosey
Keyword(s):  
Musca Domestica ◽  
Gel Filtration ◽  
Potassium Phosphate ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
A Value ◽  
Purification And Properties ◽  
Sds Page ◽  
Mammalian Enzyme ◽  
Monomeric Structure

The enzyme arylamine N-acetyltransferase (ANAT) from the housefly (Musca domestica) has been purified. The M(r) of the purified enzyme was 27,600 +/- 1700 as estimated by gel filtration. SDS/PAGE yielded a value of 26,000 +/- 300, clearly indicating a monomeric structure. The purified enzyme had apparent Km values for acetyl-CoA and tyramine of 8.4 microM and 8.8 microM respectively, a pH optimum of 7.2 in 10 mM potassium phosphate buffer and an apparent pI of 5.8. ANAT activity showed a strong dependency on the presence of 2-mercaptoethanol during the purification stages. The enzyme could be completely inactivated by treatment with p-chloromercuribenzoate although the enzyme activity was protected by preincubation with acetyl-CoA. One or more cysteine residues are clearly required for catalytic activity, as demonstrated for the mammalian enzyme. In contrast, partial sequencing of the enzyme has yielded a number of peptide sequences, including the N-terminal sequence, which show no similarity with those reported for the mammalian and avian enzymes.

scholarly journals Purification and properties of a phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279

2012 ◽  
Vol 3 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Abd El Aziem Farouk ◽  
Ralf Greiner ◽  
Anis Shobirin Meor Hussin
Keyword(s):  
Relative Rate ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enterobacter Sakazakii ◽  
Degrading Enzyme ◽  
Purification And Properties ◽  
Sds Page ◽  
Rate Of Hydrolysis

An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM).

scholarly journals Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

1994 ◽  
Vol 299 (3) ◽  
pp. 895-902 ◽  
Author(s):  
R M Jones ◽  
P M Jordan
Keyword(s):  
Arabidopsis Thaliana ◽  
Gel Filtration ◽  
Chain Termination ◽  
Porphobilinogen Deaminase ◽  
Ph Optimum ◽  
Heat Stable ◽  
Purification And Properties ◽  
Sds Page ◽  
Plant Arabidopsis Thaliana

Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

Production and biochemical and molecular characterization of a keratinolytic serine protease from chicken feather-degradingBacillus licheniformisRPk

2009 ◽  
Vol 55 (4) ◽  
pp. 427-436 ◽  
Author(s):  
Nahed Fakhfakh ◽  
Safia Kanoun ◽  
Laila Manni ◽  
Moncef Nasri
Keyword(s):  
Serine Protease ◽  
Gel Filtration ◽  
Chicken Feather ◽  
Feather Degradation ◽  
Subtilisin Carlsberg ◽  
Degrading Bacterium ◽  
Step Procedure ◽  
Sds Page ◽  
Optimum Ph ◽  
Optimum Ph And Temperature

A novel feather-degrading bacterium was isolated from a polluted river and identified as Bacillus licheniformis RPk. The isolate exhibited high proteinase production when grown in chicken-feather media. Complete feather degradation was achieved during cultivation. Maximum protease activity (4150 U/mL with casein as a substrate and 37.35 U/mL with keratin as a substrate) was obtained when the strain was grown in a medium containing 7.5 g/L chicken feathers, 2 g/L yeast extract, 0.5 g/L NaCl, 0.1 g/L MgSO4·7H2O, 0.7 g/L KH2PO4, and 1.4 g/L K2HPO4for 48 h with agitation of 200 rev/min at 37 °C. The major protease produced by B. licheniformis RPk was purified to homogeneity by a 3-step procedure. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS–PAGE and gel filtration. The optimum pH and temperature for the caseinolytic activity were around 11.0 and 65 °C, respectively. The optimum pH and temperature for the keratinolytic activity were 9.0 and 60 °C, respectively. The activity of the enzyme was totally lost in the presence of phenylmethylsulfonyl fluoride, which suggests that the purified enzyme is a serine protease. The thermostability of the enzyme was considerably enhanced in the presence of Ca2+at temperatures >50 °C. The kerRP gene, which encodes the keratinolytic protease, was isolated, and its DNA sequence was determined. The deduced amino acid sequence revealed that the keratinase KerRP differs from KerA of B. licheniformis PWD-1, subtilisin Carlsberg, and keratinase of B. licheniformis by 2, 4, and 62 amino acids, respectively.

Purification and partial biochemical–genetic characterization of trehalose 6-phosphate synthase from muscles of adult femaleAscaris suum

2012 ◽  
Vol 87 (2) ◽  
pp. 212-221 ◽  
Author(s):  
M. Dmitryjuk ◽  
M. Dopieralska ◽  
E. Łopieńska-Biernat ◽  
R.J. Frączek
Keyword(s):  
Genetic Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sds Page ◽  
Optimum Ph ◽  
Genes Encoding ◽  
Ammonium Sulphate Fractionation ◽  
Biochemical Genetic

AbstractTrehalose 6-phosphate (T6P) synthase (TPS;EC2.4.1.15) was isolated from muscles ofAscaris suumby ammonium sulphate fractionation, ion-exchange DEAE SEPHACELTManion exchanger column chromatography and Sepharose 6B gel filtration. On sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), 265-fold purified TPS exhibited a molecular weight of 66 kDa. The optimum pH and temperature of the purified enzyme were 3.8–4.2 and 35°C, respectively. The isoelectric point (pI) of TPS was pH 5.4. The studied TPS was not absolutely substrate specific. Besides glucose 6-phosphate, the enzyme was able to use fructose 6-phosphate as an acceptor of glucose. TPS was activated by 10 mMMgCl2, 10 mMCaCl2and 10 mMNaCl. In addition, it was inhibited by ethylenediaminetetra-acetic acid (EDTA), KCl, FeCl3and ZnCl2. Two genes encoding TPS were isolated and sequenced from muscles of the parasite. Complete coding sequences fortps1(JF412033.2) andtps2(JF412034.2) were 3917 bp and 3976 bp, respectively. Translation products (AEX60788.1 and AEX60787.1) showed expression to the glucosyltransferase-GTB-type superfamily.

PURIFICATION AND CHARACTERIZATION OF A PROTEIN C ACTIVATOR FROM THE VENOM OF AGKISTRODON CONTORTRIX CONTORTRIX

1987 ◽  
Author(s):  
Carolyn L Orthner ◽  
Prabir Bhattacharya ◽  
Dudley K Strikland
Keyword(s):  
Protein C ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Agkistrodon Contortrix ◽  
Purification And Properties ◽  
Sds Page ◽  
Single Protein ◽  
Agkistrodon Contortrix Contortrix

There are two recent reports on the purification and properties of a protein C activator (PCA) from the venom of the Southern copperhead snalce. The purification of a 37,000 Mr nonenzymatic PCA (Martinoli and Stocker, Thrcmb. Res. 43, 253, 1976) as well as of a 20,000 Mr thrombin-like enzyme (Klein and Walker, Biochem. ,25, 4175, 1986) have been described. The purpose of this investigation was to purify and further characterize the PCA(s) from this vencm. A PCA has been isolated by sulphopropyl-Sephadex followed by gel filtration chromatography resulting in approximately a 100-fold purification with a 50% yield. PCA appeared as a single band on SDS-PAGE with an estimated Mr of 32,000 or 37,000 in the absence or presence of β-mercaptoethanol, respectively. High pressure gel permeation cinematography of PCA in Tris-buffered saline, pH 7.5 resulted in a single protein peak with a Mr of 39,000 which was coincident with activity. PCA was a potent activator of human protein C (PC) with a Km for PC of 0.6uM and a Vm of 0.02 sec-1. In addition, PCA catalyzed the arnidolysis of Tosyl-gly-pro-arg-p-nitroanilide (TGPRpNA) with a Km of 1.1 irM and a Vim of 66 sec-1. The rate of arnidolysis of five other pept idyl-arginyl-pNA substrates each tested at 1.0 mM was < 10% that of TGPRpNA. PCA was inhibited by nitrophenylguanidi-nobenzoate (NPGB), phenylmethylsulphonylflouride, D-phe-pro-arg-chloromethyi_ketone (PPACK) and soybean trypsin inhibitor indicating that PCA is a serine protease. The active site concentration of PCA as measured by NPGB titration was 90% that of the protein concentration. Measurement of the rate of PCA inhibition at varying levels of PPACK indicated that it had a Ki of 34uM .and an aUcylation rate constant of 0.09 min-1. PCA activation of PC was completely inhibited by CaC12 with an apparent Ki of 99uM. Since neither PCA arnidolysis of TGPRpNA nor inhibition by PPACK was affected by Ca2+, the effect of this metal was likely on the substrate PC. In summary, a PCA has been purified to homogeneity and has properties which are distinct from those reported. PCA premises to be a useful enzyme in studies of PC and its activation.

scholarly journals Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2

1999 ◽  
Vol 66 (2) ◽  
pp. 257-270 ◽  
Author(s):  
MAEVE McDONNELL ◽  
PAUL BOUCHIER ◽  
RICHARD J. FITZGERALD ◽  
GERARD O'CUINN
Keyword(s):  
Lactococcus Lactis ◽  
Gel Filtration ◽  
Alternative Mechanism ◽  
Single Polypeptide Chain ◽  
Complementary Action ◽  
Sds Page ◽  
Wide Range ◽  
N Terminus ◽  
Single Polypeptide ◽  
Dipeptidyl Aminopeptidase

A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native Mr of 50 000–55 000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4°C and its stability was greatly improved when 10 ml glycerol/l and 2 mm-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro–Leu–Gly–NH2, Pro–Leu–Gly–Gly and Pro–His–Pro–Phe–His–Leu–Phe–Val–Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr–Pro–Phe–Pro–Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.

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