Characterization of organomercury-decomposing activity in cell extract of mercury-resistant Clostridium cochlearium T-2P

1982 ◽  
Vol 6 (1) ◽  
pp. 82-88 ◽  
Author(s):  
Hidemitsu S.K. Pan-Hou ◽  
Yukiko Kajikawa ◽  
Nobumasa Imura
Keyword(s):  
Cell Extract ◽  
Clostridium Cochlearium

scholarly journals Characterization of an Exceedingly Active NADH Oxidase from the Anaerobic Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 189 (8) ◽  
pp. 3312-3317 ◽  
Author(s):  
Xianqin Yang ◽  
Kesen Ma
Keyword(s):  
Nadh Oxidase ◽  
Specific Activity ◽  
Ph Value ◽  
Thermotoga Maritima ◽  
Cell Extract ◽  
Hyperthermophilic Bacterium ◽  
Its Gene ◽  
Molecular Masses ◽  
New Type

ABSTRACT An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80°C. The Vmax was 230 ± 14 μmol/min/mg (k cat/Km = 548,000 min−1 mM−1), and the Km values for NADH and oxygen were 42 ± 3 and 43 ± 4 μM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima.

scholarly journals Identification and Characterization of an Archaeon-Specific Riboflavin Kinase

2008 ◽  
Vol 190 (7) ◽  
pp. 2615-2618 ◽  
Author(s):  
Zahra Mashhadi ◽  
Hong Zhang ◽  
Huimin Xu ◽  
Robert H. White
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Biosynthetic Pathway ◽  
Recombinant Expression ◽  
Cell Extract ◽  
Flavin Mononucleotide ◽  
E Coli ◽  
Gene Recombinant ◽  
Identification And Characterization

ABSTRACT The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the MJ0056 gene. Recombinant expression of the MJ0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

scholarly journals Purification and Characterization of a Novel 5-Oxoprolinase (without ATP-Hydrolyzing Activity) fromAlcaligenes faecalis N-38A

1999 ◽  
Vol 65 (2) ◽  
pp. 712-717 ◽  
Author(s):  
Atsuhisa Nishimura ◽  
Yasunori Ozaki ◽  
Hiroshi Oyama ◽  
Takashi Shin ◽  
Sawao Murao
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alcaligenes Faecalis ◽  
Cell Extract ◽  
Homogeneous State ◽  
Amino Terminal ◽  
A Cell ◽  
Terminal Amino

ABSTRACT A novel type of 5-oxoprolinase was found in a cell extract of strain N-38A, which was later identified as Alcaligenes faecalis. The enzyme in the cell extract was purified to a homogeneous state with a yield of 16.6%. The molecular weight of the purified enzyme was estimated to be 47,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, suggesting that the enzyme is a monomeric protein. The enzyme specifically catalyzed a decyclization of l-pyroglutamate without hydrolyzing ATP and also without any requirements for metal ions such as Mg2+ and K+. The optimal pH for the decyclization was 7.4. The reaction was reversible. The equilibrium constant of the reaction, K eq = [l-glutamate]/[l-pyroglutamate], was evaluated to be approximately 0.035, which indicates that the reaction tends to form l-pyroglutamate. The amino-terminal amino acid sequence of the enzyme was H-Glu-Pro-Arg-Leu-Asp-Thr-Ser-Gln-Leu-Tyr-Ala-Asp-Val-His-Phe-. No protein with a similar sequence was found in the DNASIS database. Based on these data, it was strongly suggested that the enzyme described here is a novel type of 5-oxoprolinase.

scholarly journals Purification and Characterization of Gallic Acid Decarboxylase from Pantoea agglomerans T71

1998 ◽  
Vol 64 (12) ◽  
pp. 4743-4747 ◽  
Author(s):  
Mitsuhiro Zeida ◽  
Marco Wieser ◽  
Toyokazu Yoshida ◽  
Tsuyoshi Sugio ◽  
Toru Nagasawa
Keyword(s):  
Gallic Acid ◽  
Cell Extract ◽  
Spectrophotometric Analysis ◽  
Acid Decarboxylase ◽  
Purification And Characterization ◽  
Resting Cell ◽  
Oxygen Sensitive ◽  
A Cell ◽  
Plasma Emission Spectroscopy

ABSTRACT Oxygen-sensitive gallic acid decarboxylase from Pantoea(formerly Enterobacter) agglomerans T71 was purified from a cell extract after stabilization by reducing agents. This enzyme has a molecular mass of approximately 320 kDa and consists of six identical subunits. It is highly specific for gallic acid. Gallic acid decarboxylase is unique among similar decarboxylases in that it requires iron as a cofactor, as shown by plasma emission spectroscopy (which revealed an iron content of 0.8 mol per mol of enzyme subunit), spectrophotometric analysis (absorption shoulders at 398 and 472 nm), and inhibition of the enzyme activity by 2,2′-bipyridyl, o-phenanthroline, and EDTA. Another interesting feature of this strain is the fact that it contains a tannase, which is used together with the gallic acid decarboxylase in a two-enzyme resting cell bioconversion to synthesize valuable pyrogallol from readily available tannic acid.

scholarly journals Suppression of IgE antibody production in SJL mice. III. Characterization of a suppressor substance extracted from normal SJL spleen cells.

1977 ◽  
Vol 145 (6) ◽  
pp. 1501-1510 ◽  
Author(s):  
N Watanabe ◽  
Z Ovary
Keyword(s):  
Antibody Production ◽  
High Titer ◽  
Gel Filtration ◽  
Keyhole Limpet Hemocyanin ◽  
Cell Extract ◽  
Nippostrongylus Brasiliensis ◽  
Spleen Cells ◽  
Ige Antibody ◽  
Third Stage

SJL mice were immunized with 1 microng dinitrophenylated keyhole limpet hemocyanin in 1 mg Al(OH)3. The mice were infected 21 days later with 750 third stage larvae of Nippostrongylus brasiliensis. On day 35, 14 days after infection, they were injected with 1 microng DNP-N, brasiliensis extract (Nb) in 1 mg Al(OH)3. In order to obtain high titer and persistent anti-DNP IgE antibody the mice were irradiated (540 R) 1 day after injection of DNP-Nb. Suppression of anti-DNP IgE antibody production was induced by spleen cells from normal SJL mice. Suppression of IgE antibody response is also obtained by an extract from normal SJL spleen cells. The suppressor substance from normal SJL spleen cell extract is a heat-labile protein, and is not absorbed by anti-mouse immunoglobulin. The mol wt of this substance is larger than 300,000 daltons as determined by gel filtration on Sephadex G-200, but after ultracentifugation, the supernate still has suppressive activity on IgE antibody production.

scholarly journals A characterization of cytostatic factor activity from Xenopus eggs and c-mos-transformed cells.

1991 ◽  
Vol 114 (2) ◽  
pp. 329-335 ◽  
Author(s):  
I Daar ◽  
R S Paules ◽  
G F Vande Woude
Keyword(s):  
Xenopus Oocytes ◽  
Constitutive Expression ◽  
Germinal Vesicle ◽  
Cell Extract ◽  
3T3 Cells ◽  
Cytostatic Factor ◽  
Nih 3T3 ◽  
Hand Mouse ◽  
Nih 3T3 Cells

In Xenopus oocytes, the mos proto-oncogene product is required during meiosis I for the activation of maturation promoting factor (MPF) and the subsequent breakdown of the germinal vesicle (GVBD). In addition, the mos product has been shown to be a candidate "initiator" of meiotic maturation and is an active component of cytostatic factor (CSF), an activity responsible for metaphase II arrest. Here we demonstrate that pp39mos is required throughout oocyte maturation. We found that in progesterone stimulated oocytes, depletion of mos RNA immediately before GVBD terminally decreased MPF. Likewise, oocytes depleted of mos RNA and induced to mature with crude MPF proceeded through GVBD but lacked the MPF activity required to arrest mature oocytes at metaphase II. Thus, during maturation the mos product is required, directly or indirectly, to sustain MPF activity. On the other hand, mouse NIH/3T3 cells transformed by the constitutive expression of pp39mosxc possessed CSF activity but lacked constitutive levels of MPF or its associated histone H1 kinase activity. Moreover, cytosols prepared from transformed NIH/3T3 cells or Xenopus eggs had similar levels of CSF activity, but pp39mos levels were greater than 40-fold higher in the transformed cell extract. These analyses show that maintenance of CSF during interphase does not result in the maintenance of MPF.

Functional characterization of the putative FAD synthase from Mycoplasma hyopneumoniae

2021 ◽  
Vol 368 (3) ◽  
Author(s):  
Amanda Malvessi Cattani ◽  
Camila Vieira Pinheiro ◽  
Irene Silveira Schrank ◽  
Franciele Maboni Siqueira
Keyword(s):  
Drug Targets ◽  
Functional Characterization ◽  
3D Structure ◽  
Mycoplasma Hyopneumoniae ◽  
Cell Extract ◽  
Enzymatic Assays ◽  
Cell Extracts ◽  
Potential Drug Targets

ABSTRACT In bacteria, the biosynthesis of the cofactor flavin adenine dinucleotide (FAD), important in many physiological responses, is catalyzed by the bifunctional enzyme FAD synthase (FADSyn) which converts riboflavin into FAD by both kinase and adenylylation activity. The in silico 3D structure of a putative FADSyn from Mycoplasma hyopneumoniae (MhpFADSyn), the etiological agent of enzootic pneumonia was already reported, nevertheless, the in vitro functional characterization was not yet demonstrated. Our phylogenetic analysis revealed that MhpFADSyn is close related to the bifunctional FADSyn from Corynebacterium ammoniagenes. However, only the domain related to adenylylation was assigned by InterPro database. The activity of MhpFADSyn was evaluated through in vitro enzymatic assays using cell extracts from IPTG-inducible heterologous expression of MhpFADSyn in Escherichia coli. The flavoproteins were analyzed by HPLC and results showed that IPTG-induced cell lysate resulted in the formation of twofold increased amounts of FAD if compared to non IPTG-induced cells. Consumption of riboflavin substrate was also threefold greater in IPTG-induced lysate compared to non IPTG-induced cell extract. Thus, the recombinant MhpFADSyn protein could be associated to FAD biosynthesis. These findings contribute to expand the range of potential drug targets in diseases control and unveil metabolic pathways that could be attribute to mycoplasmas.

scholarly journals Heterologous Expression and Characterization of the Manganese-Oxidizing Protein from Erythrobacter sp. Strain SD21

2014 ◽  
Vol 80 (21) ◽  
pp. 6837-6842 ◽  
Author(s):  
Katherine Nakama ◽  
Michael Medina ◽  
Ahn Lien ◽  
Jordan Ruggieri ◽  
Krystle Collins ◽  
...  
Keyword(s):  
Calcium Binding ◽  
Sequence Similarity ◽  
Cell Extract ◽  
Binding Domain ◽  
Full Length ◽  
Truncated Protein ◽  
Content Type ◽  
Heme Peroxidase ◽  
Calcium Binding Domain

ABSTRACTThe manganese (Mn)-oxidizing protein (MopA) fromErythrobactersp. strain SD21 is part of a unique enzymatic family that is capable of oxidizing soluble Mn(II). This enzyme contains two domains, an animal heme peroxidase domain, which contains the catalytic site, followed by a C-terminal calcium binding domain. Different from the bacterial Mn-oxidizing multicopper oxidase enzymes, little is known about MopA. To gain a better understanding of MopA and its role in Mn(II) oxidation, the 238-kDa full-length protein and a 105-kDa truncated protein containing only the animal heme peroxidase domain were cloned and heterologously expressed inEscherichia coli. Despite having sequence similarity to a peroxidase, hydrogen peroxide did not stimulate activity, nor was activity significantly decreased in the presence of catalase. Both pyrroloquinoline quinone (PQQ) and hemin increased Mn-oxidizing activity, and calcium was required. TheKmfor Mn(II) of the full-length protein in cell extract was similar to that of the natively expressed protein, but theKmvalue for the truncated protein in cell extract was approximately 6-fold higher than that of the full-length protein, suggesting that the calcium binding domain may aid in binding Mn(II). Characterization of the heterologously expressed MopA has provided additional insight into the mechanism of bacterial Mn(II) oxidation, which will aid in understanding the role of MopA and Mn oxidation in bioremediation and biogeochemical cycling.

Sign in / Sign up

Export Citation Format

Share Document