CLONING, EXPRESSION, BIOCHEMICAL PARTIAL CHARATERIZATION AND MODELING OF ESTERASE FROM ACINETOBACTER CALCOACETICUS SP DSM587

2013 ◽  
Vol 6 ◽  
Author(s):  
Inmaculada Navarro-González
Keyword(s):  
Molecular Mass ◽  
Acyl Chain ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Catalytic Triad ◽  
Maximum Activity ◽  
Structure Modeling ◽  
Gel Filtration Chromatography ◽  
Aminoacid Sequence ◽  
Chain Lengths

The aim of this paper has been to clone, express, purify and characterization the EstB from<br />Acinetobacter calcoaceticus encoded by the gen X8895. The esterase was cloned in Pet28a and<br />partially purified. The molecular mass of the purified enzyme was 36 kDa (by SDS) and 68.9<br />KDa (by gel filtration chromatography). The EstB showed a maximum activity at 35ºC and pH 8<br />and towards shorter acyl chain lengths (PNPA, PNPB, PNPC) and showed activity about Smethyltiobutanoate,<br />too. The catalytic triad has been predicted by aminoacid sequence<br />alignment and the structure modeling was performed used esterase 1QoR as template.

scholarly journals Purification and Characterization of 1-Naphthol-2-Hydroxylase from Carbaryl-Degrading Pseudomonas Strain C4

2007 ◽  
Vol 189 (7) ◽  
pp. 2660-2666 ◽  
Author(s):  
Vandana P. Swetha ◽  
Aditya Basu ◽  
Prashant S. Phale
Keyword(s):  
Molecular Mass ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Substrate Inhibition ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sole Source ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Phenol Derivatives

ABSTRACT Pseudomonas sp. strain C4 metabolizes carbaryl (1-naphthyl-N-methylcarbamate) as the sole source of carbon and energy via 1-naphthol, 1,2-dihydroxynaphthalene, and gentisate. 1-Naphthol-2-hydroxylase (1-NH) was purified 9.1-fold to homogeneity from Pseudomonas sp. strain C4. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme is a homodimer with a native molecular mass of 130 kDa and a subunit molecular mass of 66 kDa. The enzyme was yellow, with absorption maxima at 274, 375, and 445 nm, indicating a flavoprotein. High-performance liquid chromatography analysis of the flavin moiety extracted from 1-NH suggested the presence of flavin adenine dinucleotide (FAD). Based on the spectral properties and the molar extinction coefficient, it was determined that the enzyme contained 1.07 mol of FAD per mol of enzyme. Although the enzyme accepts electrons from NADH, it showed maximum activity with NADPH and had a pH optimum of 8.0. The kinetic constants Km and V max for 1-naphthol and NADPH were determined to be 9.6 and 34.2 μM and 9.5 and 5.1 μmol min−1 mg−1, respectively. At a higher concentration of 1-naphthol, the enzyme showed less activity, indicating substrate inhibition. The Ki for 1-naphthol was determined to be 79.8 μM. The enzyme showed maximum activity with 1-naphthol compared to 4-chloro-1-naphthol (62%) and 5-amino-1-naphthol (54%). However, it failed to act on 2-naphthol, substituted naphthalenes, and phenol derivatives. The enzyme utilized one mole of oxygen per mole of NADPH. Thin-layer chromatographic analysis showed the conversion of 1-naphthol to 1,2-dihydroxynaphthalene under aerobic conditions, but under anaerobic conditions, the enzyme failed to hydroxylate 1-naphthol. These results suggest that 1-NH belongs to the FAD-containing external flavin mono-oxygenase group of the oxidoreductase class of proteins.

scholarly journals Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

1996 ◽  
Vol 314 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Antonio del CASTILLO-OLIVARES ◽  
Miguel A. MEDINA ◽  
Ignacio NÚÑEZ de CASTRO ◽  
Javier MÁRQUEZ
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ammonium Sulphate Precipitation ◽  
Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

scholarly journals Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain

1990 ◽  
Vol 270 (3) ◽  
pp. 715-719 ◽  
Author(s):  
A Höer ◽  
D Höer ◽  
E Oberdisse
Keyword(s):  
Enzyme Activity ◽  
Molecular Mass ◽  
Kinetic Data ◽  
Gel Filtration ◽  
Reaction Products ◽  
Inhibitory Effects ◽  
Gel Filtration Chromatography ◽  
Pyrimidine Nucleotides ◽  
High Affinity ◽  
Marked Inhibition

We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.

scholarly journals Purification and characterization of benzaldehyde dehydrogenase I from Acinetobacter calcoaceticus

1989 ◽  
Vol 263 (3) ◽  
pp. 913-919 ◽  
Author(s):  
R M Chalmers ◽  
C A Fewson
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Acinetobacter Calcoaceticus ◽  
Polyacrylamide Gel ◽  
Prosthetic Group ◽  
Gel Filtration Chromatography ◽  
Ph Optimum ◽  
Benzaldehyde Dehydrogenase

Benzaldehyde dehydrogenase I was purified from Acinetobacter calcoaceticus by DEAE-Sephacel, phenyl-Sepharose and f.p.l.c. gel-filtration chromatography. The enzyme was homogeneous and completely free from the isofunctional enzyme benzaldehyde dehydrogenase II, as judged by denaturing and non-denaturing polyacrylamide-gel electrophoresis. The subunit Mr value was 56,000 (determined by SDS/polyacrylamide-gel electrophoresis). Estimations of the native Mr value by gel-filtration chromatography gave values of 141,000 with a f.p.l.c. Superose 6 column, but 219,000 with Sephacryl S300. Chemical cross-linking of the enzyme subunits indicated that the enzyme is tetrameric. Benzaldehyde dehydrogenase I was activated more than 100-fold by K+, Rb+ and NH4+, and the apparent Km for K+ was 11.2 mM. The pH optimum in the presence of K+ was 9.5 and the pI of the enzyme was 5.55. The apparent Km values for benzaldehyde and NAD+ were 0.69 microM and 96 microM respectively, and the maximum velocity was approx. 110 mumol/min per mg of protein. Various substituted benzaldehydes were oxidized at significant rates, and NADP+ was also used as cofactor, although much less effectively than NAD+. Benzaldehyde dehydrogenase I had an NAD+-activated esterase activity with 4-nitrophenol acetate as substrate, and the dehydrogenase activity was inhibited by a range of thiol-blocking reagents. The absorption spectrum indicated that there was no bound cofactor or prosthetic group. Some of the properties of the enzyme are compared with those of other aldehyde dehydrogenases, specifically the very similar isofunctional enzyme benzaldehyde dehydrogenase II from the same organism.

scholarly journals Human platelet heparanase: purification, characterization and catalytic activity

1998 ◽  
Vol 330 (3) ◽  
pp. 1341-1350 ◽  
Author(s):  
Craig FREEMAN ◽  
R. Christopher PARISH
Keyword(s):  
Molecular Mass ◽  
Tumour Cell ◽  
Human Platelet ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Cell Aggregation ◽  
Capillary Endothelium ◽  
Gel Filtration Chromatography ◽  
Average Molecular Mass ◽  
Tumour Metastasis

Heparan sulphate (HS) is an important component of the extracellular matrix (ECM) and the vasculature basal lamina (BL) which functions as a barrier to the extravasation of metastatic and inflammatory cells. Platelet-tumour cell aggregation at the capillary endothelium results in activation and degranulation of platelets. Cleavage of HS by endoglycosidase or heparanase activity produced in relatively large amounts by the platelets and the invading cells may assist in the disassembly of the ECM and BL, and thereby facilitate cell migration. Using a recently published rapid, quantitative assay for heparanase activity towards HS [Freeman, C. and Parish, C. R. (1997), Biochem. J., 325, 229-237], human platelet heparanase has now been purified 1700-fold to homogeneity in 19% yield by a five column procedure, which consists of concanavalin A-Sepharose, Zn2+-chelating-Sepharose, Blue A-agarose, octyl-agarose and gel filtration chromatography. The enzyme, which was shown to be an endoglucuronidase that degrades both heparin and HS, has a native molecular mass of 50 kDa when analysed by gel filtration chromatography and by SDS/PAGE. Platelet heparanase degraded porcine mucosal HS in a stepwise fashion from a number average molecular mass of 18.5 to 13, to 8 and finally to 4.5 kDa fragments as determined by gel filtration analysis. Bovine lung heparin was degraded from 8.9 to 4.8 kDa while porcine mucosal heparin was degraded from 8.1 kDa to 3.8 and finally to 2.9 kDa fragments. Studies of the enzyme's substrate specificity using modified heparin analogues showed that substrate cleavage required the presence of carboxyl groups, but O- and N-sulphation were not essential. Inhibition studies demonstrated an absolute requirement for the presence of O-sulphate groups. Platelet heparanase was inhibited by heparin analogues which also inhibited tumour heparanase, suggesting that sulphated polysaccharides which inhibit tumour metastasis may act to prevent both tumour cell and platelet heparanase degradation of endothelial cell surface HS and the basal laminar.

scholarly journals Characterization of a Novel Thermostable Carboxylesterase from Geobacillus kaustophilus HTA426 Shows the Existence of a New Carboxylesterase Family

2009 ◽  
Vol 191 (9) ◽  
pp. 3076-3085 ◽  
Author(s):  
Silvia Montoro-García ◽  
Irene Martínez-Martínez ◽  
José Navarro-Fernández ◽  
Hideto Takami ◽  
Francisco García-Carmona ◽  
...  
Keyword(s):  
Acyl Chain ◽  
Three Dimensional ◽  
Catalytic Triad ◽  
Dimensional Structure ◽  
E Coli ◽  
Optimum Growth Temperature ◽  
Geobacillus Kaustophilus ◽  
Chain Lengths

ABSTRACT The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65°C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new α/β hydrolase family different from IV and VI.

scholarly journals Analysis of serum lipoproteins by high performance gel filtration chromatography.

1996 ◽  
Vol 45 (1) ◽  
pp. 103-106 ◽  
Author(s):  
Takashi KITAMURA ◽  
Seiji ITO ◽  
Yoshio KATO ◽  
Keiko SASAMOTO ◽  
Mitsuyo OKAZAKI
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Serum Lipoproteins ◽  
Gel Filtration Chromatography

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

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