Purification, properties and induction of a specific benzoate-4-hydroxylase from Aspergillus niger (UBC 814)

An inducible benzoate-4-hydroxylase has been partially purified from crude extracts of the mycelial felts of Aspergillus niger. This enzyme catalyzes the transformation of benzoate to p-hydroxybenzoate with equimolar consumption of NADPH and O2. It requires tetrahydropteridine as a prosthetic group. The optimum activity was found at pH 6.2 with a Km value at 30°C of 1.6 · 10−4 M for NADPH and 1.3 · 10−4 M for benzoate. Fe2+ (iron) is required for the enzyme activity. The enzyme is stabilized by the inclusion of benzoate, EDTA and glutathione in the extracting buffer. The enzyme is specific for benzoate as substrate. Sulfhydryl group(s) are essential for enzyme activity as indicated by p-chloromercuri-benzoate and N-ethylmaleimide inactivation. Benzoate-4-hydroxylase activity is decreased in the mycelial felts of Aspergillus niger grown in the presence of higher concentrations of benzoate. Maximum activity of the enzyme was observed at 36 h after inoculation.

Rotational cut-off and anharmonicity effects on thermodynamic properties of gases at high temperatures

The exact expressions for the partition function (Q) and the coefficient of specific heat at constant volume (Cv) for a rotating-anharmonic oscillator molecule, including coupling and rotational cut-off, have been formulated and values of Q and Cv have been computed in the temperature range of 100 to 100,000 K for O2, N2 and H2 gases. The exact Q and Cv values are also compared with the corresponding rigid-rotator harmonic-oscillator (infinite rotational and vibrational levels) and rigid-rotator anharmonic-oscillator (infinite rotational levels) values. The rigid-rotator harmonic-oscillator approximation can be accepted for temperatures up to about 5000 K for O2 and N2. Beyond these temperatures the error in Cv will be significant, because of anharmonicity and rotational cut-off effects. For H2, the rigid-rotator harmonic-oscillator approximation becomes unacceptable even for temperatures as low as 2000 K.

Purification and properties of an NADP+-specific isocitrate dehydrogenase from Rhizobium meliloti

An NADP+-specific isocitrate dehydrogenase has been purified and characterized from Rhizobium meliloti. The enzyme showed Mn++ or Mg++ requirement. The apparent Km values were 2.00×10-5 m and 1.51×10-5 m for dl-isocitrate and NADP+, respectively. The enzyme was inhibited by ATP, to a lesser extent by ADP and AMP. agr-Ketoglutarate also inhibited the enzyme activity. Oxalacetate and glyoxylate together inhibited the enzyme activity. The inhibition was competitive. Studies with thiol inhibitors suggested that the enzyme contained a sulfhydryl group at or near the active site. The enzyme has an approximate molecular weight of 60 000. Fluorescence studies suggested that the enzyme contained tryptophan.

Specific polyadenylation and purification of total messenger RNA from Escherichia coli

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages, We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese, The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA, Withdrawal of Mg2+ from the polyadenylation reaction rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes, Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species, Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis, Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Purification and characterization of assimilatory nitrite reductase from Candida utilis

Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

An NAD(P)(+)-dependent secondary-alcohol dehydrogenase of Alcaligenes eutrophus: Purification, characterization and its application for the production of chiral alcohols

A soil micro-organism identified as Alcaligenes eutrophus capable of utilizing nerolidol, a sesquiterpene alcohol as the sole source of carbon, contains an inducible NAD(P)(+)-linked secondary-alcohol dehydrogenase (SADH), The enzyme was purified 252-fold from crude cell-free extract by a combination of salt precipitation, ion-exchange and affinity-matrix chromatography, Native and SDS/PAGE PAGE of the purified enzyme showed a single protein band and the enzyme appears to be a homotetramer having an apparent molecular mass of 139 kDa comprising four identical subunits of 38.5 kDa, The isoelectric point (pi) of SADH was determined to be 6.2, Depending on pH of the reaction media, the enzyme carried out both oxidation and reductions of various terpenoids and steroids, At pH 5.5, the enzyme catalysed the stereospecific reduction of prochiral ketones to optically active (S)-alcohols and the oxidation reaction was predominated over the former at pH 9.5, NADP(+) and NADPH were respectively preferred over NAD(+) and NADH for oxidation and reduction reactions, The K-m values for testosterone, NADP(+) and NAD(+) were 11.8, 55.6, and 122 mu M respectively, Neither enzyme was significantly inhibited by metal-binding agents, but some thiol-blocking compounds inhibited it, SADH tolerates moderate concentrations of water-miscible organic solvents such as ethanol, methanol, acetone and dioxan, Some of the properties of this enzyme were found to be significantly different from those thus far described.

Purification and properties of protocatechuate-3,4-dioxygenase from Tecoma stans L

Protocatechuate-3,4-dioxygenase from the leaves of Tecoma stans was purified to near homogeneity and some of its properties studied. It was optimally active at pH 5.2 and at 40°C. Its molecular weight of approx. 150 000 was determined by gel filtration on a Sephadex G-150 column. The Km value for protocatechuate was found to be 330 μM and for ferrous sulfate, 40 μM. The enzyme was highly specific for protocatechuate and did not attack any of the substrate analogues. None of the substrate analogues tested inhibited the enzyme activity. Sulfhydryl reagents inhibited the enzyme activity which could be partially reversed by sulfhydryl compounds. The dioxygenase activity was not associated with polyphenol oxidase activity.

Purification and some of the properties of a novel secondary alcohol dehydrogenase from Alcaligenes eutrophus

Alcaligenes eutrophus utilizing nerolidol, a sesquiterpene alcohol,as the sole source of carbon contains an inducible NAD(P)+-linked secondary alcohol dehydrogenase (SADH). The enzyme was purified to homogeneity by a combination of salt precipitation, ion exchange and affinity matri chromatographies. The apparent molecular mass of the enzyme was estimated to be 139 KDa with four identical subunits of 38.5 KDa. The enzyme carried out both oxidation and reduction reactions. At pH 5.5, enzyme catalyzed the stereospecific reduction of prochiral ketones to secondary alcohols. The pH optimum for the oxidation reaction was 9.5. NADP+ and NADPH were respectively preferred over NAD+ and NADH for oxidation and reduction reactions. Some of the properties of this enzyme were found to be significantly different from those thus far described.

Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.

Expression, purification and X-ray analysis of 1,3-propanediol dehydrogenase (Aq_1145) from Aquifex aeolicus VF5

1,3-Propanediol dehydrogenase is an enzyme that catalyzes the oxidation of 1,3-propanediol to 3-hydroxypropanal with the simultaneous reduction of NADP(+) to NADPH. SeMet-labelled 1,3-propanediol dehydrogenase protein from the hyperthermophilic bacterium Aquifex aeolicus VF5 was overexpressed in Escherichia coli and purified to homogeneity. Crystals of this protein were grown from an acidic buffer with ammonium sulfate as the precipitant. Single-wavelength data were collected at the selenium peak to a resolution of 2.4 angstrom. The crystal belonged to space group P3(2), with unit-cell parameters a = b = 142.19, c = 123.34 angstrom. The structure contained two dimers in the asymmetric unit and was solved by the MR-SAD approach.

Cloning, overexpression, folding and purification of a biosynthetically derived three disulfide scorpion toxin (BTK-2) from Mesobuthus tamulus

BTK-2, a 32 residue scorpion toxin initially identified in the venom of red Indian scorpion Mesobuthus tamulus was cloned, overexpressed and purified using Cytochrome 155 fusion protein system developed in our laboratory. The synthetic gene coding for the peptide was designed taking into account optimal codon usage by Escherichia coli. High expression levels of the fusion protein enabled facile purification of this peptide. The presence of disulfide bonded isomers, occurring as distinctly populated states even in the fusion protein, were separated by gel filtration chromatography. The target peptide was liberated from the host protein by Tev protease cleavage and subsequent purification was achieved using RP-HPLC methods. Reverse phase HPLC clearly showed the presence of at least two isomeric forms of the peptide that were significantly populated. The oxidative folding of BTK-2 was achieved under ambient conditions during the course of purification. Structural characterization of the two forms, by solution homonuclear and heteronuclear NMR methods, has shown that these two forms exhibit significantly different structural properties, and represent the natively folded and a "misfolded" form of the peptide. The formation of properly folded BTK-2 as a major fraction without the use of in vitro oxidative refolding methods clearly indicate the versatility of the Cytochrome b(5) fusion protein system for the efficient production of peptides for high resolution NMR studies.

Purification, characterization and molecular cloning of a monocot mannose-binding lectin from Remusatia vivipara with nematicidal activity

A mannose-binding lectin (RVL) was purified from the tubers of Remusatia vivipara, a monocot plant by single-step affinity chromatography on asialofetuin-Sepharose 4B. RVL agglutinated only rabbit erythrocytes and was inhibited by mucin, asialomucin, asialofetuin and thyroglobulin. Lectin activity was stable up to 80A degrees C and under wide range of pH (2.0-9.3). SDS-PAGE and gel filtration results showed the lectin is a homotetramer of Mr 49.5 kDa, but MALDI analysis showed two distinct peaks corresponding to subunit mass of 12 kDa and 12.7 kDa. Also the N-terminal sequencing gave two different sequences indicating presence of two polypeptide chains. Cloning of RVL gene indicated posttranslational cleavage of RVL precursor into two mature polypeptides of 116 and 117 amino-acid residues. Dynamic light scattering (DLS) and gel filtration studies together confirmed the homogeneity of the purified lectin and supported RVL as a dimer with Mr 49.5 kDa derived from single polypeptide precursor of 233 amino acids. Purified RVL exerts potent nematicidal activity on Meloidogyne incognita, a root knot nematode. Fluorescent confocal microscopic studies demonstrated the binding of RVL to specific regions of the alimentary-tract and exhibited a potent toxic effect on M. incognita. RVL-mucin complex failed to interact with the gut confirming the receptor mediated lectin interaction. Very high mortality (88%) rate was observed at lectin concentration as low as 30 A mu g/ml, suggesting its potential application in the development of nematode resistant transgenic-crops.

Indoleacetaldoxime hydro-lyase : II. Purification and properties

The purification and some properties of the enzyme indoleacetaldoxime hydrolyase (EC 4.2.1.29) from the fungus Gibberella fujikuroi, which dehydrates indoleacetaldoxime (IAOX) to indoleacetonitrile (IAN), are described. The enzyme activity in the fungus is present only under certain culture conditions. It is a soluble enzyme, has an optimum pH at 7, shows an energy of activation of —15,670 cal/mole, and has a Michaelis constant of 1.7 × 10−4 Image at 30 °. It appears to be specific for IAOX, and 1 mole of IAN is produced per mole of IAOX utilized. The enzyme is inhibited by a number of aldoximes of which phenylacetaldoxime (PAOX) is the most potent inhibitor. Inhibition by PAOX is competitive (Ki = 2.2 × 10−8 Image ). The enzyme is inhibited by SH reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide, and by a number of SH compounds such as cysteine, β-mercaptoethanol, and 2,3-dimercaptopropanol (BAL). However, glutathione activates the enzyme. Metal chelating agents such as 8-OH-quinoline and diethyl dithiocarbamate inhibit the enzyme; the inhibition is partly reversed by ferric citrate. Ascorbic acid, and particularly dehydroascorbic acid (DHA), are good activators of the enzyme. Several other biological oxidants had either no action or had a slight effect. Potassium cyanide activates the enzyme at low concentration but inhibits at higher concentrations. Reduction of the enzyme with NaBH4 reduces activity, and the effect is partly reversed by pyridoxal phosphate and also by DHA. The above properties indicate that both an SH function and an oxidized function are required for activity.

Purification and properties of uridine hydrolase from mung-bean (Phaseolus radiatus) seedlings

The occurrence in plants of an enzyme system catalyzing the cleavage of uridine has been demonstrated. The enzyme from Phaseolus radiatus was purified about 132-fold with 24% recovery by a combination of procedures involving mild acid treatment, ammonium sulphate fractionation, negative adsorption on calcium phosphate gel and DEAE-cellulose chromatography. The enzyme cleaves uridine to uracil and ribose in the absence of phosphate indicating that the mechanism of cleavage was hydrolytic rather than phosphorolytic. The enzyme is specific to uridine and does not act on other purine and pyrimidine compounds. The enzyme shows maximum activity at pH 7.4 and has a temperature optimum of 45 °. It does not require metal ions for activity. Inhibition of the enzyme by p-chloromercuribenzoate as well as N-ethylmaleimide and the reversal of p-chloromercuribenzoate inhibition by sulfhydryl agents indicate the probable involvement of readily oxidizable sulfhydryl groups in enzyme activity.

Studies on the metabolism of o-aminophenol purification and properties of isophenoxazine synthase from Bauhenia monandra

An enzyme which catalyzes the oxidative conversion of o-aminophenol to 2-amino-3-H-isophenoxazin-3-one has been purified 396-fold by using standard fractionation procedures. The enzyme is specific for o-aminophenol and has pH and temperature optima at 6.2 and 40 °, respectively. It is insensitive to metal chelating agents but is inhibited by several reducing substances. There is no cofactor or metal ion requirement for the reaction. A competitive type of inhibition was observed with structural analogs such as anthranilic acid and 3-hydroxyanthranilic acid. There are no free sulfhydryl groups in the enzyme, but preincubation of the enzyme with substrate or substrate analogs resulted in the liberation of titratable free sulfhydryl groups. The mechanism of biosynthesis of isophenoxazine ring is discussed.