Purification and properties of the S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B

The S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B, was separated from other alkylsulphohydrolases and purified to homogeneity. Under the experimental conditions used the enzyme completely hydrolysed d-octan-2-yl sulphate (d-1-methylheptyl sulphate), but showed no activity towards the corresponding l-isomer. Additional evidence has been obtained to indicate that it is probably optically stereospecific for d-secondary alkyl sulphate esters with the ester sulphate group at C-2 and with a chain length of at least seven carbon atoms. Enzyme activity towards racemic samples of heptan-2-yl sulphate (1-methylhexyl sulphate), octan-2-yl sulphate and decan-2-yl sulphate (1-methylnonyl sulphate) increased with increasing chain length. l-Octan-2-yl sulphate is a competitive inhibitor of the enzyme, as are certain primary alkyl sulphates and primary alkanesulphonates. Inhibition by each of the last two types of compounds is characteristic of the behaviour of an homologous series. Inhibition increases with increasing chain length and plots of log K(i) values against the number of carbon atoms in each alkyl chain show the expected linear relationship. A crude preparation of the S2 secondary alkylsulphohydrolase was used to show that this particular enzyme hydrolyses l-octan-2-yl sulphate, but is probably inactive towards the corresponding d-isomer. The similarity of the S1 and S2 enzymes to the CS2 and CS1 enzymes respectively of Comamonas terrigena was established, and some comments have been made on the possible roles of these and other alkylsulphohydrolases in the biodegradation of detergents.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.     

Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B.

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000--46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C--O bond of the C--O--S ester linkage was confirmed by 18O-incorporation studies.     

Purification, properties and cellular localization of the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena.

The availability of homogeneous samples of the potassium salts of L- and D-octan-2-yl sulphate has enabled the separation of the optically stereospecific CS1 and CS2 secondary alkysulphohydrolases from extracts of cells of Comamonas terrigena. The CS2 enzyme was purified to homogeneity, and an initial study was made of its general properties, specificity, cellular localization and relationship to the CS1 enzyme. The CS2 enzyme has a molecular weight of approx. 250000 and a subunit size of approx. 58000, indicating that the molecule is a tetramer. Under the experimental conditions used the enzyme appears to be specific for (+)-secondary alkyl sulphate esters with the sulphate group at C-2 and with a chain length of at least six carbons. Enzyme activity towards racemic C-2 sulphates increases with increasing chain length up to C10, and there is some indirect evidence to suggest that activity declines when that chain length is exceeded. Other indirect evidence confirms that the CS1 enzyme exhibits similar specificity, except that only (-)-isomers can serve as substrates. Both enzymes are present in broth-grown stationary-phase cells of C. terrigena in approximately equal amounts.     

Purification and properties of phage P22 c2 repressor.

The c2 repressor of phage P22 has been purified to homogeneity. It specifically binds to lambdaimm21 and P22 DNA. Its affinity for the presumed operator mutant P22 virB is reduced. The initial dissociation rates of the complex between c2 repressor and lambdaimm21 DNA are 0.02 min-1 at 0 degrees C, 0.08 min-1 at 20 degrees C and 0.17 min-1 at 32 degrees C. The dissociation rates of complexes formed between the c2 repressor and the lambdaimm21 operators OR, OL and OR vira were measured and compared to the corresponding rates obtained with 21 cI repressor.     

P2 nucleotide receptors on C2C12 satellite cells

In developing muscle cells environmental stimuli transmitted by purines binding to the specific receptors are crucial proliferation regulators. C2C12 myoblasts express numerous purinergic receptors representing both main classes: P2X and P2Y. Among P2Y receptors we have found the expression of P2Y(1), P2Y(2), P2Y(4), P2Y(6) and P2Y(12) family members while among P2X receptors P2X(4), P2X(5) and P2X(7) were discovered. We have been able to show that activation of those receptors is responsible for ERK class kinase activity, responsible for regulation of cell proliferation pathway. We have also demonstrated that this activity is calcium dependent suggesting Ca(2+) ions as secondary messenger between receptor and kinase regulatory system. More specifically, we do suspect that in C2C12 myoblasts calcium channels of P2X receptors, particularly P2X(5) play the main role in proliferation regulation. In further development of myoblasts into myotubes, when proliferation is gradually inhibited, the pattern of P2 receptors is changed. This phenomenon is followed by diminishing of the P2Y(2)-dependent Ca(2+) signaling, while the mRNA expression of P2Y(2) receptor reminds still on the high level. Moreover, P2X(2) receptor mRNA, absent in myoblasts appears in myotubes. These data show that differentiation of C2C12 cell line satellite myoblasts is accompanied by changes in P2 receptors expression pattern.     

Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus.

A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.     

Purification and properties of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase from a thermophilic bacterium.

1. A stable ATPase complex with sensitivity to dicyclohexylcarbodiimide (TFo-F1) was purified from the membranes of the thermophilic aerobic bacterium PS3, by ion exchange chromatography in the presence of Triton X-100. 2. The ATPase of TFo-F1 was maximal at 70 degrees at pH 8.6 and was stable after monomerization in 4 M urea and 0.5% Triton X-100 at 25 degrees. The activity was dependent on Mg2+, Co2+, or Mn2+, and it became insensitive to dicyclohexylcarbodiimide when Ca2+ or Cd2+ was added instead. 3. TFo-F1 required P-lipids of this bacterium contained branched fatty acyl groups but no unsaturated groups and were stable against oxidation and heat. 4. Studies by electron microscopy, gel electrophoresis, and use of anti-ATPase antibody and [3H]acetyl-ATPase indicated that the TFo-F1 complex was composed of an ATPase moiety (TF1, five different subunits) and a hydrophobic moiety (TFo, three different subunits. TFo conferred TF1 with sensitivity to dicyclohexylcarbodiimide. 5. Vesicles catalyzing 32Pi-ATP exchange and ATP-driven enhancement of fluorescence of anilinonaphthalene sulfonate were reconstituted by dialyzing pure TFo-F1 and P-lipids together, and were active even at 50-75 degrees. The vesicles reconstituted from TFo-F1 and bacterial P-lipids were more stable than those reconstituted from TFo-F1 and soybean P-lipids.     

Purification and properties of human B cell-activating factor.

Human monocytes, when appropriately stimulated in vitro, release into the culture medium a factor (BAF) that stimulates the IgM response of T-depleted murine splenocytes to heterologous erythrocytes. The behavior of this factor on gel filtration, isoelectric focusing, ion exchange chromatography, and isopycnic centrifugation was studied. BAF appears to be a molecule of 15,000 daltons, pI 6.5, 1.33 g/ml with low solubility at low ionic strength. It is stable to acid, mild heating, and long-term storage. Activity is lost in alkali or by boiling. Papain may reduce BAF activity slightly, whereas trypsin and chymotrypsin have no significant effect. These properties are similar to those of other monokines reported to have a similar m.w.     

Immobilization of the surfactant-degrading bacterium Pseudomonas C12B in polyacrylamide gel. II. Optimizing SDS-degrading activity and stability

Conditions were established for optimizing the surfactant (SDS)-degrading activity of Pseudomonas C12B immobilized in polyacrylamide gel beads. Optimum activity was obtained by using immobilized cells derived from stationary phase of batch cultures and incubating with SDS at 30°C at pH 6.5. Half-saturation of the degradation system was achieved at an SDS concentration of 0.23 m . Biocatalyst stability was highest for beads maintained in basal salts medium, retaining 91% of initial activity after 161 d. In Tris/HCl buffer or distilled water, the stability was much lower, although in all cases the stability of immobilized cells was higher than that of free cells under equivalent conditions. Biocatalyst beads “inactivated” by sequential incubation in three batches of distilled water containing only SDS could be reactivated by transferring beads to nutrient medium. Beads packed in a glass column and operated in a continuous up-flow mode using SDS/basal salts eluant produced 100% hydrolysis when run at retention times above 60 min. The system was highly stable in the continuous flow mode; when operated at a residence time of 55 min (initially giving 98% degradation), the extent of degradation decreased only slightly to 93% over a continuous operation period of 3 weeks.     

A comparative study on the B12N12, Al12N12, B12P12 and Al12P12 fullerene-like cages

The stability, geometry and electronic structure of the title nanoclusters were compared by using density functional theory (DFT) calculations. Their electrical property analysis showed that the relative magnitude of the HOMO-LUMO gaps (eV) that are average values from the calculated results with five different DFT functionals is as follows: B12N12(7:02)>Al12N12(4.09)>B12P12(3.80)>Al12P12(3.39). Computing the standard enthalpy and the Gibbs free energy of formation, it was found that the B(12)N(12) structure is thermodynamically stable at 298 K and 1 atmosphere of pressure, while the Al(12)N(12) structure may be stable at low temperatures. Due to positive values of change of enthalpy and entropy of formation for both the B(12)P(12) and Al(12)P(12) clusters, it seems that their formation from the consisting atoms is not spontaneous at any temperature.     

Purification and characterization of the heat-labile alpha-amylase secreted by the psychrophilic bacterium TAC 240B.

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment.     

Purification and properties of two ferredoxins from the nitrogen-fixing bacterium Bacillus polymyxa

Two ferredoxins, designated FdI and FdII, have been isolated from the nitrogen-fixing bacterium Bacillus polymyxa. The two ferredoxins were readily separated on DEAE-cellulose and disc gel electrophoresis. The amino acid compositions of FdI and FdII showed them to be different protein species; the greater number of acidic amino acid residues in FdI than in FdII appears to account for separation based on electronic charge. FdI and FdII were both found to have four nonheme iron and four acid-labile sulfur groups per mole. The absorption spectra of the two ferredoxins are almost identical, with a peak in the visible region of the spectrum at 385 nm. The $\text{A}{}_{358}\text{A}{}_{280}$ absorbance ratio of both ferredoxins was 0.54–0.55. FdII was not stable under aerobic conditions, as indicated by a decrease in the visible region of the spectrum. Both FdI and FdII have nearly identical molecular weights, as judged by gel filtration and amino acid composition (approx 8800).The purified ferredoxins catalyzed the photoreduction of NADP by spinach chloroplasts with equal effectiveness. In the nitrogen-fixation reaction of B. polymyxa, FdII was more effective than FdI.     

Purification and properties of cytochrome c-556 from Agrobacterium tumefaciens B2a.

Cytochrome c-556 from Agrobacterium mefaciens B2a was isolated in a pure, homoneous state. The best purification procedure volved ammonium sulphate fractionation, delting on Sephadex G-25, column chromatographic fractionation on DEAE- and CM-cellulose, and gel filtration on Sephadex G-75 superfine. Substitution of the CM-cellulose step by isoelectric focusing was successful. The purity of the final preparation is warranted by the purity index value, the electrophoretic patterns in the absence and presence of sodium dodecylsulphate, the sedimentation profile and the N-terminal amino acid analysis (alanine). The absorption spectrum of reduced cytochrome c-556 has maxima at 318, 419, 526 and 555.5 nm. The molar extinction coefficient for the alpha-band is 20 200M-1cm-1. The isoelectric point, determined both by preparative and analytical isoelectric focusing, is 5.55 +/- 0.10. The molecular weight of cytochrome c-556 was determined by gel filtration as 12000 and by dodecylsulphate gel electrophoresis as 11 500.     

On the photosynthetic properties of marine bacterium COL2P belonging to Roseobacter clade

Aerobic anoxygenic phototrophs (AAPs) are prokaryotic microorganisms capable of harvesting light using bacteriochlorophyll-based reaction centres. Marine AAP communities are generally dominated by species belonging to the Roseobacter clade. For this reason, we used marine Roseobacter-related strain COL2P as a model organism to characterize its photosynthetic apparatus, level of pigmentation and expression of photosynthetic complexes. This strain contained functional photosynthetic reaction centres with bacteriochlorophyll a and spheroidenone as the main light-harvesting pigments, but the expression of the photosynthetic apparatus was significantly reduced when compared to truly photoautotrophic species. Moreover, the absence of peripheral light-harvesting complexes largely reduced its light-harvesting capacity. The size of the photosynthetic unit was limited to 35.4 ± 1.0 BChl a molecules supplemented by the same number of spheroidenone molecules. The contribution of oxidative phosphorylation and photophosphorylation was analysed by respiration and fluorometric measurements. Our results indicate that even with a such reduced photosynthetic apparatus, photophosphorylation provides up to three times higher electron fluxes than aerobic respiration. These results suggest that light-derived energy can provide a substantial fraction of COL2P metabolic needs.