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PubMed1985

Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments.

We have measured the 13C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D2O at pD 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D2O at pD 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The 13C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. We have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the 13C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.

Alcaligenes↗
PubMed1987

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Alcaligenes↗
PubMed1977

A 13C nuclear magnetic resonance study of gel-forming (1 goes to 3)-beta-d-glucans. Evidence of the presence of single-helical conformation in a resilient gel of a curdlan-type polysaccharide 13140 from Alcaligenes faecalis var. myxogenes IFO 13140.

A 13C nuclear magnetic resonance (NMR) study of a resilient gel of a curdlan-type polysaccharide 13140, a (1 goes to 3)-beta-D-glucan, from Alcaligenes faecalis var. myxogenes IFO 13140, was performed in an effort to understand the gel structure. It was found that very broad 13C resonance peaks of line widths ca. 150 (C-1-C-5) and 50 HZ (C-6) are able to be seen in the gel state. Because of a fivefold increase of the peak intensities with respect to those in an aqueous suspension by gelation, these peaks are unequivocally ascribed to certain regions relevant to the gel structure. Those 13 C NMR peaks, however, account for only 20-30 (as viewed from C-1-C-5) and 60% (from C-6) of the total gel, the peak areas of the rest being lost. With respect to those of the disordered low molecular weight acid degraded fraction, fraction II, and laminaran, downfield displacements of C-1, C-3, and C-4 signals are found to take place by amounts of 2.8, 3.2, and 0.9 ppm, respectively, while the remaining peaks (C-2, C-5, and C-6) are unchanged. In view of similar differences of chemical shifts between cyclodextrins and linear (1 goes to 4)-alpha-D-glucans (Colson, P., Jennings, H.J., and Smith, I.C.P. (1974), J. Am. Chem. Soc. 96, 8081-8087), the observed downfield 13C shifts of the glucosidic bonds in the gel are explained by the presence of the fixed conformation of the preferred dihedral angles, in which internal rotations around the glucosidic bonds are not allowed. Combined with the results of theoretical prediction and the downfield shift of C-4 signals, which is consistent with the presence of an O-4'...O-5 intramolecular hydrogen bond, the observed 13C peaks are ascribed to a region of single helical conformation, whereas the peak-loss portion of 13C NMR signals (70-80 and 40% from C-1 and C-6, respectively) is presumably ascribed to the multiple-helical junction zones for the gel structure and their vicinities. The variation of the line widths as well as the peak positions is also found to take place by stepwise addition of NaOH (greater than 0.22 M). The onset of the conformational transition, helix to random coil, is in good agreement with the change of viscosity, specific rotation, optical rotatory dispersion, and absorption maximum shift by complex formation with Congo Red reported by Ogawa et al. (Ogawa, K., Watanabe, T., Tsurugi, J., and Ono, S. (1972), Carbohydr. Res. 23, 399-405).

Alcaligenes↗
PubMed2001

Alternate substrate binding modes to two mutant (D98N and H255N) forms of nitrite reductase from Alcaligenes faecalis S-6: structural model of a transient catalytic intermediate.

High-resolution nitrite soaked oxidized and reduced crystal structures of two active site mutants, D98N and H255N, of nitrite reductase (NIR) from Alcaligenes faecalis S-6 were determined to better than 2.0 A resolution. In the oxidized D98N nitrite-soaked structures, nitrite is coordinated to the type II copper via its oxygen atoms in an asymmetric bidentate manner; however, elevated B-factors and weak electron density indicate that both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N delta 2 atom of Asn98 to form a hydrogen bond with the bound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIR structure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soaked H255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite binding site despite the lack of direct interaction with the substrate in the native enzyme. The resulting pentacoordinate copper site in the H255N structure also serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated to the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soaked D98N and H255N crystal structures may represent an evolutionary link between the mononuclear type I copper centers and dinuclear Cu(A) sites.

Alcaligenes↗
PubMed2002

Alcaligenes xylosoxidans dissimilatory nitrite reductase: alanine substitution of the surface-exposed histidine 139l ligand of the type 1 copper center prevents electron transfer to the catalytic center.

Nitrite reductase of Alcaligenes xylosoxidans contains three blue type 1 copper centers with a function in electron transfer and three catalytic type 2 copper centers. The mutation H139A, in which the solvent-exposed histidine ligand of the type 1 copper ion was changed to alanine, resulted in the formation of a colorless protein containing 4.4 Cu atoms per trimer. The enzyme was inactive with reduced azurin as the electron donor, and in contrast to the wild-type enzyme, no EPR features assignable to type 1 copper centers were observed. Instead, the EPR spectrum of the H139A enzyme, with parameters of g(1) = 2.347 and A(1) = 10 mT, was typical of type 2 copper centers. On the addition of nitrite, the EPR features developed spectral features with increased rhombicity, with g(1) = 2.29 and A(1) = 11 mT, arising from the type 2 catalytic site. As assessed by visible spectroscopy, ferricyanide (E degree = +430 mV) was unable to oxidize the H139A enzyme, and this required a 30-fold excess of K(2)IrCl(6) (E degree = +867 mV). Oxidation resulted in the EPR spectrum developing additional axial features with g(1) = 2.20 and A(1) = 9.5 mT, typical of type 1 copper centers. The oxidized enzyme after separation from the excess of K(2)IrCl(6) by gel filtration was a blue-green color with absorbance maxima at 618 and 420 nm. The instability of the protein prevented the precise determination of the midpoint potential, but these properties indicate that it is in the range 700-800 mV, an increase of at least approximately 470 mV compared with the native enzyme. This high potential, which is consistent with a trigonal planar geometry of the Cu ion, effectively prevents azurin-mediated electron transfer from the type 1 center to the catalytic type 2 Cu site. However, with dithionite as reductant, 20% of the activity of the wild-type enzyme was observed, indicating that the direct reduction of the catalytic site by dithionite can occur. When CuSO(4) was added to the crude extract before isolation of the enzyme, the Cu content of the purified H139A enzyme increased to 5.7 Cu atoms per trimer. The enzyme remained colorless, and the activity with dithionite as a donor was not significantly increased. The additional copper in such preparations was associated with an axial type 2 Cu EPR signal with g(1) = 2.226 and A(1) = 18 mT, and which were not changed by the addition of nitrite, consistent with the activity data.

Alanine↗
PubMed1996

Paramagnetic cobalt and nickel derivatives of Alcaligenes denitrificans azurin and its M121Q mutant. A 1H NMR study.

Using cobalt or nickel to replace copper in native azurin allows one to fingerprint the metal coordination site of the protein. The metal sites of wild type Alcaligenes denitrificans azurin and its M121Q mutant are clearly distinguishable through the paramagnetic 1H NMR spectra of the Ni(II) and Co(II) derivatives. In the wild type azurin, Gly45 coordinates to nickel or cobalt, while Met121 appears as a weak metal ligand. On the contrary, in the M121Q azurin mutant, the metal exhibits a clear preference for the Gln121, which coordinates through the side chain carbonyl oxygen, and Gly45 is not a ligand. Changes in the isotropic shifts and relaxation properties of signals from the Cys112, His46, and His117 metal ligands suggest a movement of the metal ion out of the equatorial plane, indicating that the metal site is tetrahedral. These effects are less pronounced in the Ni(II) M121Q azurin than in the Co(II) metalloderivative. The similarity between the NMR spectra of the Co(II) derivatives of stellacyanin and the M121Q azurin is in agreement with a very similar metal site in both proteins and supports the existence of a coordinated Gln in stellacyanin.

Alcaligenes↗
PubMed1999

Amino acid replacements at the H2-activating site of the NAD-reducing hydrogenase from Alcaligenes eutrophus.

The role of amino acid residues in the H(2)-activating subunit (HoxH) of the NAD-reducing hydrogenase (SH) from Alcaligenes eutrophus has been investigated by site-directed mutagenesis. Conserved residues in the N-terminal L1 (RGxE) and L2 (RxCGxCx(3)H) and the C-terminal L5 (DPCx(2)Cx(2)H/R) motifs of the active site-harboring subunit were chosen as targets. Crystal structure analysis of the [NiFe] hydrogenase from Desulfovibrio gigas uncovered two pairs of cysteines (motifs L2 and L5) as coordinating ligands of Ni and Fe. Glutamate (L1) and histidine residues (L2 and L5) were proposed as being involved in proton transfer [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E. C., Frey, M., and Fontecilla Camps, J. C. (1995) Nature 373, 580-587]. The A. eutrophus mutant proteins fell into three classes. (i) Replacement of the putative four metal-binding cysteines with serine led to the loss of H(2) reactivity and blocked the assembly of the holoenzyme. Exchange of Cys62, Cys65, or Cys458 was accompanied by the failure of the HoxH subunit to incorporate nickel, supporting the essential function of these residues in the formation of the active site. Although the fourth mutant of this class (HoxH[C461S]) exhibited nickel binding, the modified protein was catalytically inactive and unable to oligomerize. (ii) Mutations in residues possibly involved in proton transfer (HoxH[E43V], HoxH[H69L], and HoxH[H464L]) yielded Ni-containing proteins with residual low levels of hydrogenase activity. (iii) The most promising mutant protein (HoxH[R40L]), which was identified as a metal-containing tetrametric enzyme, was completely devoid of H(2)-dependent oxidoreductase activity but exhibited a remarkably high level of D(2)-H(+) exchange activity. These characteristics are compatible with the interpretation of a functional proton transfer uncoupled from the flow of electrons.

Alcaligenes↗
PubMed2001

Analysis of adsorption function of polyhydroxybutyrate depolymerase from Alcaligenes faecalis T1 by using a quartz crystal microbalance.

Enzymatic adsorption and degradation of three types of aliphatic polyester films and two types of polyolefin films by the extracellular PHB depolymerase from Alcaligenes faecalis T1 have been studied by using a quartz crystal microbalance (QCM) technique. Hydrolysis of poly[(R)-3-hydroxybutyrate] was quantitatively followed by the QCM technique. Adsorption of the enzymes to films was also quantitatively detected by the QCM. Kinetic study on enzymatic adsorption suggests that the PHB depolymerase binds to the substrates not only by hydrophobic interaction but also by specific interaction between the ester bonds of polyesters and the binding domain of the enzyme. The results show that the QCM technique is a sensitive tool to study enzymatic degradation kinetics of biodegradable polyesters.

Adsorption↗
PubMedDate not supplied

Biocatalytic transformation of [(2-Hydroxyethyl)thio]acetic acid and thiodiglycolic acid from thiodiglycol by Alcaligenes xylosoxydans ssp. xylosoxydans (SH91).

A Gram-negative bacterium, Alcaligenes xylosoxydans ssp. xylosoxydans (SH91), consumed thiodiglycol (TDG), the nontoxic hydrolysis product of sulfur mustard, as a primary carbon source and transformed TDG to commercially relevant chemical precursors, [(2-hydroxyethyl)thio]acetic acid (HETA) and thiodiglycolic acid (TDGA). Aerobic fed batch and repeated batch experiments were run to compare the molar yields of HETA and TDGA that result under different operating policies. In repeated batch experiments, 35% of the TDG was converted to HETA. Under the conventional batch process and a repeated fed batch process, the HETA yields were reduced (21% and 18%, respectively), while the yield of TDGA was increased (47% and 31%,respectively). This work demonstrated that cell growth associated biocatalytic transformations were manipulated to achieve a desired byproducts profile through an understanding of the specific reaction and cell growth kinetics and by altering the reaction operating policy accordingly.

Alcaligenes↗
PubMedDate not supplied

Transformation of thiodiglycol by resting cells of Alcaligenes xylosoxydans PGH10.

A new strain of Alcaligenes xylosoxydans able to aerobically cometabolize thiodiglycol, the primary hydrolysis product of sulfur mustard, was isolated and tested in a laboratory scale stirred tank reactor. The strain, named PGH10, cannot use TDG as sole carbon and energy source for growth, but resting cells previously grown on either rich broth or defined mineral media efficiently metabolize this compound through [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid as intermediates. Degradation of TDG by PGH10 is shown to take place at late exponential and stationary phase but is not triggered by carbon exhaustion. Cultures pregrown to saturation for 48 h in the absence of TDG can be stored and used for degradation of TDG, reducing significantly the time required to achieve the reduction of the compound concentration to undetectable levels. Degradation can take place in buffered media with no carbon source added, although best results were obtained in mineral media supplemented with citrate or fructose. Oxidation to [(2-hydroxyethyl)thio]acetic acid and thiodiacetic acid was proposed to be catalyzed by a butanol-dehydrogenase activity. Inhibition of TDG transformation in the presence of several alcohols is also shown.

Alcaligenes↗
PubMedDate not supplied

Growth kinetics, nutrient uptake, and expression of the Alcaligenes eutrophus poly(beta-hydroxybutyrate) synthesis pathway in transgenic maize cell suspension cultures.

Transgenic suspension cultures of Black Mexican Sweet maize (Zea mays L.) expressing the Alcaligenes eutrophus genes encoding enzymes of the pathway for biosynthesis of the biodegradable polymer poly(beta-hydroxybutyrate) (PHB) were established as a tool for investigating metabolic regulation of the PHB pathway in plant cells. Cultures were grown in a 2 L modified mammalian cell bioreactor and in shake flasks. Biomass doubling times for transgenic bioreactor cultures (3.42 +/- 0.76 days) were significantly higher than those for untransformed cultures (2.01 +/- 0.33 days). Transgenic expression of the bacterial enzymes beta-ketothiolase (0.140 units/mg protein) and acetoacetyl-CoA reductase (0.636 units/mg protein) was detected by enzyme assays and immunoblots. However, over the first 2 years of cultivation, reductase activity decreased to 0.120 units/mg proteins. Furthermore, the PHB synthase gene, although initially present, was not detectable after 1.5 years of cultivation in suspension culture. These facts suggest that transgenic expression of PHB pathway genes in plant cells may not be stable. A hydroxybutyrate derivative was detected via gas chromatography even after 4 years of cultivation. Although the method used to prepare samples for gas chromatography cannot directly distinguish among PHB polymer, hydroxybutyryl-CoA (HB-CoA), and hydroxybutyric acid, solvent washing experiments indicated that most or all of the signal was non-polymeric, presumably H-CoA. The synthesis of HB-CoA appeared to be linked to substrate growth limitation, with HB-CoA accumulation increasing dramatically and cell growth ceasing upon depletion of ammonium. This suggests that the PHB synthesis pathway in plants is subject to regulatory mechanisms similar to those in prokaryotic cells.

Alcaligenes↗
PubMedDate not supplied

Separation of Alcaligenes eutrophus cells containing Poly(3-hydroxybutyrate) from fermentation broth with pretreatment using Al- and Fe-based coagulants.

Alcaligenes eutrophus containing intracellular poly(3-hydroxybutyrate) was recovered from fermentation broth by centrifugation and filtration after pretreatment with Al- and Fe-based coagulants. Coagulation efficiency was largely affected by pH, and the optimum pH's for cell recovery were about 4.6-5.6 for the Al-based coagulants and about 5-8 for the Fe-based coagulants. Ammonium ions that combined with metals to form complex compounds increased the coagulant requirement, and the additional requirement of coagulant was found to be proportional to the ammonium concentration. In addition, various ligands in addition to ammonium ions contained in the culture medium interfered with the coagulation reaction and increased the coagulant requirement also. The coagulant requirement increased with the cell concentration regardless of coagulant type. The polymeric coagulants such as PACS, Hi-PAX, and Ferix-3 were more effective than nonpolymeric coagulants of aluminum sulfate and ferrous sulfate. The optimum dosages of the coagulants tested were determined over a broad range of cell concentration of 20.5-210 g/L. It was observed that the energy requirement for centrifugation could be greatly reduced with cell coagulation.

Alcaligenes↗
PubMed2006

Intersite structural rearrangement of the blue copper site induced by substrate binding: spectroscopic studies of a copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015.

A copper-containing nitrite reductase from Alcaligenes xylosoxidans NCIMB 11015 has its own unique blue or type 1 copper protein resonance Raman spectrum in the usual Cu-S(Cys) stretching region, nu(Cu-S(Cys)), with a pair of strong peaks at 412 and 420 cm(-1) and a weak peak at 364 cm(-1). The predominantly nu(Cu-S(Cys)) Raman bands at 412, 420, and 364 cm(-1) of the type 1 copper site all shifted to higher frequencies upon binding of nitrite to the type 2 copper site, and the resonance Raman difference spectra progressively intensified with the increments of nitrite ion concentration. Positive support for substrate binding to the type 2 copper is provided by the nu(Cu-S(Cys)) bands in the resonance Raman spectrum of a type 2 copper-depleted enzyme, which is insensitive to the presence of NO2-. The shift to higher frequency of the Raman bands of the type 1 copper center with the addition of nitrite ions suggests a stronger Cu-S(Cys) interaction in the substrate-bound A. xylosoxidans nitrite reductase.

Alcaligenes↗
PubMed1999

Biotransformation of lantadene A (22 beta-angeloyloxy-3-oxoolean-12-en-28-oic acid), the pentacyclic triterpenoid, by Alcaligenes faecalis.

A bacterial strain capable of biotransformation of lantadene A (22 beta-angeloyloxy-3-oxo-olean-12-en-28-oic acid), the pentacyclic hepatotoxin of lantana (Lantana camara var. aculeata) has been isolated from soil using lantadene A as the sole carbon source. The organism is Gram negative, rod shaped, motile, catalase positive and has been identified as Alcaligenes faecalis. The isolate has been found to be specific for lantadene A and did not utilize lantadene B. In studies using sucrose as an additional carbon source, A. faecalis elicited biotransformation of lantadene A to its trans isomer 22 beta-tigloyloxy-3-oxoolean-12-en-28-oic acid, designated as lantadene X and two other minor metabolites which could not be isolated in pure state.

Alcaligenes↗
PubMed2001

Metabolism of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes sp. JS867 under oxygen limited conditions.

Transformation of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes JS867 under varying degrees of oxygen limitation was examined. Complete 2,4-DNT removal was observed under oxygen excess with near stoichiometric release (83%) of nitrite. Average kinetic parameters were estimated based on a dual-Monod biokinetic model with 2,4-DNT and O2 as growth limiting substrates. The negative impact of nitrite accumulation on the reaction rate was adequately described by inclusion of a noncompetitive inhibition term for NO2-. Under aerobic conditions, mumax, KsDNT, and KiNO were 0.058 (0.004)hr(-1), 3.3(+/-1.3) mg 2,4-DNT/L, and 1.2(+/-0.2)hr(-1), respectively. At increasing oxygen limitation, rates of 2,4-DNT disappearance and nitrite production decreased and incomplete removal of 2,4-DNT commenced. JS867 was able to use NO2- as a terminal electron acceptor when grown on glucose or succinate under anaerobic conditions. However, during growth on 2,4-DNT and under O2-limited conditions, JS867 did not use released nitrite as electron acceptor. The nearly constant molar ratios of DNT removed over NO2- released under various degrees of oxygen limitation suggested that oxygenolytic denitration pathways continued. No evidence of nitroreduction was obtained under the examined oligotrophic conditions. JS867 displayed a high affinity for oxygen consumption with K(SO2) value of 0.285(+/-0.198) mg O2/L. Our results indicate that under oligotrophic conditions with 2,4-DNT as dominant carbon source, oxygen availability and nitrite accumulation may limit 2,4-DNT biomineralization, but the accumulation of reduced 2,4-DNT transformation products will be small.

Alcaligenes↗
PubMed2003

Bioreactor cultivation of Escherichia coli for production of recombinant penicillin G amidase from Alcaligenes faecalis.

The penicillin G amidase (PGA) from Alcaligenes faecalis, which has interesting properties for use in combinatorial biochemistry, was produced by recombinant expression in Escherichia coli. The corresponding gene was cloned into a multicopy vector under the strict regulatory control of the rhamnose inducible promoter. Cells were grown in a synthetic minimal medium in a bioreactor (5 l working vol.), and production of PGA was induced by repeated addition of the inducer rhamnose, that served also as a carbon source. The fermentation yield was about 4500 units PGA activity per liter of culture medium.

Alcaligenes↗
PubMed2003

Long-term analysis of diesel fuel consumption in a co-culture of Acinetobacter venetianus, Pseudomonas putida and Alcaligenes faecalis.

The dynamics of a microbial population isolated from superficial waters of Venice Lagoon and the ability to utilise diesel fuel (n-alkanes mixture C12-C28) as the sole carbon and energy source were studied in a long-term reconstruction experiment. The reconstructed microbial population consisted of three bacterial strains belonging to the species Acinetobacter venetianus, Pseudomonas putida, and Alcaligenes faecalis, which were able to oxidise n-alkanes to alkanoates, n-alkanols to alkanoates, or only n-alkanoates, respectively. Three different approaches: plate counting, cell counting by epifluorescence microscopy with DAPI staining, and by fluorescence in situ hybridisation (FISH) by using a probe conjugate with fluoresceine isothiocyanate specifically targeted towards the 16S rRNA of bacteria belonging to the genus Acinetobacter were used to monitor the growth of the bacterial population. The growth of A. venetianus was stimulated by the presence of other strains, suggesting a beneficial interaction. After the first week of growth A. venetianus cells formed aggregates, as confirmed by confocal microscopy (CLSM), which allowed them to be distinguished from free cells. A relationship between cell number and measured areas (microm2) per aggregate was found. Each cell presented an average surface of 1.21 microm2. Each aggregate was formed by a cellular monolayer biofilm consisting of up to several thousands of cells. The A. venetianus aggregates increased in number and size over time, but after two weeks fragmentation events, which had a beneficial effect on the growth of P. putida and A. faecalis, occurred.

Acinetobacter↗
PubMed2003

Purification and characterization of chitinase from Alcaligenes xylosoxydans.

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecular mass of chitinase was estimated to be 45 kDa and 44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 degrees C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l-1. Cu2+ and Na+ at 5 mM inhibited chitinase activity to 25% while Ca2+, Mg2+ and Ba2+ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.

Alcaligenes↗