Medium Optimization and Fermentation Kinetics for κ-Carrageenase Production by Thalassospira sp. Fjfst-332.

Effective degradation of κ-carrageenan by isolated Thalassospira sp. fjfst-332 is reported for the first time in this paper. It was identified by 16S rDNA sequencing and morphological observation using Transmission Electron Microscopy (TEM). Based on a Plackett-Burman design for significant variables, Box-Behnken experimental design and response surface methodology were used to optimize the culture conditions. Through statistical optimization, the optimum medium components were determined as follows: 2.0 g/L κ-carrageenan, 1.0 g/L yeast extract, 1.0 g/L FOS, 20.0 g/L NaCl, 2.0 g/L NaNO3, 0.5 g/L MgSO4·7H2O, 0.1 g/L K2HPO4, and 0.1 g/L CaCl2. The highest activity exhibited by Thalassospira sp. fjfst-332 was 267 U/mL, which makes it the most vigorous wild bacterium for κ-carrageenan production. In order to guide scaled-up production, two empirical models-the logistic equation and Luedeking-Piretequation-were proposed to predict the strain growth and enzyme production, respectively. Furthermore, we report the fermentation kinetics and every empirical equation of the coefficients (α, ß, X0, Xm and µm) for the two models, which could be used to design and optimize industrial processes.

Determination of the Molar Extinction Coefficients of the B800 and B850 Absorption Bands in Light-harvesting Complexes 2 Derived from Three Purple Photosynthetic Bacteria Rhodoblastus acidophilus, Rhodobacter sphaeroides, and Phaeospirillum molischianum by Extraction of Bacteriochlorophyll a.

The molar extinction coefficients of light-harvesting complex 2 (LH2) have been ambiguous in spite of its fame and wide utilization. Herein we determine the molar extinction coefficients of the LH2 proteins derived from the three purple photosynthetic bacteria Rhodoblastus acidophilus, Rhodobacter sphaeroides and Phaeospirillum molischianum at 298 K by direct extraction of bacteriochlorophyll (BChl) a from the lyophilized proteins, followed by estimation of BChl a amounts from their electronic absorption spectra.

Optimization of Lipase production from a novel strain Thalassospira permensis M35-15 using Response Surface Methodology.

Lipases can catalyze the hydrolysis of glycerol, esters and long chain fatty acids. A lipase producing isolate M35-15 was screened and identified as Thalassospira permensis using 16S rRNA gene sequence analysis. To our knowledge this is the first report on Thalassospira permensis producing lipases. In this paper the optimization of medium composition for the increase in bacterial lipase was achieved using statistical methods. Firstly the key ingredients were selected by Plackett-Burman experimental design, then the levels of the ingredients were optimized using central composite design of Response Surface Methodology. The predicted optimal lipase activity was 11.49 U under the conditions that medium composition were 5.15 g/l glucose, 11.74 g/l peptone, 6.74 g/l yeast powder and 22.90 g/l olive oil emulsifier.

Biochemical characterization and structural analysis of a new cold-active and salt-tolerant esterase from the marine bacterium Thalassospira sp.

A gene encoding an esterase, ThaEst2349, was identified in the marine psychrophilic bacterium Thalassospira sp. GB04J01. The gene was cloned and overexpressed in E. coli as a His-tagged fusion protein. The recombinant enzyme showed optimal activity at 45 °C and the thermal stability displayed a retention of 75 % relative activity at 40 °C after 2 h. The optimal pH was 8.5 but the enzyme kept more than 75 % of its maximal activity between pH 8.0 and 9.5. ThaEst2349 also showed remarkable tolerance towards high concentrations of salt and it was active against short-chain p-nitrophenyl esters, displaying optimal activity with the acetate. The enzyme was tested for tolerance of organic solvents and the results are suggesting that it could function as an interesting candidate for biotechnological applications. The crystal structure of ThaEst2349 was determined to 1.69 Å revealing an asymmetric unit containing two chains, which also is the biological unit. The structure has a characteristic cap domain and a catalytic triad comprising Ser158, His285 and Asp255. To explain the cold-active nature of the enzyme, we compared it against thermophilic counterparts. Our hypothesis is that a high methionine content, less hydrogen bonds and less ion pairs render the enzyme more flexible at low temperatures.

[Expression and characterization of a novel halohydrin dehalogenase from Tistrella mobilis KA081020-065].

Halohydrin dehalogenase is of great significance for biodegradation of the chlorinated pollutants, and also serves as an important biocatalyst in the synthesis of chiral pharmaceutical intermediates. A putative halohydrin dehalogenase (HheTM) gene from Tistrella mobilis KA081020-065 was cloned and over-expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA column and characterized. Gel filtration and SDS-PAGE analysis showed that the native form of HheTM was a tetramer. It exhibited the highest activity at 50 degrees C. The nature and pH of the buffer had a great effect on its activity. The enzyme maintained high stability under the alkaline conditions and below 30 degrees C. HheTM catalyzed the transformation of ethyl(S)-4-chloro-3-hydroxybutyrate in the presence of cyanide, to give ethyl (R)-4-cyano-3-hydroxybutyrate, a key intermediate for the synthesis of atorvastatin.

Statistical approach for the enhanced production of cold-active ß-galactosidase from Thalassospira frigidphilosprofundus: a novel marine psychrophile from deep waters of Bay of Bengal.

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active ß-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for ß-galactosidase production. Under optimal conditions, the production of ß-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. ß-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active ß-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active ß-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.

Optimization of polygalacturonase production from a newly isolated Thalassospira frigidphilosprofundus to use in pectin hydrolysis: statistical approach.

The present study deals with the production of cold active polygalacturonase (PGase) by submerged fermentation using Thalassospira frigidphilosprofundus, a novel species isolated from deep waters of Bay of Bengal. Nonlinear models were applied to optimize the medium components for enhanced production of PGase. Taguchi orthogonal array design was adopted to evaluate the factors influencing the yield of PGase, followed by the central composite design (CCD) of response surface methodology (RSM) to identify the optimum concentrations of the key factors responsible for PGase production. Data obtained from the above mentioned statistical experimental design was used for final optimization study by linking the artificial neural network and genetic algorithm (ANN-GA). Using ANN-GA hybrid model, the maximum PGase activity (32.54 U/mL) was achieved at the optimized concentrations of medium components. In a comparison between the optimal output of RSM and ANN-GA hybrid, the latter favored the production of PGase. In addition, the study also focused on the determination of factors responsible for pectin hydrolysis by crude pectinase extracted from T. frigidphilosprofundus through the central composite design. Results indicated 80% degradation of pectin in banana fiber at 20 °C in 120 min, suggesting the scope of cold active PGase usage in the treatment of raw banana fibers.

Cold active ß-galactosidase from Thalassospira sp. 3SC-21 to use in milk lactose hydrolysis: a novel source from deep waters of Bay-of-Bengal.

The cold active ß-galactosidase from psychrophilic bacteria accelerate the possibility of outperforming the current commercial ß-galactosidase production from mesophilic sources. The present study is carried out to screen and isolate a cold active ß-galactosidase producing bacterium from profound marine waters of Bay-of-Bengal and to optimize the factors for lactose hydrolysis in milk. Isolated bacterium 3SC-21 was characterized as marine psychrotolerant, halophile, gram negative, rod shaped strain producing an intracellular cold active ß-galactosidase enzyme. Further, based upon the 16S rRNA gene sequence, bacterium 3SC-21 was identified as Thalassospira sp. The isolated strain Thalassospira sp. 3SC-21 had shown the enzyme activity between 4 and 20 °C at pH of 6.5 and the enzyme was completely inactivated at 45 °C. The statistical method, central composite rotatable design of response surface methodology was employed to optimize the hydrolysis of lactose and to reveal the interactions between various factors behind this hydrolysis. It was found that maximum of 80.18 % of lactose in 8 ml of raw milk was hydrolysed at pH of 6.5 at 20 °C in comparison to 40 % of lactose hydrolysis at 40 °C, suggesting that the cold active ß-galactosidase from Thalassospira sp. 3SC-21 would be best suited for manufacturing the lactose free dairy products at low temperature.

[Arsenic metabolism in purple nonsulfur bacteria].

OBJECTIVE: To elucidate the arsenic metabolic pathway of purple nonsulfur bacteria (PNSB). METHODS: We investigated the distribution within their genomes, organization, composition, arrangement, core genes and coding proteins of arsenic gene clusters found in complete genome from 17 strains of PNSB by comparing the genomes analysis, and studied the arsenic metabolism in 3 members of PNSB under anaerobic conditions by UV-Vis and HPLC-ICP-MS. RESULTS: Arsenate reduction and arsenite methylation pathways mediated by ars operon are the dominating arsenic metabolic processes. The arsenic gene clusters differ vastly in composition and arrangement. Some members of PNSB evolved two independently families of arsenate reduction genes (arsC). The cells of Rhodopseudomonas palustris CQV97, Rhodobacter azotoformans 134K20 and Rhodobacter capsulatus XJ-1 could reduce As (V) to As (III), whereas As (III) could not be transformed back to As (V). Higher concentration phosphate competitively inhibited arsenate toxicity to cells. CONCLUSION: Our investigations shed light on the evolution and functional implications in arsenic gene clusters of PNSB, and support the notion that arsenate reduction and arsenite methylation appears to be the dominant process in PNSB.

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Photobiological hydrogen-producing systems.

Hydrogen photoproduction by micro-organisms combines the photosynthetic properties of oxygenic and non-oxygenic microbes with the activity of H2-producing enzymes in nature: hydrogenases and nitrogenases. The overall efficiency of the process depends on the separate efficiencies of photosynthesis and enzymatic catalysis. This tutorial review discusses the biochemical pathways for H2 production in different organisms, barriers to be overcome, and possible suggestions for integrating photobiological H2 production with fermentative, anaerobic systems for a potentially more efficient process.

Static and dynamic protein impact on electronic properties of light-harvesting complex LH2.

A comparative analysis of the temperature dependence of the absorption spectra of the LH2 complexes from different species of photosynthetic bacteria, i.e., Rhodobacter sphaeroides, Rhodoblastus acidophilus, and Phaeospirillum molischianum, was performed in the temperature range from 4 to 300 K. Qualitatively, the temperature dependence is similar for all of the species studied. The spectral bandwidths of both B800 and B850 bands increases with temperature while the band positions shift in opposite directions: the B800 band shifts slightly to the red while the B850 band to the blue. These results were analyzed using the modified Redfield theory based on the exciton model. The main conclusion drawn from the analysis was that the spectral density function (SDF) is the main factor underlying the strength of the temperature dependence of the bandwidths for the B800 and B850 electronic transitions, while the bandwidths themselves are defined by the corresponding inhomogeneous distribution function (IDF). Slight variation of the slope of the temperature dependence of the bandwidths between species can be attributed to the changes of the values of the reorganization energies and characteristic frequencies determining the SDF. To explain the shift of the B850 band position with temperature, which is unusual for the conventional exciton model, a temperature dependence of the IDF must be postulated. This dependence can be achieved within the framework of the modified (dichotomous) exciton model. The slope of the temperature dependence of the B850 bandwidth is then defined by the value of the reorganization energy and by the difference between the transition energies of the dichotomous states of the pigment molecules. The equilibration factor between these dichotomous states mainly determines the temperature dependence of the peak shift.

Purification and characterization of alkaline serine proteinase from photosynthetic bacterium, Rubrivivax gelatinosus KDDS1.

In order to reduce the protein content of wastewater, photosynthetic bacteria producing proteinases were screened from wastewater of various sources and stocked in culture. An isolated strain, KDDS1, was identified as Rubrivivax gelatinosus, a purple nonsulfur bacterium that secretes proteinase under micro-aerobic conditions under light at 35 degrees C. Molecular weight of the purified enzyme was estimated to be 32.5 kDa. The enzyme showed the highest activity at 45 degrees C and pH 9.6, and the activity was completely inhibited by phenylmethyl sulfonyl fluoride (PMSF), but not by EDTA. The amino-terminal 24 amino acid sequence of the enzyme showed about 50% identity to those of serine proteinases from Pseudoalteromonas piscicida strain O-7 and Burkholderia pseudomallei. Thus, the enzyme from Rvi. gelatinosus KDDS1 was thought to be a serine-type proteinase. This was the first serine proteinase characterized from photosynthetic bacteria.

Nitrate reductase from the magnetotactic bacterium Magnetospirillum magnetotacticum MS-1: purification and sequence analyses.

We purified the nitrate reductase from the soluble fraction of Magnetospirillum magnetotacticum MS-1. The enzyme was composed of 86- and 17-kDa subunits and contained molybdenum, non-heme iron, and heme c. These properties are very similar to those of the periplasmic nitrate reductase found in Paracoccus pantotrophus. The M. magnetotacticum nap locus was clustered in seven open reading frames, napFDAGHBC. The phylogenetic analyses of NapA, NapB, and NapC suggested a close relationship between M. magnetotacticum nap genes and Escherichia coli nap genes, which is not consistent with the 16S rDNA data. This is the first finding that the alpha subclass of Proteobacteria possesses a napFDAGHBC-type nap gene cluster. The nap gene cluster had putative fumarate and nitrate reduction regulatory protein (Fnr) and NarL protein binding sites. Furthermore, we investigated the effect of molybdate deficiency in medium on the total iron content of the magnetosome fraction and discussed the physiological function of nitrate reductase in relation to the magnetite synthesis in M. magnetotacticum.

Characterization of aldehyde ferredoxin oxidoreductase gene defective mutant in Magnetospirillum magneticum AMB-1.

A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA21, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis. Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes. The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation. AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1. Iron uptake and growth of NMA21 were lower than wild type. Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced. These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M. magneticum AMB-1 under microaerobic respiration.

Novel domain packing in the crystal structure of a thiosulphate-oxidizing enzyme.

A key component of the oxidative biogeochemical sulphur cycle involves the utilization by bacteria of reduced inorganic sulphur compounds as electron donors to photosynthetic or respiratory electron transport chains. The SoxAX protein of the photosynthetic bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that is involved in the oxidation of thiosulphate and sulphide. The recently solved crystal structure of the SoxAX complex represents the first structurally characterized example of a productive electron transfer complex between haemoproteins where both partners adopt the c-type cytochrome fold. The packing of c-type cytochrome domains both within SoxA and at the interface between the subunits of the complex has been compared with other examples and found to be unique.

Production of luciferase-magnetic particle complex by recombinant Magnetospirillum sp. AMB-1.

Luciferase-bacterial magnetic particle (BMP) complexes were produced by recombinant Magnetospirillum sp. AMB-1. We constructed plasmids pKML and pNELM, respectively, by fusing luc to the 5' and 3' terminal of magA, encoding an integral iron translocating protein situated in the BMP membrane, of AMB-1. In addition, we produced bifunctional active-fusion proteins on BMPs by using a plasmid pAcML. In this plasmid, acetate kinase and luciferase genes were fused to the N-terminus and the C-terminus of MagA, respectively. Bacterial magnetic particles isolated from transconjugants for pKML, pNELM and pAcML exhibited luciferase activity. Bacterial magnetic particles isolated from transconjugants for pAcML also exhibited acetate kinase activity. Fed-batch culture of pKML transconjugant yielded 2.6 mg BMPs per liter of culture, and 95% conversion of iron into magnetite was obtained, at a nitrate concentration of 1.4 mM. Continuous feeding of iron as ferric quinate significantly enhanced growth and total magnetic production. Final cell concentration of 1.8 x 10(9) cells/mL and 6 mg per liter of culture was obtained. Magnetite production by fed-batch culture of AMB-1 was about 3 times that obtained by batch culture. There were no significant differences in BMPs yield between recombinant AMB-1 cultivated by fed-batch culture and wild type of AMB-1.

Heme-copper oxidase family structure of Magnetospirillum magnetotacticum 'cytochrome a1'-like hemoprotein without cytochrome c oxidase activity.

The genes encoding 'cytochrome a1'-like hemoprotein of Magnetospirillum magnetotacticum were identified and sequenced. Three ORFs, mcalI, mcaI and hosA, were included in the sequenced region. The six histidine residues which were predicted to associate with the prosthetic cofactors of heme-copper oxidase superfamily were conserved in the hemoprotein. However, none of the amino acid residues which were proposed to participate in the oxygen-reducing and the coupled proton pumping reactions in cytochrome c oxidase were at all conserved in the hemoprotein.

Iron reductase for magnetite synthesis in the magnetotactic bacterium Magnetospirillum magnetotacticum.

Ferric iron reductase was purified from magnetotactic bacterium Magnetospirillum (formerly Aquaspirillum) magnetotacticum (ATCC 31632) to an electrophoretically homogeneous state. The enzyme was loosely bound on the cytoplasmic face of the cytoplasmic membrane and was found more frequently in magnetic cells than in nonmagnetic cells. The molecular mass of the purified enzyme was calculated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be about 36 kDa, almost the same as that calibrated by gel filtration analysis. The enzyme required NADH and flavin mononucleotide (FMN) as optimal electron donor and cofactor, respectively, and the activity was strongly inhibited by Zn2+ acting as a partial mixed-type inhibitor. The Km values for NADH and FMN were 4.3 and 0. 035 microM, respectively, and the Ki values for Zn2+ were 19.2 and 23.9 microM for NADH and FMN, respectively. When the bacterium was grown in the presence of ZnSO4, the magnetosome number in the cells and the ferric iron reductase activity declined in parallel with an increase in the ZnSO4 concentration of the medium, suggesting that the ferric iron reductase purified in the present study may participate in magnetite synthesis.

Interaction site for soluble cytochromes on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center mapped by site-directed mutagenesis.

The crystallographic structure of the Blastochloris (formerly called Rhodopseudomonas) viridis tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) suggests that all four hemes are located close enough to the surface of the protein to accept electrons from soluble cytochrome c2. To identify experimentally the site of this reaction we prepared site-directed mutants of Rubrivivax gelatinosus RCs with surface charge substitutions in the bound cytochrome subunit and studied the kinetics of their reduction by soluble cytochromes (mitochondrial horse cytochrome c, Blc. viridis cytochrome c2, and Rvi. gelatinosus cytochrome c8). In comparison with the wild-type, the mutants E79K (glutamate-79 substituted by lysine), E93K (glutamate-93 substituted by lysine), and E85K (glutamate-85 substituted by lysine) located near the solvent-exposed edge of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll, exhibited decreased second-order rate constants for the reaction between the tetraheme subunit and the soluble cytochromes. Double charge substitutions in this region: E79K/E85K (glutamate-79 and -85 both replaced by lysine) and E93K/E85K (glutamate-93 and -85 both replaced by lysine) appeared to show an additive inhibitory effect. Mutations in other charged regions did not alter the kinetics of electron transfer between bound and soluble cytochromes. In light of the available structural information on Blc. viridis RC, these results indicate that the cluster of acidic residues immediately surrounding the distal heme 1 of the RC-bound tetraheme subunit forms an electrostatically favorable binding site for soluble cytochromes. Thus, all four hemes in the subunit seem to be directly involved in the electron transfer toward the photo-oxidized special pair of bacteriochlorophyll. On the basis of these findings, a model is proposed for the hypothetical cytochrome c2-RC transient complex for Blc. viridis.