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The biogenic magnetite nanoparticles presented here had a high capacity of adsorbing metal cations, which was approximately 30- to 40-fold greater than commercially available magnetite. These results suggest the potential application of microbial magnetite formation in the removal of toxic metal cations from water., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Genetically modified bacterial biosensors can detect specific environmental compounds. Here, we attempted to establish a fluorescent microplate method to detect arsenic using recombinant Escherichia coli cells transformed with plasmids harboring three tandem copies of the ars promoter/operator-the gene for green fluorescent protein (gfp). In the biosensors, one copy of arsR, whose transcription is autoregulated by the ars promoter/operator and ArsR in the genome of E. coli, was placed in trans in another plasmid under the control of isopropyl-1-thio-beta-D-galactopyranoside-inducible promoter. First, this manipulation enabled regulation of the arsR expression at an adequate level. Second, the copy number of reporter unit also affected signal and noise. When the plasmid harboring three copies of the reporter unit was used, the signal-to-noise ratio doubled and the detection limit decreased from 20 to 7.5 microg L(-1) As(III), compared to the use of the plasmid harboring one copy of the ars promoter/operator-arsR-gfp. Thus, segregation of arsR from the ars promoter/operator-gfp using two plasmids is effective in regulating the signal-to-noise ratio and the detection limit with the different functions.

We found that a newly isolated Burkholderia sp. produced (R)-2-amino-1-phenylethanol from 2-aminoacetophenone, showing the high stereospecificity. NADPH-dependent 2-aminoacetophenone reductase purified to homogeneity was a dimer with a molecular mass of 65,000. The purified enzyme did not reduce acetophenone and 1-phenyl-1-propanone. The purified enzyme converted 2-aminoacetophenone to only (R)-2-amino-1-phenylethanol.

The gene encoding N-benzyl-3-pyrrolidinol dehydrogenase (DDBJ/EMBL/GenBank accession no. AB294179), a useful biocatalyst for producing (S)-N-benzyl-3-pyrrolidinol, was cloned from the genomic DNA of Geotrichum capitatum JCM 3908. The gene contained an open reading frame consisting of 1023 nucleotides corresponding to 340 amino acid residues. The subunit molecular weight was calculated to be 39,000. The predicted amino acid sequence did not have significant similarity to those of N-benzyl-3-pyrrolidinone reductases reported previously. From 30 mM N-benzyl-3-pyrrolidinone, (S)-N-benzyl-3-pyrrolidinol was obtained with a yield >99.9% and an enantiomeric excess >99.9% in 1-h and 2-h reactions without NADH addition by the resting cells of Escherichia coli HB 101 strains harboring the expression plasmids pSG-POBS and pSF-POBS that possess the glucose dehydrogenase gene and formate dehydrogenase gene as an NADH-reproducing system, respectively, besides the N-benzyl-3-pyrrolidinol dehydrogenase gene. N-Benzyl-3-pyrrolidinol dehydrogenase activity (0.56 U/mg) was observed in E. coli (pSG-POBS), which was 17-fold the specific activity observed in G. capitatum JCM 3908.

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.

Diverse bacteria and fungi oxidize Mn(II) enzymatically and produce insoluble Mn(III, IV) oxides, and these organisms are considered to be the primal agents for the occurrence of natural Mn oxide phases in most environments. Biogenic Mn oxides have a high sorption capacity for metal cations and an ability to oxidize numerous inorganic and organic compounds, owing to their structural and redox features. Thus, the microbial process is of significance in both biogeochemical and biotechnological contexts. In this article we summarize the enzymatic Mn(II) oxidation and interactions of biogenic Mn oxides with toxic metal and metalloid ions. Although Mn oxide formation by fungi has not been fully characterized yet, recent researches with ascomycetes emphasize the similarity between the bacterial and fungal Mn(II) oxidation with respect to the involved catalyst (i.e., multicopper oxidase-type enzymes) and the reaction product [i.e., layer-type Mn(IV) oxides]. Laboratory cultures of bacterial and fungal Mn oxidizers are expected to provide fundamental knowledge in their potential use for remediation of environments and effluents contaminated with toxic metal(loid) ions.

Sterigmatomyces elviae DSM 70852 produced 12 g/l (S)-1,2,4-butanetriol (enantiomeric excess >99.9%) from 20 g/l racemate in 82 h. From the results of the inversion of an (R)-isomer to an (S)-isomer and GC-MS, it was suggested that (R)-1,2,4-butanetriol is oxidized to 1,4-dihydroxy-2-butanone, which is reduced to an (S)-isomer.

We investigated the production of manganese (Mn) oxides using repeated-batch bioreactors maintained over long periods under laboratory conditions. Freshwater epilithic biofilms were used as the initial inocula. The bioreactors yielded suspended solids that could remove 0.1 mM dissolved Mn(II) within a few days. Chemical titration, X-ray absorption near-edge structure spectroscopy, and X-ray diffraction analysis revealed that the Mn(II) had been converted to poorly crystallized layer-type Mn(IV) oxides, which were similar to known biogenic Mn oxides from pure bacterial cultures. Spherical or rod-shaped Mn microconcretions occurred in the suspended solids; transmission electron microscopy showed that these structures likely resulted from the microbial activity but not represent living cells. Instead, the presence of encapsulated, sheathed, and hyphal budding cells in the suspended solids indicated that a range of Mn-depositing bacteria contributed to the Mn oxide formation. To our knowledge, our data represent the first observation of production of such Mn oxides in a laboratory microcosm wherein a range of Mn-depositing bacteria coexist. The fact that sorption of trace Zn(II) and Ni(II) ions onto the suspended solids co-occurred with the removal of dissolved Mn(II) emphasizes the important role of Mn-oxidizing microorganisms in the fates of trace or contaminant metals in the aquatic environment.

(S)-N-Benzyl-3-pyrrolidinol is widely used in the synthesis of pharmaceuticals as a chiral building block. We produced 30 mM (S)-N-benzyl-3-pyrrolidinol (enantiometric excess > 99.9%) from the corresponding ketone N-benzyl-3-pyrrolidinone with more than 99.9% yield in 28 h of the resting-cell reaction of Geotrichum capitatum JCM 3908. NAD(+)-dependent alcohol dehydrogenase reducing N-benzyl-3-pyrrolidinone from G. capitatum JCM 3908 was purified to homogeneity by ammonium sulfate fractionation and a series of DEAE-Toyopearl, Butyl-Toyopearl, Superdex 200, and Hydroxyapatite column chromatographies. The results of SDS-PAGE and HPLC showed the enzyme to be a dimer with a molecular mass of 78 kDa. The purified enzyme produced (S)-N-benzyl-3-pyrrolidinol (e.e.>99.9%) from N-benzyl-3-pyrrolidinone. The enzyme reduced 2,3-butanedione, 2-hexanone, cyclohexanone, propionaldehyde, n-butylaldehyde, n-hexylaldehyde, n-octylaldehyde, n-valeraldehyde, and benzylacetone more effectively than it did N-benzyl-3-pyrrolidinone. No activity was detected towards N-benzyl-2-pyrrolidinone or 2-pyrrolidinone. The activity towards (R)-N-benzyl-3-pyrrolidinol was not detected under the assay conditions employed. The oxidizing activity of the enzyme was higher towards 2-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol, and 1-phenyl-2-propanol than towards (S)-N-benzyl-3-pyrrolidinol. The K(m) values for N-benzyl-3-pyrrolidinone reduction and (S)-N-benzyl-3-pyrrolidinol oxidation were 0.13 and 8.47 mM, respectively. To our knowledge, this is the first time that an N-benzyl-3-pyrrolidinol/N-benzyl-3-pyrrolidinone oxidoreductase was purified from a eukaryote; moreover, this is the first report of (S)-N-benzyl-3-pyrrolidinol dehydrogenase activity in microorganisms. This enzyme showed features different from those of known prokaryotic N-benzyl-3-pyrrolidinone reductases. This enzyme will be very useful for the production of chiral compounds.

Optically active alcohol is an important building block as a versatile chiral synthon for the asymmetric synthesis of pharmaceuticals and agrochemicals. We purified and characterized glycerol dehydrogenase from Hansenula ofunaensis and prepared optically active 1,2-octanediol using a recombinant Escherichia coli harboring the glycerol dehydrogenase gene. The deduced amino acid sequence was investigated for identities with those of other alcohol dehydrogenases in the NCBI databank. The identification of the unknown product of a resting-cell reaction was performed by GC-MS. In the deduced amino acid sequence composed of 376 residues, the NAD(H) binding pattern and cysteine residues that correspond to the cysteine ligands at the zinc atom were conserved as they are in alcohol dehydrogenases from other origins. Glycerol dehydrogenase from Hansenula polymorpha DL-1 (Pichia angusta, DDBJ/EMBL/GenBank accession no. BAD32688) had the highest identity to our enzyme, showing 73% identity. Our glycerol dehydrogenase catalyzed the NAD(+)-dependent oxidation of long-chain secondary alcohols such as 1,2-pentanediol, 1,2-hexanediol, 1,2-heptanediol, and 1,2-octanediol. Activities toward 2,4-pentanediol and 2,5-hexanediol were hardly detected. From these results, it was confirmed that our enzyme requires two hydroxyl groups on adjacent carbon atoms for oxidation. 2,3-Pentanedione, 2,3-hexanedione, and 3,4-hexanedione were significantly reduced. The transformants oxidized only (R)-1,2-octanediol in 50 mM racemate (R:S=52:48), and produced (S)-1,2-octanediol (24 mM, <99.9% e.e.) after 24 h of incubation. The reaction product was suggested to be 1-hydroxy-2-octanone by GC-MS, which showed secondary hydroxyl groups oxidized. Glycerol dehydrogenase from H. ofunaensis could be useful for the production of long-chain optically active secondary alcohols.

Bacterial communities in crude-oil samples from Japanese oil stockpiles were investigated by 16S rRNA gene cloning, followed by denaturing gradient gel electrophoresis (DGGE) analysis. 16S rRNA genes were successfully amplified by PCR after isooctane treatment from three kinds of crude-oil sample collected at four oil stockpiles in Japan. DGGE profiles showed that bacteria related to Ochrobactrum anthropi, Burkholderia cepacia, Stenotrophomonas maltophilia, Propionibacterium acnes, and Brevundimonas diminuta were frequently detected in most crude-oil samples. The bacterial communities differed in the sampling time and layer. Among the predominant bacteria detected in the crude oil, only three species were found for bacteria isolated on agar plates and were related to Burkholderia, Stenotrophomonas, and Propionibacterium, while Ochrobactrum sp. could not be isolated although this species seemed to be the most abundant bacterium in crude oil from the DGGE profiles. Using an archaea-specific primer set, methanogens were found in crude-oil sludge but not in crude-oil samples, indicating that methanogens might be involved in sludge formation in oil stockpiles.

Two putative fructosyl-amino acid oxidase genes, FAP1 and FAP2, found in the Schizosaccharomyces pombe genome were cloned and expressed. Both of the gene products (Fap1 and Fap2) were flavoproteins and have no activity for fructosyl-amino acids. It was suggested that Fap1 and Fap2 are an L-pipecolic acid oxidase and L-saccharopine oxidase, respectively.

Poly(sodium acrylate) (PSA) can be degraded by consortia of several bacterial species. We investigated the degradation mechanism for PSA (average molecular weight, 2100) by consortium no. L7-98. PSA was used as the sole carbon source in a mineral salt medium. After cultivation, the PSA had a range of molecular weights, including low-molecular-weight compounds, which were purified by gel-permeation and reversed-phase column chromatography. One purified compound, B1, with the molecular weight of 200, had a carbonyl group next to the terminus, according to 1H and 13C nuclear magnetic resonance spectrometry and X-ray analysis of the crystal structure. Two categories of metabolites of PSA were detected in the culture by electrospray ionization mass spectrometry. Results of high-resolution mass spectrometry (HR-MS) suggested that one kind of compounds had a carbonyl group and that the other kind of compounds had an aldehyde group and a double bond. Compounds having the molecular weights of 200 and 272 were rapidly produced from an acrylic acid oligomer with the molecular weight of 258 by resting cells of the consortium. HR-MS showed that a methylene group at the terminal unit was oxidized to a carbonyl group and that the compound with the molecular weight of 200 was compound B1. From these results, we propose that the degradation pathway of PSA involves (i) oxidation of a methylene group to a carbonyl group next to the terminus, (ii) decarboxylation to form an aldehyde group and dehydrogenation to form a double bond between the terminal unit and the next unit, and (iii) oxidation of the aldehyde group to a carboxyl group followed by elimination of an acetic acid.

The possible involvement of serC in vitamin B6 (B6) biosynthesis in Bacillus subtilis was investigated and compared with that in Escherichia coli. The genes of E. coli and B. subtilis were disrupted with pBEN66 and pMutin1-derived integration vectors, respectively. Nutrient requirement analyses showed that the serC-disrupted E. coli mutant required pyridoxine (PN) and L-serine, and lacked the ability to synthesize B6. Glycolaldehyde (GA), a confirmed precursor of B6, could replace PN and support the growth of the disruptant. However, the serC-disrupted E. coli mutant grown in a minimal medium supplemented with L-serine and GA synthesized B6 at a level less than 20% of that synthesized by the wild type. In contrast to E. coli, the serC-disrupted B. subtilis mutant required L-serine or glycine for growth, but did not require PN. The serC disruptant retained its ability for B6 biosynthesis and produced almost the same amount of PN as the wild type. GA had no effect on the growth and level of B6 biosynthesis of both the wild type and the serC disruptant. These results lead to the conclusion that serC is directly involved in B6 biosynthesis in E. coli, but not in B. subtilis.

We show that yaaD and yaaE are involved in vitamin B6 (B6) biosynthesis in Bacillus subtilis. This is the first report which identifies genes involved in B(6) biosynthesis in B. subtilis. Based on homology, yaaD and yaaE belong to the highly conserved SNZ and SNO families, respectively. Disruptants of yaaD and yaaE required pyridoxal (PL) or pyridoxine (PN), and grew in the same way as the wild type in a minimal medium supplemented with 0.05 mM PL. The SNZ family is considered to be involved in singlet-oxygen resistance. Singlet-oxygen quenchers, L(+)-ascorbic acid and reduced glutathione, did not support the growth of these disruptants. Both yaaD and yaaE were transcribed at the highest level during the middle- to late-exponential phase and at a much lower level during the stationary phase. Neither PL nor PN affected the transcriptional rates of yaaD and yaaE. It is concluded that yaaD and yaaE are involved in B6 biosynthesis in B. subtilis, and are transcribed at the highest level during the middle- to late-exponential phase.

Resting cells of Fusarium moniliforme strain MS31 produced (R)-1-phenylpropanol from propylbenzene. The components of the medium and the reaction conditions were adjusted to increase the specific activity of the hydroxylating enzyme involved. Glucose and sodium nitrate were selected as carbon and nitrogen sources, respectively. The substrate, propylbenzene, inhibited fungal growth and the activity of the enzyme. Acetoin added to the medium increased both growth and activity of the enzyme, and hydroxylation of propylbenzene increased by 1.4-fold. Maximum bioconversion of propylbenzene by resting cells of the fungus was at 25-30 degrees C and pH 7.0 with cells at concentration of 40 mg (dry) per milliliter of reaction mixture. Conversion was accelerated as soon as propylbenzene was added; slowing 2 h later. In the end, F. moniliforme strain MS31 produced (R)-1-phenylpropanol with an enantiomeric excess of 98% at the concentration of 16 mM (2.2 mg.ml(-1)).

Oxygenase is useful when oxygen is to be introduced at a nonactivated carbon-hydrogen bond to give an optically active center. To obtain such an enzyme from microorganisms, we screened soil samples for organisms that assimilated methylethylketone as their sole carbon source. Yeasts and molds that converted ethylbenzene and propylbenzene into their respective oxygenated products during incubation together as resting cells were isolated. One particularly potent strain was identified as Fusarium moniliforme. The fungus oxidized the side chains of ethylbenzene and propylbenzene selectively at the benzylic position. The products were found to be 1-phenylethanol and 1-phenylpropanol, respectively, by GC-MS and HPLC with a chiral column, with 100% enantiomeric excess of the (R-(+)-form.

Resting cells of Fusarium moniliforme strain MS31 convert propylbenzene to 1-phenylpropanol with high regio- and stereospecificity. To elucidate the scope of substrate acceptability by the fungus, we used various aromatic compounds for the bioconversion. The fungus hydroxylated various alkylbenzenes at the benzylic position to produce optically active alcohols. Butylbenzene was converted to nonbenzylic alcohols. In all cases, the R absolute configuration of products was more abundant. Aromatic compounds with linear side chains and (1-methylethyl)benzene were converted to their corresponding alcohols with an enantiomeric excess of 94% to 100%. Further oxidation of the alcohols was detected, but it was weak.

To remove sulfur compounds in petroleum, we isolated sulfate-reducing bacteria that could degrade dibenzothiophene in the presence of only nitrogen gas. Among the 19 strains isolated, some could grow in the presence of 10% (v/v) kerosene and of which two strains were identified as Desulfomicrobium escambium and Desulfovibrio longreachii. Gas chromatography of the ethyl-acetate extract of bacterial cultures, in which 10% or more of the dibenzothiophene initially present was degraded, gave five unknown peaks as the presumable degradation products. Thus, desulfurization of dibenzothiophene could be carried out without oxygen or hydrogen in a pathway different from the anaerobic one already reported, in which biphenyl is detected as the main product.

The cyst cells of Haematococcus pluvialis were separated into fractions of relatively uniform size by sucrose density gradient centrifugation. The fraction at the bottom of the centrifuge tube with the largest specific gravity from density gradients of mature cysts mainly consisted of enlarged, red cyst cells and had the highest astaxanthin content. To examine the relationship between cell size and astaxanthin content of cysts, formation of the fluorescent dichlorofluorescein (DCF) from 2',7'-dichlorohydrofluorescein diacetate of cyst cells in each fraction from density-gradient centrifugation under oxidative stress caused by methyl viologen (1.0 mM) was studied. The formation of DCF in cyst cells was decreased with larger cell diameter. This decrease was also correlated with increases in astaxanthin content. Therefore, both cell diameter and the fluorescent DCF content of cyst cells would be good parameter to select astaxanthin-hyperproducing strains from native populations of H. pluvialis.

Glycolaldehyde (GA) was shown to be a precursor of vitamin B6 (B6), and to be formed from glycolate by glycolaldehyde dehydrogenase (GADH) in Escherichia coli. In this study, we show the glycolaldehyde-forming route in B6 biosynthesis in Bacillus subtilis. In the crude extract of B. subtilis, the oxidizing activity of GADH was detected. However, coexisting NADH/NADPH oxidase activity interfered with the determination of the reducing (GA-forming) activity of GADH. NADH/NADPH oxidase was purified and identified as the product of ahpF. In an ahpF disruptant, NADH/NADPH activity was almost eliminated, but the reducing activity of GADH was not detected. We also investigated another possible GA-forming enzyme, glyoxal reductase (GR). GR was partially purified and identified as the product of yvgN. yvgN disruptant did not require B6, and retained the ability to synthesize the same amount of B6 as the wild-type strain. From these results, we concluded that neither GADH nor GR is involved in B6 biosynthesis in B. subtilis.

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.

Four methanol-utilizing yeasts, Candida boidinii, Hansenula polymorpha, Hansenula ofunaensis, and Pichia pinus, produced polyols from corresponding sugars in a methanol medium. H. polymorpha produced larger amounts of xylitol than the other yeasts. Productivity was the highest at pH 8 when 5 g (dry)/l cultured cells were incubated with 2.5 g/l urea as the nitrogen source in a medium containing 1% (v/v) methanol and 1 g/l MgSO4.7H2O. Under these conditions, 57 g/l xylitol was obtained from 110 g/l D-xylose after 3 d of cultivation. The largest amount of xylitol (58 g/l; yield, 0.62 g/g) was produced from 125 g/l, D-xylose and 5% (w/v) glycerol instead of methanol after 4 d of cultivation.

A flavinogenic mutant was derived from Candida boidinii by mutagenesis. The mutant was smaller than the wild type, did not grow on a minimal medium, and required l-tryptophan, l-leucine, inositol, and nicotinate for growth. The mutant was defective in the oxidative pentose phosphate pathway, lacking glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The specific activities of the transaldolase and transketolase of the mutant were higher than those of the wild type. These high activities might direct the flux of the carbon source to the nonoxidative pathway with formation of a large amount of pentose phosphates, increasing riboflavin synthesis. Under microaerobic conditions at 25 degrees C, 90 mg/l riboflavin was obtained.

Flow cytometry used in combination with single-cell sorting is a powerful technique for the identification and isolation of microbial cells with particular characteristics, especially when such cells grow more slowly than other cells in a large heterogeneous population. Many applications of flow cytometry with cell sorting, originally used by specialists studying mammalian cells, have been modified so that microorganisms also can be evaluated. The methods can now be used more widely because of the increasing availability of the expensive equipment. There are means for the fluorescence detection of a wide variety of properties, such as amounts of various cell components, specific sequences of peptides and nucleotides, cell functions, and enzyme activities. From the extensive literature, representative reports of an assortment of uses of flow cytometry with cell sorting are reviewed in this article, intended to introduce the technique and its many advantages to microbiologists.

An intracellular enzyme having reduction activity towards dihydroxyacetone (DHA), and that was induced by DHA, was purified and characterized from a methanol-grown yeast, Hansenula ofunaensis. After harvesting cells grown in a 1% methanol medium until the early stationary phase, the enzyme was purified through ammonium sulfate fractination and a series of ion-exchange, hydrophobic, and gel-filtration column chromatographies. SDS-PAGE and HPLC showed the enzyme to be a homo dimer composed of two identical subunits, each with a molecular mass of 38 kDa. The optimum pHs for DHA reduction and glycerol oxidation were 6.0 and 7.0, respectively. The optimum temperature for enzyme activity was 55 degrees C. The enzyme reduced several other compounds, including acetaldehyde, acetol, 2-butanone and 3-methyl-2-butanone, more effectively than it did DHA, while its oxidation activity was higher towards ethanol, 2-propanol, 1,2-propanediol, 2,3-butanediol and 1,3-butanediol than towards glycerol. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The purified enzyme had high K(m) values for glycerol and DHA and low K(m) values for 2-butanol and butanone, although physiologically it had a role in DHA metabolism. There were similarities between the purified enzyme and sec-alcohol dehydrogenases reported previously in their behavior towards inhibitors and metal ions, as well as in their K(m) values for 2-butanol and 2-butanone, but differences in their subunit molecular masses and activities for ethanol. At pH 9.8, the oxidative activity of the purified enzyme for l-2-butanol was about eleven times higher than that for d-2-butanol.