Identification and functional analysis of fructosyl amino acid-binding protein from Gram-positive bacterium Arthrobacter sp.

AIM: Fructosyl amino acid-binding protein (FABP) is a substrate-binding protein (SBP), which recognizes fructosyl amino acids (FAs) as its ligands. Although FABP has been shown as a molecular recognition tool of biosensing for glycated proteins, the availability of FABP is still limited and no FABP was reported from Gram-positive bacteria. In this study, a novel FABP from Gram-positive bacteria, Arthrobacter spp., was reported. METHOD AND RESULTS: BLAST analysis revealed that FABP homologues exist in some of Arthrobacter species genomes. An FABP homologue cloned from Arthrobacter sp. FV1-1, FvcA, contained a putative lipoprotein signal sequence, suggesting that it is a lipoprotein anchored to the bacterial cytoplasmic membrane, which is a typical characteristic for SBPs from Gram-positive bacteria. In contrast, FvcA also exhibits high amino acid sequence similarity to a known Gram-negative bacterial FABP, which exists as a free periplasmic protein. FvcA, without the N-terminal anchoring region, was then recombinantly produced as soluble protein and was found to exhibit Nα-FA-specific binding activity by intrinsic fluorescent measurement. CONCLUSION: This study identified a novel FABP from a Gram-positive bacterium, Arthrobacter sp., which exhibited Nα-FA-specific binding ability. This is the first report concerning an FABP from a Gram-positive bacterium, suggesting that FABP-dependent FA catabolism system is also present in Gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel FABP exhibits the ability to specifically bind to Nα-FA with a high affinity. This selectivity is beneficial for applying FABP in HbA1c sensing. The successful preparation of water-soluble, functionally expressed Gram-negative bacterial FABP may make way for future applications for a variety of SBPs from Gram-positive bacteria employing the same expression strategy. The results obtained here enhance our understanding of bacterial FA catabolism and contribute to the improved development of FABP as Nα-FA-sensing molecules.

Fluorescence resonance energy transfer from pyrene to perylene labels for nucleic acid hybridization assays under homogeneous solution conditions.

We characterized the fluorescence resonance energy transfer (FRET) from pyrene (donor) to perylene (acceptor) for nucleic acid assays under homogeneous solution conditions. We used the hybridization between a target 32 mer and its complementary two sequential 16 mer deoxyribonucleotides whose neighboring terminals were each respectively labeled with a pyrene and a perylene residue. A transfer efficiency of approximately 100% was attained upon the hybridization when observing perylene fluorescence at 459 nm with 347-nm excitation of a pyrene absorption peak. The Förster distance between two dye residues was 22.3 A (the orientation factor of 2/3). We could change the distance between the residues by inserting various numbers of nucleotides into the center of the target, thus creating a gap between the dye residues on a hybrid. Assuming that the number of inserted nucleo-tides is proportional to the distance between the dye residues, the energy transfer efficiency versus number of inserted nucleotides strictly obeyed the Förster theory. The mean inter-nucleotide distance of the single-stranded portion was estimated to be 2.1 A. Comparison between the fluorescent properties of a pyrene-perylene pair with those of a widely used fluorescein-rhodamine pair showed that the pyrene-perylene FRET is suitable for hybridization assays.

Enzymatic synthesis of a novel trehalose derivative, 3,3'-diketotrehalose, and its potential application as the trehalase enzyme inhibitor.

We reported the preparation of a novel trehalose derivative based on enzymatic oxidation of trehalose by water-soluble glucose-3-dehydrogenase (G3DH) from marine bacterium Halomonas sp. alpha-15 cells. The product of G3DH enzymatic conversion was 3,3'-diketotrehalose (3,3'dkT), a novel trehalose derivative of which both third hydroxy groups of glucopyranosides were oxidized. 3,3'dkT was revealed to show an inhibitory effect toward pig-kidney and Bombyx mori trehalases. The IC(50) values of 3,3'dkT were 0.8 and 2.5 mM and K(i) values were 0.2 and 0.6 mM for pig-kidney and for B. mori trehalases, respectively. In addition, 3,3'dkT did not show any inhibitory effect on both maltase and mannosidase activities. Therefore, 3,3'dkT was a specific inhibitor of trehalases.

Thermostable chimeric PQQ glucose dehydrogenase.

The thermal stability of PQQ glucose dehydrogenases (PQQGDHs) which were chimeras with more than 95% made up of the N-terminal region of Escherichia coli PQQGDH and the rest made up of the C-terminal region of Acinetobacter calcoaceticus PQQGDH was investigated. Among the chimeric PQQGDHs, E97A3 (E. coli 97% and A. calcoaceticus 3%) and E95A5 were found to possess higher thermal stability than parental E. coli PQQGDH. Further detailed characterization of the thermal stability was carried out, focusing on E97A3. E97A3 showed a more than 3-fold and 12-fold increase in half life time at 40 degrees C, compared with the PQQGDHs of E. coli and A. calcoaceticus, respectively. Using transition state theory, the increase in the free energy of inactivation observed in E97A3 was compared with those of the E. coli and A. calcoaceticus parental enzymes. The region responsible for this stabilization was also discussed.

Identification of proteins encoded in Escherichia coli hydA, hydB and analysis of the hydA locus.

The hydB gene of Escherichia coli, which is related with the expression of hydrogenase activity, was cloned into the plasmid (pES1). Using the maxicell protein-labeling method, the molecular weight of hydB gene product was estimated. Comparing between the gene products from the mutant strains and that of the hydB genes cloned strains, the molecular weight of the gene product was 35,000 Mr. Similarly, the molecular weight of the gene product of hydA, which had been previously cloned, was determined by maxicell analysis. The molecular weight of hydA gene product was estimated to be 80,000 Mr. Using deletion analysis and Tn1000 insertional inactivation of hydA's function, the hydA coding region was estimated between 2.2 kb and 2.8 kb in a 3.1 kb EcoRI-MluI fragment on the recombinant plasmid pEH3.

Production of gamma-linolenic acid from the marine green alga Chlorella sp. NKG 042401.

gamma-Linolenic acid (GLA) production using a high GLA producing marine green alga, Chlorella sp. NKG 042401, was studied. GLA was presented in the galactolipid fraction (37.9%/total fatty acids). The effects of growth conditions on GLA production were studied. Optimum salinity for GLA production was 5 g l-1, at which salinity the highest cell concentration was achieved, resulting in a 1.6-fold increase in GLA productivity. Total fatty acid, however, was not drastically affected by change of salinity. Nitrogen starvation decreased the ratio of unsaturated fatty acids, and consequently GLA ratio in total fatty acid decreased. The urea adduct method was used to concentrate GLA from crude extract. As a result, after 5 sequential concentration procedures, GLA was concentrated 5-fold with a yield of 49%.

Development of acetylcholine sensor using carbon fiber (amperometric determination).

An enzyme sensor is developed using carbon fiber to measure acetylcholine concentration. The mechanism is based on the detection of H2O2 which is a product of the sequential enzyme reactions of acetylcholinesterase and choline oxidase. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzymes are co-immobilized in polyvinyl alcohol containing styryl pyrydinium (photo-crosslinkable polymer). A fast response time of 0.8 min is obtained. A linear correlation is observed between 0.2 and 1.0 mM. Other optimal operational conditions with respect to pH, temperature and stability are discussed. The use of carbon fiber containing co-immobilized enzymes could offer several novel advantages especially in neuroscience research. In conclusion, the aims of the present work are centered on carbon fiber electrode fabrication, immobilization and electrochemical measurements.

A novel microbial sensor using luminous bacteria.

A novel microbial sensor system that uses luminous bacteria was developed for the determination of both glucose and toxic compounds. The sensor system consisted of a membrane with luminous bacteria immobilized upon it and a photomultiplier. Measurements were based on the in vivo intensity of the light emitted by the bacteria, as this is affected by their environment. A linear relationship was observed between increased luminescence and concentrations of glucose between 0.05 mM and 0.55 mM. The relative standard deviation was 10% for 0.55 mM glucose (n = 10). Toxic compounds such as benzalkonium chloride, sodium dodecyl sulphate and chromium(VI) were also detected by measuring the decrease in luminescence in their presence.

Foreign gene expression in marine cyanobacteria under pseudo-continuous culture.

Foreign gene expression in a marine cyanobacterium, Synechococcus NKBG 15041c, has been carried out in both batch and pseudo-continuous culture, using chloramphenicaol acetyltransferase (CAT) as a model peptide and the broad host range vector pKT230. CAT has been successfully expressed in marine cyanobacteria under kanamycin resistance gene promoter. Pseudo-continuous culture of these recombinant marine cyanobacteria has been done by varying the dilution rate. At a dilution rate between 0.01 to 0.02 h-1, highest productivity was achieved. Under these conditions, long-term pseudo-continuous culture of recombinant marine cyanobacteria was achieved for more than 600 h with CAT productivity 18-fold greater than that observed in batch culture.

Effect of PQQ glucose dehydrogenase overexpression in Escherichia coli on sugar-dependent respiration.

Pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) was overexpressed in Escherichia coli, and its impact on sugar-dependent respiration was investigated. Sugar-dependent respiration patterns under PQQGDH overexpression can be devided into two types. The first type involves D-glucose and D-mannose, which are utilized by the phosphotransferase system (PTS) and are also the substrates of PQQGDH. As a result of PQQGDH overexpression, the apparent Km value of sugar-dependent respiration shifted to higher concentration compared with E. coli parental cells. The second type included D-xylose and D-galactose, which are the substrates of PQQGDH, but not the PTS sugars. PQQGDH overexpressing cells showed much higher respiration than parental cells. These results suggested that PQQGDH overexpression may alter sugar utilization preferences in E. coli, suggesting further possible applications in metabolic engineering for carbon source utilization.

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.

Increased production of recombinant pyrroloquinoline quinone (PQQ) glucose dehydrogenase by metabolically engineered Escherichia coli strain capable of PQQ biosynthesis.

We have previously shown that the production of recombinant Escherichia coli PQQGDH was greatly improved by using a medium supplemented with the cofactor PQQ, which is not synthesized in E. coli. We show here that the increase in the accumulated PQQGDH is due to the increased stability of the holo-enzyme over apo-enzyme, using recombinant Acinetobacter calcoaceticus PQQGDH. In order to achieve cost-effective PQQGDH production, we incorporated the genes for PQQ biosynthetic pathway from Klebsiella pneumoniae into E. coli, which as a result allowed E. coli to produce PQQ. Using this metabolically engineered E. coli strain as a host, a 10-fold increase in the production of recombinant A. calcoaceticus PQQGDH was achieved, compared to the condition without PQQ and MgCl2.

Screening and characterization of fructosyl-valine-utilizing marine microorganisms.

We describe the isolation of microorganisms utilizing fructosyl-amine (Amadori compound) from the marine environment and of fructosyl-amine oxidase from a marine yeast. Using fructosyl-valine (Fru-Val), a model Amadori compound for glycated hemoglobin, we isolated 12 microbial strains that grow aerobically in a minimal medium supplemented with Fru-Val as the sole nitrogen source. Among these strains, a yeast strain identified as Pichia sp. N1-1, produced a Fru-Val-oxidizing enzyme. The enzyme was purified in its active form, a single-polypeptide water-soluble protein of 54 kDa by gel electrophoresis, producing H(2)O(2) with the oxidation of Fru-Val. By its substrate specificity, the enzyme was categorized as a novel fructosyl-amine oxidase. This is the first study on the isolation of microorganisms utilizing fructosyl-amine in the marine environment and of fructosyl-amine oxidase from budding yeast.

Basic studies of hydrogen evolution by Escherichia coli containing a cloned Citrobacter freundii hydrogenase gene.

Citrobacter freundii genes that complemented Escherichia coli hyd-(hydrogenase activity) mutation were cloned in plasmids pCBH4 (6.2 kb) and pCBH6(5.7 kb). Hydrogen evolution by the transformant E. coli HK-8(pCBH4 or pCBH6) was investigated. The optimum culture temperature of recombinant E. coli cells for hydrogen evolution from glucose was in the neighborhood of 18 degrees C. The recombinant E. coli cells cultured at this condition showed a several-fold increase of hydrogen evolution, as compared with that of the wild-type cells. The plasmid-retention stability of this recombinant E. coli was extremely high, especially plasmid pCBH4, which was completely retained during 2 wk without any restriction. Hydrogen production by immobilized recombinant E. coli was then investigated using cells cultured at 18 degrees C. The hydrogen evolution rate from glucose and Lennox-broth were about twofold higher than that of E. coli C600, and this high hydrogen evolution rate was maintained for more than 1 mo.

Cloning of a marine cyanobacterial promoter for foreign gene expression using a promoter probe vector.

A marine cyanobacterial promoter was cloned to allow efficient foreign gene expression. This was carried out using chloramphenicol acetyl transferase (CAT) as a marker protein. For rapid and simple measurement of CAT activity, a method based on a fluorescently labeled substrate was improved by utilizing HPLC equipped with a flow-through fluorescent spectrophotometer. This method was used in conjunction with a newly constructed promoter probe vector. Cyanobacterial transformants, harboring plasmid containing a cloned 2-kbp marine cyanobacterial genomic fragment, showed a 10-fold higher CAT activity, compared with that achieved using the kanamycin-resistant gene promoter. From the sequence analysis of the cloned fragment, a putative promoter region was found.

Biodegradation of formaldehyde by a formaldehyde-resistant bacterium isolated from seawater.

A formaldehyde-tolerant bacterium designated as a DM-2 strain was used to biodegrade formaldehyde. The cells, precultivated in the presence of 400 ppm of formaldehyde, were able to degrade formaldehyde in a minimal medium supplemented with up to 400 ppm of formaldehyde in the presence of 3% NaCl. The rate of formaldehyde degradation achieved in this study was 45 ppm/h when the DM-2 culture's optical density at 660 nm was 1.2.

Site-directed mutagenesis study on the thermal stability of a chimeric PQQ glucose dehydrogenase and its structural interpretation.

We have previously reported that a chimeric pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH), E97A3, which was made up of 97% of Escherichia coli PQQGDH sequence and 3% of Acinetobacter calcoaceticus PQQGDH, showed increased thermal stability compared with both parental enzymes. Site-directed mutagenesis studies were carried out in order to investigate the role of amino-acid substitution at the C-terminal region, Ser771, of a chimeric PQQGDHs on their thermal stability. A series of Ser771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction governs the thermal stability of the chimeric enzymes. Comparison of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97A3 acquired stability at the first step of denaturation, which is reversible, and where no significant secondary structure change was observed. These results suggested that the interaction between C-terminal and N-terminal regions may play a crucial role in maintaining the overall structure of beta-propeller proteins.

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase.

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphonothioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the Ki value of each enantiomer toward electric eel acetylcholinesterase. R-EPN (Ki = 6 microM) inhibited acetylcholinesterase much more effectively than S-EPN (Ki = 52 microM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The Km value for R-EPN decreased from 0.24 to 0.03 mM, and the Vmax value increased from 0.25 to 0.60 U/mg of protein. Vmax/Km values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Facile isolation of endo-pectate lyase from Erwinia carotovora based on electrostatic interaction.

Endo-pectate lyase (PATE) from Erwinia carotovora was selectively cosedimented with extracellularly produced lipopolysaccharide-lipid complex (LPSLC) through dialysis of the cell free culture broth. The selective isolation of PATE was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The cosedimentation of the PATE with LPSLC was initiated by decreasing conductivity of the solution and terminated at approx 1 m siemens (mScm-1). As much as 62% of PATE activity in the culture broth was removed by precipitation. PATE was isolated from the precipitate by gel chromatography. The cosedimentation of PATE with LPSLC was remarkably affected by pH or ionic strength. The addition of polycationic peptide polymyxin B sulfate or a metal chloride affected the interaction. The cosedimentation was diminished by acetylation of the free amino groups of PATE. From these results, it was confirmed that the cosedimentation was induced by electrostatic interaction.

Purification of a marine bacterial glucose dehydrogenase from Cytophaga marinoflava and its application for measurement of 1,5-anhydro-D-glucitol.

A novel glucose dehydrogenase (GDH) from a marine bacterium Cytophaga marinoflava IFO 14170 was isolated from its membrane fraction. This GDH catalyzes the oxidation of a hydroxy group of glucose, but does not react in its C-1 position. This enzyme is composed of a single peptide with a mol wt of 67,000. The GDH can react under high salinity. The optimum pH is around 8.0, showing typical property of marine bacterial enzymes. Using this novel enzyme, and enzymatic determination of 1,5-anhydro-D-glucitol (1,5AG) utilizing 2,6-dichrolophenolindophenol (DCIP) and phenazine methosulfate (PMS) as electron mediators was carried out. A good linear correlation was observed from 0.5 mM to 4 mM of 1,5AG.