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In this study, we developed a green light-regulated T7 RNA polymerase expression system (T7 RNAP system), to provide a novel and versatile high-expression system for cyanobacteria without using any chemical inducer, realizing high expression levels comparable with previously reported for recombinant gene expression in cyanobacteria. The T7 RNAP system was constructed and introduced into Synechocystis sp. PCC6803. T7 RNAP was inserted downstream of the cpcG2 promoter, which is recognized and activated by the CcaS/CcaR two-component green-light-sensing system, to compose a vector plasmid, pKT-CS01, to achieve the induction of T7 RNAP expression only under green light illumination, with repression under red light illumination. The reporter gene, superfolder green fluorescent protein (sfGFP), was inserted downstream of the T7 promoter. Transcriptional analyses revealed that T7 RNAP was induced under green light but repressed under red light. Expression of the sfGFP protein derived from pKT-CS01 was observed under green light illumination and was approximately 10-fold higher than that in the control transformant, which expressed sfGFP directly under the cpcG2 promoter, which is directly regulated by CcaS/CcaR, under green light illumination. Comparison with the strong promoter expression systems Pcpc560 and PtrcΔlacO revealed that the expression of sfGFP by the T7 RNAP system was comparable with the levels obtained with strong promoters. These results demonstrated that the green light-regulated T7 RNAP gene expression system will be a versatile tool for future technological platform to regulate gene expression in cyanobacterial bioprocesses. [ABSTRACT FROM AUTHOR]

An important feature offered by marine cyanobacterial strains over freshwater strains is the capacity to grow in seawater, replacing the need for often-limited freshwater. However, there are only limited numbers of marine cyanobacteria that are available for genetic manipulation and bioprocess applications. The marine unicellular cyanobacteria Synechococcus sp. strain NKBG 15041c (NKBG15041c) has been extensively studied. Recombinant DNA technologies are available for this strain, and its genomic information has been elucidated. However, an investigation of carbohydrate production, such as glycogen production, would provide information for inevitable biofuel-related compound production, but it has not been conducted. In this study, glycogen production by marine cyanobacterium NKBG15041c was investigated under different cultivation conditions. NKBG15041c yielded up to 399 μg/ml/OD730 when cells were cultivated for 168 h in nitrogen-depleted medium (marine BG11ΔN) after medium replacement (336 h after inoculation). Cultivation under nitrogen-limited conditions also yielded an accumulation of glycogen in NKBG15041c cells (1 mM NaNO3, 301 μg/ml/OD730; 3 mM NaNO3, 393 μg/ml/OD730; and 5 mM NaNO3, 328 μg/ml/OD730) under ambient conditions. Transcriptional analyses were carried out for 13 putative genes responsible for glycogen synthesis and catabolism that were predicted based on homology analyses with Synechocystis sp. PCC 6803 (PCC6803) and Synechococcus sp. PCC7002 (PCC7002). The transcriptional analyses revealed that glycogen production in NKBG15041c under nitrogen-depleted conditions can be explained by the contribution of both increased carbon flux towards glycogen synthesis, similar to PCC6803 and PCC7002, and increased transcriptional levels of genes responsible for glycogen synthesis, which is different from the conventionally reported phenomenon, resulting in a relatively high amount of glycogen under ambient conditions compared to PCC6803 and PCC7002. [ABSTRACT FROM AUTHOR]

Cyanobacteria are one of the most attractive hosts for biofuel production; however, genetic approaches to regulate specific chromosomal genes in cyanobacteria remain limited. With the aim of developing a novel method to regulate chromosomal gene expression in cyanobacteria, we focused on riboregulatory technology. Riboregulators are composed of two RNA fragments whose interaction leads to target gene regulation with high specificity. In this study, we inserted a riboregulator sequence upstream of the chromosomal gene encoding AbrB-like transcriptional regulator, cyAbrB2, to investigate the utility of this tool. The inserted riboregulator was able to regulate cyabrB2 gene expression, with a high ON-OFF ratio up to approximately 50-fold. The transcription levels of several genes for which cyAbrB2 acts as a transcriptional upregulator were also decreased . Further, the cyAbrB2 expression-repressed mutant showed high glycogen accumulation, equivalent to that in the cyabrB2 deletion mutant (Δ cyabrB2). Phenotypic similarities between the cyabrB2 expression-repressed mutant and the Δ cyabrB2 mutant suggest that the riboregulator can potentially be used as a new chromosomal gene regulation tool in cyanobacteria. [ABSTRACT FROM AUTHOR]

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme's catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding. Protein crystal growth experiments in a microgravity environment provide insight into the design of an engineered direct electron transfer type glucose dehydrogenase with increase thermal stability for glucose sensor applications. [ABSTRACT FROM AUTHOR]

A green-light-regulated gene expression system derived from Synechocystis sp. PCC 6803 was constructed and introduced into the marine cyanobacterial strain Synechococcus sp. NKBG 15041c. The regulation system was evaluated using gfp as a reporter gene under red-light illumination and under simultaneous red- and green-light illumination. Expression of the reporter gene was effectively repressed under red-light illumination and increased over 10-fold by illuminating with green light. Control vectors missing either the ccaS sensor histidine kinase gene or the ccaR response regulator gene showed no detectable induction of GFP expression. Green-light induction of gfp expression was further confirmed by quantitative reverse transcription PCR. The constructed system was effective at regulating the recombinant expression of a target gene using green light in a marine cyanobacterial strain that does not naturally possess such a green-light regulation system. Thus, constructed green-light-regulated gene expression system may be used as a core platform technology for the development of marine cyanobacterial strains in which bioprocesses will be regulated by light. [ABSTRACT FROM AUTHOR]

Objective: To improve the stability of E. coli-produced non-glycosylated fungal FA d-glucose dehydrogenase induced a disulfide bond by site-directed mutagenesis based on structural comparisons with glucose oxidases. Results: The FAD-glucose dehydrogenase (GDH) mutant Val149Cys/Gly190Cys, which was constructed based on a comparison with the three dimensional structure of glucose oxidase, showed a 110 min half-life of thermal inactivation at 45 °C, which is 13-fold greater than that of the wild-type enzyme. The considerable increase in thermal stability was further supported by Eyring plot analysis. The kinetic parameters of Val149Cys/Gly190Cys ( k = 760 s, K = 35 mM, and catalytic efficiency ( k/K) = 22 smM) were almost identical to those of the wild-type enzyme ( k = 780 s, K = 35 mM, k/K = 22 smM). The substrate specificity of Val149Cys/Gly190Cys is indistinguishable from that of the wild type. Conclusion: The constructed mutant, Val149Cys/Gly190Cys, had significantly increased structural stability without changing the catalytic activity and kinetic parameters of FAD-GDH, including its characteristic substrate specificity. [ABSTRACT FROM AUTHOR]

Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain Δ adhE/PCK. The strain produced succinate from glucose with a 60.5 % yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions. [ABSTRACT FROM AUTHOR]

Cyanobacteria are attractive host bacteria for biofuel production because they can covert CO to biofuel lipids using only sunlight, water, and inorganic ions. For genetically engineering an ideal cyanobacterium, a synthetic biological approach is promising but few genetic components have been characterized in cyanobacteria. Here for controlling cyanobacterial protein expression, we constructed riboregulators, that one of the post-transcriptional regulators composed of RNAs. Riboregulators harboring a ribosome-binding site suitable for Synechocystis sp. were designed by trial and error using Escherichia coli as host bacteria. The designed riboregulators were effective in Synechocystis sp. as well as E. coli with slight interference on growth only observed in E. coli. They will therefore be useful tools for controlling target gene expression. [ABSTRACT FROM AUTHOR]

The recently discovered fructosyl peptide oxidase from Phaeosphaeria nodorum (PnFPOX) was demonstrated to react with the glycated hexapeptide measurement standard of hemoglobin A1c, fVHLTPE. The highly reactive Coniochaeta FPOX (FPOX-C) showed no detectable activity with the hexapeptide. Two loop regions were identified as having important effects on the enzymatic properties of FPOX. The first loop has a strong influence on the ability to bind larger glycated peptides, while the second loop has a significant effect on catalytic activity. Loop-substitution mutants showed that the highest activity against fVHLTPE resulted from the combination of the first loop from PnFPOX and the second loop from FPOX-C. The most promising engineered FPOX created, which showed 17-fold greater dehydrogenase activity against fVHLTPE than wild-type PnFPOX, was the FPOX-C mutant with a PnFPOX-derived loop 1 region and an Asn56Ala substitution. [ABSTRACT FROM AUTHOR]

Fructosamine-6-kinases (FN6Ks) that catalyze phosphorylation of glycated amino acids, i.e., fructosyl amino acids (FAs), have been shown as a potential recognition element for glycated protein detection. However, there are only two available FN6Ks: those from Escherichia coli which is specific for ε-fructosyl lysine (ε-FK) and Bacillus subtilis which recognizes both ε-FK and α-FA as substrates. In this study, we characterized an FN6K homologue isolated from Arthrobacter, some of whose species are reported to assimilate FA. The BLAST searches of Arthrobacter genomic database, using the bacterial FN6K primary structure information, revealed the presence of an FN6K homologue in Arthrobacter aurescens TC1 strain. Indeed, enzymatic assays confirmed that the putative FN6K from A. aurescens is an FN6K that is specific for ε-FK, although the primary sequence alignments showed similarity of A. aurescens FN6Ks with FN6Ks from B. subtilis and E. coli at the same level. In this study, we describe for the first time the presence of FN6K in Arthrobacter spp. and ε-FK-specific degradation pathway from Gram-positive bacteria, providing important information for the development of FA-recognizing molecules as well as for the FA assimilation system in bacteria. [ABSTRACT FROM AUTHOR]

A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold. [ABSTRACT FROM AUTHOR]

Current enzymatic methods for the analysis of glycated proteins use flavoenzymes that catalyze the oxidative deglycation of fructosyl peptides, designated as fructosyl peptidyl oxidases (FPOXs). However, as FPOXs are oxidases, the signals derived from electron mediator-type electrochemical monitoring based on them are affected by dissolved O. Improvement of dye-mediated dehydrogenase activity of FPOXs and its application to enzyme electrode construction were therefore undertaken. Saturation mutagenesis study on Asn56 of FPOX from Phaeosphaeria nodorum, produced mutants with marked decreases in the catalytic ability to employ O as the electron acceptor, while showing higher dye-mediated dehydrogenase activity employing artificial electron acceptors than the parental enzyme. Thus constructed virtually fructosyl peptide dehydrogenase, Asn56Ala, was then applied to produce an enzyme electrode for the measurement of fructosyl- N-valyl-histidine (f-Val-His), the protease-digested product of HbA1c. The enzyme electrode could measure f-Val-His in the physiological target range in air. [ABSTRACT FROM AUTHOR]

Aspergillus-derived FAD-dependent glucose dehydrogenases (FADGDHs) were screened from fungal genomic databases, primarily by searching for putative homologues of the Aspergillus niger-derived glucose oxidase (GOD). Focusing on a GOD active-site motif, putative proteins annotated as belonging to the glucose methanol choline (GMC) oxidoreductase family were selected. Phylogenetic analysis of these putative proteins produced a GOD clade, which includes the A. niger and Penicillium amagasakiens GODs, and a second clade made up of putative proteins showing 30-40% homology with GOD. The genes encoding the proteins from the second clade were functionally expressed in Escherichia coli, resulting in dye-mediated glucose dehydrogenase (GDH) activity but not GOD activity. These results suggest that the putative proteins belonging to the second clade are FADGDHs. The 3D structure models of these FADGDHs were compared with the 3D structure of GOD. [ABSTRACT FROM AUTHOR]

Motif analysis of PGa4 indicated that eight consecutive guanine residues were most important for pyrroloquinoline quinine glucose dehydrogenase (PQQGDH) recognition. Because the truncated mutants minimized unexpected interactions between PGa4 and the target-binding aptamer, they will now facilitate the construction of a highly sensitive detection system using a PQQGDH-labeled diagnostic marker-binding aptamer. [ABSTRACT FROM AUTHOR]

The flavoenzyme fructosyl amino acid oxidase (FAOD) catalyzes the oxidative deglycation of fructosyl amino acids, model compounds of glycated proteins. The high oxygen reactivity of FAODs limits their potential utility in amperometric enzyme sensors employing artificial electron mediators. To alter their electron acceptor availability, site-directed mutagenesis was carried out on conserved residues predicted to be involved in the proton relay system (PRS) of two eukaryotic FAODs, the FAOD from the marine yeast Pichia sp. N1-1 and amadoriase II from the fungus Aspergillus fumigatus. The substitution of a single conserved Asn residue in the putative PRS, Asn47Ala of N1-1 FAOD and Asn52Ala of amadoriase II, resulted in significant loss in the catalytic ability to employ O2 as the electron acceptor, while having little effect on the dye-mediated dehydrogenase activity employing artificial electron acceptors instead of O2. [ABSTRACT FROM AUTHOR]

A wireless biosensor system was developed for the continuous measurement of blood glucose levels in flatfish. The biosensor was implanted in the interstitial fluid under the scleral surface of the eyeball (EISF) to investigate the relationship between EISF and blood glucose levels. EISF glucose levels were found to be correlated with those in the blood and to be approximately the same as blood glucose levels in the range of 7–25 mg dl−1. A needle-type biosensor was prepared for the continuous EISF glucose monitoring in flatfish. A working electrode was constructed using platinum iridium wire, and glucose oxidase was immobilized to the electrode. The biosensor was inserted into the EISF of flatfish for sensor implantation. A 650-mV potential (vs. Ag/AgCl) was applied by a wireless potentiostat to the working electrode for the amperometric glucose measurement. We investigated whether glucose in the EISF can be determined in vivo. The estimated glucose levels using a one-point calibration method were correlated with actual blood glucose levels. In conclusion, using a wireless biosensor system, we were able to monitor blood glucose levels in flatfish under free-swimming conditions for 16 h. [ABSTRACT FROM AUTHOR]

Cellobiose dehydrogenase (CDH; EC 1.1.99.18) is an extracellular glycosylated protein composed of two distinct domains, a C-terminal catalytic flavin domain and an N-terminal cytochrome-b-type heme domain, which transfers electrons from the flavin domain to external electron acceptors. The soluble flavin domain of the Phanerochaete chrysosporium CDH was successfully expressed in Escherichia coli. The enzyme showed dye-mediated CDH activity higher than that of the complete CDH, composed of flavin domain and heme domain, prepared using Pichia pastoris as the host microorganism. The ability to conveniently express the recombinant CDH flavin domain in E. coli provides great opportunities for the molecular engineering of the catalytic properties of CDH. [ABSTRACT FROM AUTHOR]

α-Synuclein is a native, unfolded protein that causes several neurodegenerative diseases such as dementia with Lewy bodies and Parkinson’s disease. We have now identified the first DNA aptamers against α-synuclein using native PAGE applied to the SELEX method. We call this aptamer “M5-15”; it is the α-synuclein-bound aptamer and was isolated after four cycles of screening. M5-15 is composed of three stem-loop structures that may play an important role in the binding to α-synuclein. Moreover, M5-15 specifically binds to the α-synuclein monomer and oligomer. We expect that this aptamer will become a useful tool in α-synuclein analysis and diagnosis. [ABSTRACT FROM AUTHOR]

We have detected PCR products from Salmonella spp. and Influenza A virus using Zn finger protein Zif268 and Sp1, respectively. Previously, we demonstrated a novel method of rapid and specific detection of PCR products from Legionella pneumophila genome using Zn finger protein Sp1. In principle, this methodology might be applied to the detection of most bacteria and viruses using various Zn finger proteins. Here, to demonstrate the wider applicability of our method, we detected PCR products from Salmonella spp. and the Influenza A virus. BLAST data indicated the Zif268 and Sp1 recognition sequence were located on the gyrB gene of Salmonella spp. and the nucleoprotein gene of Influenza A virus, respectively. The PCR products from the oligonucleotide corresponding to the gyrB gene of Salmonella spp. or the nucleoprotein gene of the Influenza A virus could be specifically detected by ELISA or fluorescence depolarization measurement using Zif268 or Sp1. These results indicate the wide applicability of our novel methodology. [ABSTRACT FROM AUTHOR]

A FAD-dependent glucose dehydrogenase (FADGDH) mutant with narrow substrate specificity was constructed by site-directed mutagenesis. Several characteristics of FADGDH, such as high catalytic activity and high electron transfer ability, make this enzyme suitable for application to glucose sensors. However, for further applications, improvement of the broad substrate specificity is needed. In this paper, we mutated two residues, Asn475 and Ala472, which are located near the putative active site of the catalytic subunit of FADGDH and have been predicted from the alignment with the active site of glucose oxidase. Of the 38 mutants constructed, Ala472Phe and Asn475Asp were purified and their activities were analyzed. Both mutants showed a higher specificity toward glucose compared to the wild type enzyme. [ABSTRACT FROM AUTHOR]

Enzyme biofuel cells utilizing glucose dehydrogenase as an anode enzyme were constructed. The glucose dehydrogenase is composed of a catalytic subunit, an electron transfer subunit, and a chaperon-like subunit. Cells, constructed using either a glucose dehydrogenase catalytic subunit or a glucose dehydrogenase complex, displayed power outputs that were dependent on the glucose concentration. The catalytic subunit in the anode maintained its catalytic activity for 24 h of operation. The biofuel cell which composed of glucose dehydrogenase complex functioned successfully even in the absence of an electron mediator at the anode cell. These results indicate the potential application of this thermostable glucose dehydrogenase for the construction of a compartment-less biofuel cell. [ABSTRACT FROM AUTHOR]

A novel glucose-sensing molecule was created based on galactose/glucose-binding protein (GGBP). GGBP mutants at Asp14, a residue interacting with the 4th hydroxyl group of the sugar molecule, were constructed by mutagenesis to improve the ligand specificity of GGBP. The autofluorescence-based analysis of the binding abilities of these engineered GGBPs showed that the GGBP mutants Asp14Asn and Asp14Glu bound only to glucose in a concentration-dependent manner, without being affected by the presence of galactose. The Phe16Ala mutation, which leads to an increase in the K d value toward glucose, was then introduced into these two glucose-specific mutant GGBPs. One of the constructed GGBP double-mutants, Asp14Glu/Phe16Ala, had a glucose specificity with a K d value of 3.9 mM, which makes it suitable for use in the measurement of the physiological glucose concentration. Our results demonstrate that it is possible to construct a GGBP which specifically recognizes glucose and has a higher K d value and use it as a molecular recognition element of blood glucose monitoring systems by combining two different mutations based on the 3D structure of GGBP. [ABSTRACT FROM AUTHOR]

Two DNA aptamers against a tumor marker protein, human vascular endothelial growth factor (VEGF165) were identified. In the screening process, another protein was used as the competitor to isolate those aptamers that have high specificity for the target. In addition, we evaluated the affinities of the enriched library by means of aptamer blotting. The isolated aptamers bound to VEGF165 with a K d value in the range of a few hundred nanomoles, and did not bind to the competitor. This selection method enabled us to efficiently select the specific aptamers against the target protein. These specific aptamers would be useful sensor elements for cancer diagnosis. [ABSTRACT FROM AUTHOR]

We have developed an aptameric enzyme subunit (AES) for immunoglobulin E (IgE) sensing. AES is an artificial enzyme subunit constructed from two different aptamers and does not require any modification. Using the AES, the target molecule can be detected by measuring enzymatic activity in homogeneous solution. We connected IgE-binding aptamer and its complementary strand to split thrombin-inhibiting aptamer. The hybrid of these two oligonucleotides inhibited thrombin activity and it decreased in the presence of IgE. We were able to detect IgE by using this AES in homogeneous solution with a detection limit of 50 pmol. [ABSTRACT FROM AUTHOR]

We have developed an aptameric enzyme subunit (AES) which can detect the DNA in a homogeneous solution. The AES is an artificial enzyme subunit composed of an enzyme-inhibiting aptamer bearing a target-molecule binding site. We connected a probe DNA to a thrombin-inhibiting aptamer at its 5′ or 3′ end. The inhibitory activity of the thrombin-inhibiting aptamer bearing the probe DNA decreased compared to that of the original aptamer; however, it recovered upon hybridization with the target DNA. Using this AES, we were able to detect target DNAs by measuring the thrombin activity in a homogeneous solution. [ABSTRACT FROM AUTHOR]

Using an evolution-mimicking algorithm (EMA), we have recently identified DNA aptamers that inhibit Taq DNA polymerase. In the present study, we have attempted to improve further the inhibitory activities of aptamers, as well as to characterize those aptamers with the most potent inhibitory activities. To characterize the most potent aptamer and demonstrate its applicability, the abilities to inhibit Tth DNA polymerase and to modulate specific amplification in PCR were investigated. This aptamer inhibited both Tth DNA polymerase and Taq DNA polymerase and improved the specificity of detection of a low-copy-number target gene in PCR using these DNA polymerases. [ABSTRACT FROM AUTHOR]

We previously identified a thrombin-inhibiting DNA aptamer that was presumed to form a G-quartet structure with a duplex. To investigate the importance of the sequences in the duplex region and to obtain aptamers with higher inhibitory activities, we randomized the sequences of the duplex region of this aptamer and carried out selection based on inhibitory activity using a genetic algorithm. This method consisted of selection via an inhibition assay, crossover, and mutation in silico. After two cycles, we obtained ligands with greater inhibitory activities than that of the original aptamer. In addition, the duplex sequences were found to contribute to the inhibitory activities of aptamers. [ABSTRACT FROM AUTHOR]

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V max K value for fructosyl valine, the V max K value for fructosyl-ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-ε N-lysine. [ABSTRACT FROM AUTHOR]

Aptamers are oligonucleotide ligands with a high affinity to, and specificity for, various target molecules and they are expected to be powerful tools for proteomic analysis. To select aptamers that bind to a specific unidentified protein in tissues for protein analysis, a screening method was developed using chicken skeletal muscle as a model. Target proteins in the target mixture were separated by electrophoresis and transferred to a membrane, and a DNA library was added onto it. The aptamers that bound to the target protein were visualized by chemiluminescence and collected by cutting out the visualized band. The specific aptamers to the target protein were selected by only one round of selection using this screening, suggesting this screening method might be useful for selecting aptamers for proteome analysis. [ABSTRACT FROM AUTHOR]

A general molecular imprinting approach is proposed to synthesize artificial enzymes to mimic the family of cyclic amide amidohydrolases which share similar active site and catalytic mechanism. The artificial enzymes were constructed by co-polymerizing 4(5)-vinylimidazole-Co2+-methacrylic acid clusters with divinylbenzene micro-spheres in the presence of corresponding substrates. The artificial enzymes mimicked creatininase and hydantoinase by showing specific affinity towards the corresponding substrates in buffer. The artificial hydantoinase also showed specific affinity towards corresponding substrate in organic solvent, and catalyzed the hydrolysis of hydantoin. [ABSTRACT FROM AUTHOR]

In order to maximize the efficiency of methane fermentation on short-chain fatty acids, growth media containing acetic acid and butyric acid as major carbon sources were supplied to a thermophilic down-flow anaerobic packed-bed reactor. The organic loading rate (OLR) to the reactor ranged from 0.2 to 169 kg-dichromate chemical oxygen demand(CODcr)/m3-reactor/day, corresponding to a hydraulic retention time (HRT) of between 1.4 h and 20 days. Stable methane production was maintained at HRTs as short as 2 h (OLR=120 kg-CODcr/m3/day), with the short-chain fatty acids in the feed almost completely removed during the process. The apparent substrate removal efficiency, determined from the total CODcr values in the influent and effluent, was 75% at short HRTs. However, the actual substrate removal efficiency must have been greater than 75%, since a fraction of substrate was also utilized in microbial cell synthesis, and these cells were part of the measured total CODcr. [ABSTRACT FROM AUTHOR]

AnArthrobactersp. was isolated that, when induced by fructosyl-valine, expressed a fructosyl-amine oxidase (FAOD) that was specific fora-glycated amino acids. TheN-terminal amino acid sequence of the purified oxidase was determined and used to design oligonucleotides to amplify the gene by inverse PCR. Expression of the gene inEscherichia coliproduced 0.23 units FAOD per mg protein, over 30-fold greater than native expression levels, with properties almost indistinguishable from the native enzyme. The presence of FAOD was confirmed in otherArthrobacterssp. [ABSTRACT FROM AUTHOR]

The gene encoding the fructosyl-amine oxidase (FAOD) from the marine yeast Pichia sp. N1-1 was cloned and expressed in Escherichia coli. Partial amino acid sequence analysis of the Pichia sp. N1-1 FAOD allowed the design of oligonucleotide primers for the amplification of the gene by inverse polymerase chain reaction. The FAOD gene was found to be devoid of introns and to encode a 48-kDa protein composed of 429 amino acid residues. The FAD-binding consensus sequence GXGXXG and the FAD covalent attachment-site cysteine residue have been identified within the predicted amino acid sequence. Comparisons with the amino acid sequences of other eukaryotic FAODs showed only 30% to 40% identities, establishing that the isolated Pichia N1-1 gene encodes a unique FAOD. Recombinant FAOD expression levels in E. coli reached 0.48 U/mg of soluble protein, which is considerably greater than native expression levels by inducing Pichia sp. N1-1 with fructosyl-valine (f-Val). The kinetic properties of the recombinant enzyme were almost indistinguishable from those of the native enzyme. We previously reported on the construction of a number of effective Pichia sp. N1-1 FAOD-based biosensors for measuring f-Val, a model compound for glycated hemoglobin. The further development of these biosensor systems can now greatly benefit from protein engineering and recombinant expression of the FAOD from Pichia N1-1. [ABSTRACT FROM AUTHOR]

The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification. The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH). During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme. After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample. Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability. Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process. [ABSTRACT FROM AUTHOR]

An artificial phosphotriesterase (PTE) was constructed by co-polymerization of 4(5)-vinylimidazole-Zn2+-methacrylic acid cluster with a divinylbenzene polymer. Compared with the spontaneous hydrolysis, the resulting polymer catalyst caused 105-fold rate acceleration towards the hydrolysis of diethyl p-nitrophenyl phosphate (Paraoxon). The catalytic activity of the polymer catalyst could be enhanced for 30% using molecular imprinting technique and the molecularly-imprinted catalyst (MIC) showed a turnover rate of 7.4×10-2 s-1 towards the hydrolysis of Paraoxon. The MIC also hydrolyzed thiophosphates and phosphorothiolate triester pesticides. Construction of an amperometric sensor employing the MIC as catalyst achieved a detection limit of 0.1 mM Paraoxon. [ABSTRACT FROM AUTHOR]

Water-soluble quinoprotein glucose dehydrogease (PQQGDH-B) is a dimeric enzyme whose application for glucose sensing is the focus of much attention. We attempted to increase the thermal stability of PQQGDH-B by introducing a disulfide bond at the dimer interface. The Ser residue at position 415 was selected for substitution with Cys, as structural information revealed that its side chains face each other at the dimer interface of PQQGDH-B. PQQGDH-B with Ser415Cys showed 30-fold greater thermal stability at 55°C than did the wild-type enzyme without any decrease in catalytic activity. After incubation at 70°C for 10 min, Ser415Cys retained 90% of the GDH activity of the wild-type enzyme. Disulfide bond formation between the mutant subunits was confirmed by analyses with sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence and absence of reductants. Our results indicate that the introduction of one Cys residue in each monomer of PQQGDH-B resulted in formation of a disulfide bond at the dimer interface and thus achieved a large increase in the thermal stability of the enzyme. [ABSTRACT FROM AUTHOR]

A new approach in altering the substrate specificity of enzyme is proposed using glucose dehydrogenase, with pyrroroquinoine quinone (PQQGDH) as co-factor, as the model. This approach is based on the selection of random peptide phage displayed library. Using an M13 phage-display random peptide library, we have selected peptide ligands. Among the peptide ligands, a 7-mer peptide, composed of Thr-Thr-Ala-Thr-Glu-Tyr-Ser, caused PQQGDH substrate specificity to decrease significantly toward disaccharides, such as maltose and lactose, while a smaller effect was observed toward glucose. Consequently, this peptide narrowed the substrate specificity of PQQGDH, without a significant loss of the enzyme activity. [ABSTRACT FROM AUTHOR]

An enzyme-sensor system with flow-injection analysis (FIA) has been developed for the detection of fructosyl amine compounds; the sensor utilizes fructosyl amine oxidase isolated from the marine yeast Pichia sp. N1-1 strain. With this FIA system 0.2 to 10 mmol L–1 fructosyl valine can be determined. The sensor is approximately five times more sensitive to fructosyl valine, a model compound for glycated hemoglobin HbA1c, than to N ε-fructosyl lysine, a model compound for glycated albumin. This FIA system can also be used to detect fructosyl dipeptides. The operational stability of the sensor enabled more than 120 consecutive sample injections over a period of approximately 20 h. [ABSTRACT FROM AUTHOR]

A formaldehyde-tolerantbacterium 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. [ABSTRACT FROM AUTHOR]

A multi-chimeric enzyme was constructed by combining the protein regions responsible for the enzymatic properties of Escherichia coli and Acinetobacter calcoaceticus pyrroloquinoline quinone glucose dehydrogenase (PQQGDH). The constructed multi-chimeric PQQGDH showed increased co-factor binding stability, thermal stability, an alteration in substrate specificity and a 10-fold increase in the Km value for glucose compared with the wild-type E. coli PQQGDH. The cumulative effect of each introduced protein region on the improvement of enzymatic properties was observed. The application of the multi-chimeric PQQGDH in amperometric glucose sensor construction achieved an expanded dynamic range together with increased operational stability and narrower substrate specificity. The glucose sensor can measure glucose from 5 to 40 mM, suggesting its potential for the direct measurement of high blood-glucose levels in diabetic patients. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae, which produces PQQ and is available for use with a conventional expression vector system, was selected as the host strain for soluble PQQ glucose dehydrogenase (PQQGDH-B) production. The recombinant K. pneumoniae expressed PQQGDH-B in its holo-form at about 18 000 U l-1, equal to that achieved in recombinant Escherichia coli. The signal sequence of recombinant PQQGDH-B produced by K. pneumoniae was correctly processed. K. pneumoniae can become an alternative host microorganism not only for PQQGDH-B production but also for recombinant PQQ enzymes production. [ABSTRACT FROM AUTHOR]

A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. α-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion of 1,5-anhydro- d-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol (3-ketoAG), which was identified by 13C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L·h), and the electrochemical yield of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%. [ABSTRACT FROM AUTHOR]

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 phenylphosphono-thioate (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 K i value of each enantiomer toward electriceel acetylcholinesterase. R-EPN ( K i=6 μ M) inhibited acetylcholinesterase much more effectively than S-EPN ( K i=52 μ M) 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 K is value for R-EPN decreased from 0.24 to 0.03 m M, and the V max value increased from 0.25 to 0.60 U/mg of protein. V max/ K is values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2. [ABSTRACT FROM AUTHOR]

Using the structural gene of a homo dimeric enzyme, the water-soluble pyrroloquinoline quinone glucose dehydrogenase (PQQGDH-B), a gene consisting of two identical subunits linked together by a DNA segment coding linker peptide region was constructed. Using the constructed gene, a linked-dimeric PQQGDH-B was produced in Escherichia coli as the active soluble enzyme. Linked-dimeric PQQGDH-B showed a larger increase in thermal stability than the native dimeric enzyme. During incubation over 45 °C, the residual activity of linked-dimeric PQQGDH-B was more than twice that of the native dimeric enzyme. The potential application of linked-dimeric PQQGDH-B for glucose enzyme sensor is also discussed. [ABSTRACT FROM AUTHOR]

A hetero-oligomeric glucose dehydrogenase (GDH) from a moderate thermophilic bacterium, SM4 was cross-linked with glutaraldehyde (GA) and it now showed only one optimum temperature for reaction at around 65 °C, which approximately follows the Arrhenius equation. The native enzyme had shown optima at both 45 °C and 75 °C. In addition to the alteration of the optimum temperature for reaction, GA cross-linked GDH retained more than 90% of its initial activity even after 30 min of incubation at 65 °C. [ABSTRACT FROM AUTHOR]

Halomonas (Deleya) sp. α-15 produces new co-factor binding soluble glucose 3-dehydrogenase (G3DH), which oxidizes the third hydroxy group of pyranose. This study investigated the condition of efficient production of G3DH using Halomonas (Deleya) sp. α-15. This enzyme was inducible, and α-methyl- D-glucoside, isopropyl-thioga lactopyranoside (IPTG) and lactose were revealed to be suitable carbon sources for G3DH induction. Maximum G3DH production was achieved by using minimal medium containing 0,8% (w/v) lactose with a productivity of 470U/1. [ABSTRACT FROM AUTHOR]

In Escherichia coli, classified as a mixed-acid producer in fermentation, D-lactate is one of the final metabolites from pyruvate. In order to achieve a high efficiency of bacterial hydrogen production from glucose, we have constructed an E. coli strain deficient in fermentative lactate dehydrogenase (LDH-A) by P1 transduction. The mutant, designated as MC13-4, entirely lost LDH-A activity while retaining whole formate hydrogenlyase activity. This mutation resulted in an increase in hydrogen production based on glucose consumed. The effect of uptake hydrogenases on the hydrogen production was also discussed. [ABSTRACT FROM AUTHOR]

We previously reported a novel glucose dehydrogenase (GDH) showing two peaks in the optimum temperature for the reaction at around 45°C and at around 75°C. Each peak derived from hetero-oligomeric enzyme, constructed from two distinct peptides with an α-subunit (MWs 67,000) and β-subunit (MWs 43,000), and a single peptide enzyme containing an α-subunit alone. The function of the two subunits in the thermostable co-factor binding GDH was investigated. The results of spectroscopic analyses indicated that the α-subunit contained an unknown co-factor showing specific fluorescence spectra like pyrroloquinoline quinone (PQQ), and the β-subunit was cytochrome c. Moreover, the results of a urea denaturation and reconstitution experiment suggested that the dissociation of the hetero-oligomeric complex to a single peptide was reversible. The kinetic parameter analyses for glucose and the electron mediator also suggested that the β-subunit was responsible for electron transfer from the catalytic center of the α-subunit to the electron mediator. [ABSTRACT FROM AUTHOR]