Ken-ichi Yoshida
Kobe University, Department of Agrobioscience, Faculty Member
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Research Interests: Genetics, English Literature, Genomics, Biology, Horizontal Gene Transfer, and 15 moreMultidisciplinary, Nature, Gram Positive, Genome, Gene, Gene Duplication, Secondary Metabolites, Enzyme, Gram Positive Bacteria, Bacillus subtilis, Gene Family, Carbon Source, Prophage, Molecular Sequence Data, and Transfer-RNA
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Background Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer’s disease. This conversion comprises two reactions catalyzed by... more
Background Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer’s disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. Results The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies o...
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Background Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from... more
Background Geobacillus kaustophilus is a thermophilic Gram-positive bacterium. Methods for its transformation are still under development. Earlier studies have demonstrated that pLS20catΔoriT mobilized the resident mobile plasmids from Bacillus subtilis to G. kaustophilus and transferred long segments of chromosome from one cell to another between B. subtilis. Results In this study, we applied mobilization of the B. subtilis chromosome mediated by pLS20catΔoriT to transform G. kaustophilus. We constructed a gene cassette to be integrated into G. kaustophilus and designed it within the B. subtilis chromosome. The pLS20catΔoriT-mediated conjugation successfully transferred the gene cassette from the B. subtilis chromosome into the G. kaustophilus allowing for the desired genetic transformation. Conclusions This transformation approach described here will provide a new tool to facilitate the flexible genetic manipulation of G. kaustophilus.
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BackgroundGeobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, grows on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes... more
BackgroundGeobacillus kaustophilus HTA426, a thermophilic Gram-positive bacterium, grows on inositol as its sole carbon source, and an iol gene cluster required for inositol catabolism has been postulated with reference to the iol genes in Bacillus subtilis. The iol gene cluster consists of two tandem operons induced in the presence of inositol; however, the mechanism underlying the induction remains unclear. B. subtilis iolQ is known to be involved in the regulation of iolX encoding a scyllo-inositol dehydrogenase, and its homolog in HTA426 was found two genes upstream of the first gene (gk1899) of the iol gene cluster and termed as iolQ in G. kaustophilus.ResultsWhen iolQ was inactivated, not only the myo-inositol dehydrogenase activity in the cell due to the expression of gk1899 but also the transcription of the two iol operons became constitutive. IolQ was produced and purified as a C-terminal His-tag fusion in Escherichia coli and subjected to the in vitro gel mobility shift as...
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Phytic acid, known as myo-inositol (MI) hexakisphosphate, is the principal storage form of phosphorus in many plant tissues, especially bran and seeds. Phytases comprise a group of phosphatases that can trim inorganic phosphates from... more
Phytic acid, known as myo-inositol (MI) hexakisphosphate, is the principal storage form of phosphorus in many plant tissues, especially bran and seeds. Phytases comprise a group of phosphatases that can trim inorganic phosphates from phytic acid. B. subtilis laboratory standard strain 168 and its derivatives exhibit no phytase activity, whereas a natto starter secretes significant phytase actively. The natto phytase gene was cloned into strain RIK1285, a protease-defective derivative of 168, to construct a random library of its N-terminal fusions with 173 different signal peptides identified in the 168 genome. The library was screened to assess the efficiency of phytase secretion based on clear zones around colonies on plates, which appeared when phytic acid was hydrolyzed. The pbp signal peptide enhanced the secretion of the natto phytase most efficiently, i.e., twice that of the original signal peptide. The secreted phytase can be one of the enzymatic tools to liberate MI from phy...
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Background: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5′-nucleotidases that hydrolyze the phosphate group of 5′-nucleotides. In Bacillus subtilis, genes encoding 5′-nucleotidase have remained to be... more
Background: In Escherichia coli, nagD, yrfG, yjjG, yieH, yigL, surE, and yfbR encode 5′-nucleotidases that hydrolyze the phosphate group of 5′-nucleotides. In Bacillus subtilis, genes encoding 5′-nucleotidase have remained to be identified. Results: We found that B. subtilis ycsE, araL, yutF, ysaA, and yqeG show suggestive similarities to nagD. Here, we expressed them in E. coli to purify the respective His6-tagged proteins. YcsE exhibited significant 5′-nucleotidase activity with a broader specificity, whereas the other four enzymes had rather weak but suggestive activities with various capacities and substrate specificities. In contrast, B. subtilis yktC shares high similarity with E. coli suhB encoding an inositol monophosphatase. YktC exhibited inositol monophosphatase activity as well as 5′-nucleotidase activity preferential for GMP and IMP. The ycsE, yktC, and yqeG genes are induced by oxidative stress and were dispensable, although yqeG was required to maintain normal growth ...
The objective of this research was to evaluate the PGPR effect on nodulation and nitrogen-fixing efficiency of soybean (Glycine max (L.) Merr.) by co-inoculation with Bradyrhizobium diazoefficiens USDA110. Co-inoculation of Bacillus... more
The objective of this research was to evaluate the PGPR effect on nodulation and nitrogen-fixing efficiency of soybean (Glycine max (L.) Merr.) by co-inoculation with Bradyrhizobium diazoefficiens USDA110. Co-inoculation of Bacillus velezensis S141 with USDA110 into soybean resulted in enhanced nodulation and N2-fixing efficiency by producing larger nodules. To understand the role of S141 on soybean and USDA110 symbiosis, putative genes related to IAA biosynthesis were disrupted, suggesting that co-inoculation of USDA110 with S141ΔyhcX reduces the number of large size nodules. It was revealed that yhcX may play a major role in IAA biosynthesis in S141 as well as provide a major impact on soybean growth promotion. The disruption of genes related to cytokinin biosynthesis and co-inoculation of USDA110 with S141ΔIPI reduced the number of very large size nodules, and it appears that IPI might play an important role in nodule size of soybean–Bradyrhizobium symbiosis. However, it was poss...
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A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis... more
A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions. Changing the pI of a bacterial serine...
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Research Interests: Biochemistry, Microbiology, Biology, Medicine, Multidisciplinary, and 8 moreHumans, Bacteria, PLoS one, Fermentation, Feces, Microbiota, Taurine, and Colon
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Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorizes red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires... more
Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorizes red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%...
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Background A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound... more
Background A stereoisomer of inositol, scyllo-inositol, is known as a promising therapeutic agent for Alzheimer's disease, since it prevents the accumulation of beta-amyloid deposits, a hallmark of the disease. However, this compound is relatively rare in nature, whereas another stereoisomer of inositol, myo-inositol, is abundantly available. Results Bacillus subtilis possesses a unique inositol metabolism involving both stereoisomers. We manipulated the inositol metabolism in B. subtilis to permit the possible bioconversion from myo-inositol to scyllo-inositol. Within 48 h of cultivation, the engineered strain was able to convert almost half of 10 g/L myo-inositol to scyllo-inositol that accumulated in the culture medium. Conclusions The engineered B. subtilis serves as a prototype of cell factory enabling a novel and inexpensive supply of scyllo-inositol.
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Research Interests: Microbiology, Biology, Medicine, Escherichia coli, Gene, and 3 moreBacteriocin, Bacillus subtilis, and Plasmid
Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription... more
Genes involved in the same cellular process are often clustered together in an operon whose expression is controlled by an upstream promoter. Generally, the activity of the promoter is strictly controlled. However, spurious transcription undermines this strict regulation, particularly affecting large operons. The negative effects of spurious transcription can be mitigated by the presence of multiple terminators inside the operon, in combination with an antitermination system. Antitermination systems modify the transcription elongation complexes and enable them to bypass terminators. Bacterial conjugation is the process by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugation involves many genes that are mostly organized in one or a few large operons. It has recently been shown that many conjugation operons present on plasmids replicating in Gram-positive bacteria possess a bipartite antitermination system that allows not only many terminators i...
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Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In... more
Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony forma...
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We report here the complete genome sequence of nitrogen-fixing Paenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related to Paenibacillus rhizophilus 7197, a... more
We report here the complete genome sequence of nitrogen-fixing Paenibacillus sp. strain URB8-2, isolated from the rhizosphere of wild grass in Kobe, Japan, revealing that this bacterium is related to Paenibacillus rhizophilus 7197, a novel species collected recently in Inner Mongolia, China, and that it possesses two gene clusters for distinct types of nitrogenases.
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Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been... more
Upon binding to ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) is activated to form a heterodimer with an aryl hydrocarbon receptor nuclear translocator (Arnt) and binds to DNA. It has been shown that the binding of AhR to DNA depends on the dioxin response element (DRE) and controls xenobiotic-response genes. AhR-binding DNA fragments from mouse hepatoma Hepa-1c1c7 cells stimulated with TCDD were once enriched in a chromatin immunoprecipitation (ChIP) DNA library and screened through a high-throughput southwestern chemistry-based enzyme-linked immunosorbent assay (SW-ELISA). After screening 1700 fragments, the ChIP-SW-ELISA screening strategy allowed us to isolate 77 fragments tightly interacting with AhR in the presence of TCDD. Only 39 of the 77 fragments appeared to contain a typical DRE, indicating that in some cases the DRE was dispensable for AhR-binding, while 75 fragments were located within promoter-distal regions. Genomic mapping of the 77 fragments enabled us to estimate 121 potential AhR targets including known targets such as Cyp1A1 and Cyp1B1, but only a limited number exhibited an altered expression dependent on TCDD. This study revealed the fact that TCDD-activated AhR frequently binds to promoter-distal regions even without a DRE and is not always involved in transcriptional regulation, suggesting that within the genome DNA-binding of AhR could take place often in many regions without cis-regulatory elements and might not be a key determinant to establish its regulatory function.
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The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, rho 11, and then in Escherichia coli plasmids (pBR322 and pUC118). The nucleotide sequence of the idh gene, which... more
The Bacillus subtilis inositol dehydrogenase (Idh)-encoding gene (idh) was cloned in the B. subtilis temperate phage, rho 11, and then in Escherichia coli plasmids (pBR322 and pUC118). The nucleotide sequence of the idh gene, which consists of 344 codons and whose product has an Mr of 38,351, was determined. E. coli, bearing pIOL05d15, in which expression of the idh gene is under the control of the lac promoter of pUC118, overproduced an active Idh to approx. 20% of total protein upon addition of isopropyl-beta-D-thiogalactopyranoside. This overproduced enzyme cross-reacted with an anti-Idh antibody, and exhibited the same Mr and substrate specificity as those of the B. subtilis enzyme.
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Research Interests: Genetics, Tobacco, Intelligent Transport System, Arabidopsis thaliana, DNA, and 12 moreEscherichia coli, Polymerase Chain Reaction, Inositol, Molecular cloning, Bacillus subtilis, Auxin, Genome sequence, Heat Shock Protein, Base Sequence, Open Reading Frame, Clostridium perfringens, and Aldehyde Dehydrogenase
Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a... more
Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 23-kb chromosomal segment, which covers the region between the iol and hut operons, has been cloned and sequenced, creating a 99-kb contig from the gnt operon to the wapA locus. This region (23351 bp) contains 25 complete open reading frames (ORFs; genes) including deoR, dra, nupC and pdp and two partial ones. The region (5140 bp) containing these four genes, being also sequenced by H. H. Saxild et al., was sequenced by subjecting a long polymerase chain reaction product to random sequencing using phage M13mp19. However, we could detect no conflict, between two independently determined sequences, which could be attributed to our sequencing method. A homology search for the 24 newly identified gene products revealed significant homology to known proteins in 14 of them. It was notable that three proteins, encoded by the successive genes (yxeMNO), exhibited meaningful homology to the E. coli GlnHPQ products constituting a periplasmic ATP-dependent transport system for glutamine.
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The demand for D-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the L-form of racemic D,L-2-phenylglycine to... more
The demand for D-2-phenylglycine used to synthesize semisynthetic antibiotics and pesticides is increasing. We have isolated a Chryseobacterium sp. that selectively transformed the L-form of racemic D,L-2-phenylglycine to (2S)-2-acetylamide-2-phenylacetic acid with a molar yield of 50% and an enantiomer excess of >99.5% under optimal culture conditions, consequently resulting in 99% pure D-2-phenylglycine remaining in the culture. The enantioselective N-acetylation was catalyzed by an acetyl-CoA-dependent N-acetyltransferase whose synthesis was induced by L-2-phenylglycine. The enzyme differed from previously reported bacterial arylamine N-acetyltransferases in molecular mass and substrate specificity. The relative activity ratio of the enzyme with the substrates L-2-phenylglycine, D-2-phenylglycine, 2-(2-chlorophenyl)glycine, and 5-aminosalicylic acid (a good substrate of arylamine N-acetyltransferase) was 100:0:56.9:5.49, respectively. The biotransformation by the N-acetyltransferase-producing bacterium reported here could constitute a new preparative route for the enzymatic resolution of D,L-2-phenylglycine.
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Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli.... more
Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.
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Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was... more
Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.
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Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known... more
Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE.
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Bacillus subtilis IolT is the major myo-inositol transporter for growth, while IolF is a minor one unable to support growth. We found that either IolT or IolF was sufficient for moderate growth using D-chiro-inositol. Conversely to IolT,... more
Bacillus subtilis IolT is the major myo-inositol transporter for growth, while IolF is a minor one unable to support growth. We found that either IolT or IolF was sufficient for moderate growth using D-chiro-inositol. Conversely to IolT, IolF transported D-chiro-inositol more preferentially than myo-inositol. These results indicate that IolT and IolF are different in substrate specificity.
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Diabetes mellitus is a complex disease that is characterized by the defection of insulin sensitivity in such peripheral tissues as skeletal muscle, adipose tissue and liver. We have previously demonstrated that certain inositol... more
Diabetes mellitus is a complex disease that is characterized by the defection of insulin sensitivity in such peripheral tissues as skeletal muscle, adipose tissue and liver. We have previously demonstrated that certain inositol derivatives stimulated glucose uptake accompanied by the translocation of glucose transporter 4 (GLUT4) to the plasma membrane in L6 myotubes. We investigated in this present study whether an oral intake of D-pinitol (PI) and myo-inositol (MI) would affect GLUT4 translocation in the skeletal muscle of mice. PI or MI at 1 g/kg BW administered orally to mice 30 min before a post-oral injection of glucose at 2 g/kg BW resulted in both PI and MI increasing GLUT4 translocation in the skeletal muscle and lowering the plasma glucose and insulin levels. PI and MI, therefore, have the potential to prevent diabetes mellitus by reducing the postprandial blood glucose level and stimulating GLUT4 translocation in the skeletal muscle.
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A number of 2',3',4'-trihydroxy-2-phenylacetophenone derivatives were synthesized and examined for growth inhibition... more
A number of 2',3',4'-trihydroxy-2-phenylacetophenone derivatives were synthesized and examined for growth inhibition of several kinds of bacteria. 2',3',4'-Trihydroxy-2-phenylacetophenone itself exhibited no antibacterial activity, but some of its derivatives showed various antibacterial activities depending on functional groups introduced on the 2-phenyl ring. Eighteen out of 24 compounds synthesized in this study appeared to possess antibacterial activities against at least two Gram-positive strains of Bacillus subtilis and Staphylococcus aureus, 2-(biphenyl-4-yl)-2',3',4'-trihydroxyacetophenone being the most active with LC(50) of 5.8 muM and 5.6 muM respectively. However, none of the synthesized compounds exhibited inhibitory effects on Gram-negative strains, such as Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica, suggesting that anti-Gram-positive specificity of the antibacterial compounds.
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Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members... more
Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-β-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a β-glucosidase resulted in the unexpected generation of a thermostable, highly specific β-fucosidase, concomitant with large increases in β-glucosidase, β-cellobiosidase, α-arabinofuranosidase, β-mannosidase, β-glucuronidase, β-xylopyranosidase, and β-fucosidase activities and a dramatic decline in 6-phospho-β-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-β-glycosidase and to generate a thermostable β-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.