Bacillus coagulans SANK 70258 suppresses Enterobacteriaceae in the microbiota of ulcerative colitis in vitro and enhances butyrogenesis in healthy microbiota.

The aim of this study was to clarify the effect of the spore-forming and lactic acid-producing probiotic strain, Bacillus coagulans SANK 70258, on human colonic microbiota of healthy subjects and ulcerative colitis patients. A model culture system was employed to construct the in vitro human colonic microbiota, to retain the bacterial species richness and simulate the patient's disordered composition, from the fecal inoculum. Bacterial 16S rRNA gene sequencing confirmed that administration of B. coagulans SANK 70258 (at an initial concentration of 4 × 107-total cells/mL) suppressed bacteria related to the family Enterobacteriaceae in the microbiota models for both healthy subjects (P = 0.016) and ulcerative colitis patients (P = 0.023). In addition, administration of B. coagulans SANK 70258 increased bacteria related to the family Lachnospiraceae (P = 0.031), thereby enhancing butyrate production (P = 0.031) in the microbiota models of healthy subjects. However, these changes were not observed in the microbiota models of ulcerative colitis patients, likely owing to the low abundance of Lachnospiraceae species. This study demonstrates the potential of B. coagulans SANK 70258 to exhibit antimicrobial activity against harmful organisms in patients with ulcerative colitis, while improving the intestinal microenvironment by increasing butyrogenesis in healthy persons. KEY POINTS: • B. coagulans SANK 70258 treatment reduced colonic Enterobacteriaceae species. • B. coagulans SANK 70258 treatment enhanced butyrogenesis in healthy individuals. • B. coagulans SANK 70258 treatment increased Lachnospiraceae in healthy persons. • B. coagulans SANK 70258 improves the colonic microenvironment in ulcerative colitis.

Effect of the Ratio of Magnetite Particle Size to Microwave Penetration Depth on Reduction Reaction Behaviour by H2.

In this study, we investigated reduction of magnetite by H2 during microwave irradiation. This process combines the advantages of microwave irradiation and using H2 as a reducing agent to mitigate CO2 emissions during the ironmaking process. Weight change measurements showed that a reduction of 75% was achieved after treatment under H2 for 60 min. For better understanding of the effective parameters in microwave chemistry, scanning electron microscopy, combined with energy-dispersive X-ray spectroscopy (SEM-EDX), was performed, which demonstrated a greater reduction of large particles (>40 µm) than small particles. This behaviour could be attributed to the higher microwave absorption capability of large particles with a higher ratio of particle size to penetration depth (d/δ). Small particles behave as transparent material and are heated via conduction and/or convection; thus, there is no contribution from the catalytic effect of microwaves to the reduction reaction. Moreover, the reduction of Fe3O4 to Fe0.94O, followed by transformation to Fe, seems to proceed from the surface toward the centre of the particle despite the volumetric microwave heating. This could be due to the higher gas accessibility of iron oxide on the particle surface than in the particle centre.

Structure basis for the regulation of glyceraldehyde-3-phosphate dehydrogenase activity via the intrinsically disordered protein CP12.

The reversible formation of a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-CP12-phosphoribulokinase (PRK) supramolecular complex, identified in oxygenic photosynthetic organisms, provides light-dependent Calvin cycle regulation in a coordinated manner. An intrinsically disordered protein (IDP) CP12 acts as a linker to sequentially bind GAPDH and PRK to downregulate both enzymes. Here, we report the crystal structures of the ternary GAPDH-CP12-NAD and binary GAPDH-NAD complexes from Synechococcus elongates. The GAPDH-CP12 complex structure reveals that the oxidized CP12 becomes partially structured upon GAPDH binding. The C-terminus of CP12 is inserted into the active-site region of GAPDH, resulting in competitive inhibition of GAPDH. This study also provides insight into how the GAPDH-CP12 complex is dissociated by a high NADP(H)/NAD(H) ratio. An unexpected increase in negative charge potential that emerged upon CP12 binding highlights the biological function of CP12 in the sequential assembly of the supramolecular complex.

Purification, characterization and amino acid sequence of a novel enzyme, D-threo-3-hydroxyaspartate dehydratase, from Delftia sp. HT23.

D-threo-3-hydroxyaspartate dehydratase (D-THA DH) was purified from the cell-free extract of the soil-isolated bacterium Delftia sp. HT23. The enzyme exhibited dehydratase activity towards D-threo-3-hydroxyaspartate, l-threo-3-hydroxyaspartate, l-erythro-3-hydroxyaspartate and d-serine. Absorption of the purified enzyme at 412 nm suggests that it contains pyridoxal 5'-phosphate (PLP) as a cofactor. The NH(2)-terminal and internal amino acid sequences showed significant similarity to hypothetical alanine racemase of genome-sequenced Delftia acidovorans SPH-1; however, the purified enzyme showed no alanine racemase activity. Using the sequence information of D. acidovorans SPH-1, the gene encoding d-THA DH was cloned. The deduced amino acid sequence, which belongs to the alanine racemase family, shows significant (26-36%) similarity to d-serine dehydratase of both Saccharomyces cerevisiae and chicken. In order to obtain purified d-THA DH efficiently, the gene was expressed in Escherichia coli. The recombinant enzyme was highly activated by divalent cations, such as Mn(2+), Co(2+) and Ni(2+). Site-directed mutagenesis experiment revealed that lysine 43 is an important residue involved in PLP binding and catalysis. This is the first reported enzyme that acts on d-THA. In addition, this enzyme is the first example of a prokaryotic dehydratase belonging to the fold-type III PLP-dependent enzyme family.

Gene cloning and expression of pyridoxal 5'-phosphate-dependent L-threo-3-hydroxyaspartate dehydratase from Pseudomonas sp. T62, and characterization of the recombinant enzyme.

L-threo-3-Hydroxyaspartate dehydratase (L-THA DH, EC 4.3.1.16), which catalyses the cleavage of L-threo-3-hydroxyaspartate (L-THA) to oxalacetate and ammonia, has been purified from the soil bacterium Pseudomonas sp. T62. In this report, the gene encoding L-THA DH was cloned and expressed in Escherichia coli, and the gene product was purified and characterized in detail. A 957-bp nucleotide fragment was confirmed to be the gene encoding L-THA DH, based on the agreement of internal amino acid sequences. The deduced amino acid sequence, which belongs to the serine/threonine dehydratase family, shows similarity to YKL218c from Saccharomyces cerevisiae (64%), serine racemase from Schizosaccharomyces pombe (64%) and Mus musculus (36%), and biodegradative threonine dehydratase from E. coli (38%). Site-directed mutagenesis experiments revealed that lysine at position 53 is an important residue for enzymatic activity. This enzyme exhibited dehydratase activity specific only to L-THA [K(m) = 0.54 mM, V(max) = 39.0 micromol min(-1) (mg protein)(-1)], but not to other 3-hydroxyaspartate isomers, and exhibited no detectable serine/aspartate racemase activity. This is the first report of an amino acid sequence of the bacterial enzyme that acts on L-THA.