Dipeptides have unique physiological functions. This study focused on the salt-taste-enhancing dipeptide Met-Gly. BL00235, an l-amino acid ligase from Bacillus licheniformis NBRC12200, synthesizes Met-Gly as a major product as well as Met-Met as a by-product. To alter the substrate specificity of BL00235 and synthesize Met-Gly selectively, we chose to alter Pro85 residue based on the BL00235 crystal structure. We predicted that Met might be not recognized as a C-terminal substrate by occupying the space around C-terminal substrate. Pro85 was replaced with Phe, Tyr, and Trp, which have bulky aromatic side chains, by site-directed mutagenesis. These mutants lost the capacity to synthesize Met-Met, during the synthesis of Met-Gly. Furthermore, they did not synthesize Met-Met, even when methionine was used as a substrate. These results show that the amino acid residue at position 85 has a key role in C-terminal substrate specificity.

Results of the conductivity and breakdown strength measured for several biodegradable polymers are reported. At room temperature, poly-L-lactic acid (PLLA) and polyethylene terephthalate succinate (PETS) have relatively low conductivity values that are comparable to low density poIyethylene (LDPE). However, when the fact that PLLA and PETS are in the glass state is taken into consideration, the conductivity values of PLLA and PETS should be considered to be relatively high. Moreover, PLLA and PETS show rapid increase in the conduction current around their glass transition temperatures. The other three biodegradable polymers that are in the rubber state at room temperature, namely E-polycaprolactone butylene succinate (PCL-BS), polybutylene succinate (PBS), and polybutylene succinate adipate (PBSA), have much higher conductivities. Although the impulse breakdown strength is relatively similar in all the samples, PLLA and PETS have higher breakdown strengths in temperature regions around their glass transition temperatures. This apparently anomalous behavior is explainable by assuming that part of the energy supplied by the applied impulse voltage was consumed due to the glass transition. As for dc or ac breakdown strength at room temperature, PLLA and PETS show a relatively higher strength than PCL-BS and PBS.

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Many dipeptides have unique physiological functions such as antihypertensive effects and taste enhancing effects. In this study, we focused on the taste of dipeptides and conducted screening for dipeptides as salt taste enhancers. Dipeptides were synthesized using TabS, an L-amino acid ligase (Lal) from Pseudomonas syringae NBRC14081. Six kinds of amino acids, which were easily released by the hydrolysis of proteins or peptides, reacted with 20 proteogenic amino acids, and the reaction mixtures were evaluated using sensory evaluation. In the first screening, the reaction mixture or L-Leu-L-Ser, a known salt taste enhancing dipeptide, was added to a salt solution containing ATP and panelists judged the salt taste intensities. In the second screening, the reaction mixture or residual substrate amino acids was added to a salt solution containing ATP and subjected to sensory evaluation. LMet-Gly was identified as a candidate salt taste enhancer. In addition to sensory evaluation, the salt taste enhancing effect of L-Met-Gly was evaluated using a taste sensor. Taste sensor analysis showed that L-Met-Gly had a salt taste enhancing effect, and the relative sensor response for L-Met-Gly was equal to or higher than that for L-Leu-L-Ser. This is the first report that L-Met-Gly is a salt taste enhancing dipeptide. Furthermore, we propose that the screening method using reaction mixtures of Lal is applicable for the taste evaluation of other dipeptides.

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