Haruyuki Yamashita

An Alternative Menaquinone Biosynthetic Pathway Operating in Microorganisms

In microorganisms, menaquinone is an obligatory component of the electron-transfer pathway. It is derived from chorismate by seven enzymes in Escherichia coli. However, a bioinformatic analysis of whole genome sequences has suggested that some microorganisms, including pathogenic species such as Helicobacter pylori and Campylobacter jejuni, do not have orthologs of the men genes, even though they synthesize menaquinone. We deduced the outline of this alternative pathway in a nonpathogenic strain of Streptomyces by bioinformatic screening, gene knockouts, shotgun cloning with isolated mutants, and in vitro studies with recombinant enzymes. As humans and commensal intestinal bacteria, including lactobacilli, lack this pathway, it represents an attractive target for the development of chemotherapeutics.

Neoculin as a New Taste-modifying Protein Occurring in the Fruit of Curculigo latifolia

A unique taste-modifying activity that converts the sense of sourness to the sense of sweetness occurs in the fruit of the plant Curculigo latifolia, intrinsic to West Malaysia. The active component, known as curculin, is a protein consisting of two identical subunits. We have found a new taste-modifying protein, named neoculin, of the same origin. Both chemical analysis and cDNA cloning characterized neoculin as a heterodimeric protein consisting of an acidic, glycosylated subunit of 113 amino acid residues and a basic subunit that is the monomeric curculin itself.

Heterologous Mevalonate Production in Streptomyces lividans TK23

Mevalonate is a ubiquitous biosynthetic intermediate of terpenoids and is used as a moisturizer in cosmetics and a chemical for biochemical research. In this study, we have achieved a heterologous production of this useful compound by expression in Streptomyces lividans TK23 of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase genes, which were cloned from Streptomyces sp. strain CL190.

Molecular cloning of curculin, a novel taste-modifying protein with a sweet taste

cDNA clones for curculin, a novel taste-modifying protein, were isolated and sequenced. The encoded prepro-curculin was composed of 158 amino acid residues including a signal sequence of 22 residues and a carboxy-terminal extension peptide of 22 residues. Northern blot analysis showed that the mRNA for curculin was first detected in Curculigo latifolia fruits at 2 weeks after pollination and remained at a constant level for the following 4 weeks.

Surface plasmon resonance analysis on interactions of food components with a taste epithelial cell model.

A new device for evaluating the continuity of taste was developed with the use of surface plasmon resonance (SPR). The model of lingual cells was constructed with liposomes immobilized onto an L1 sensor chip for SPR. Using this device, we classified food components into three categories according to the sensorgram pattern and residual ratio on lipid bilayer. Samples in group A strongly interacted with lipid bilayer, those in group B poorly interacted, and those in group C belong to neither group A nor group B. Sweet proteins and gymnemic acids that prolonged sweet perception were categorized in group A. Almost all the carbohydrates investigated and aspartame, of which the taste perception does not continue, belonged to group B. This device made it possible to detect the interaction with lipid bilayer and dissected the mechanism of taste continuity.

Identification of bitterness-masking compounds from cheese.

Bitterness-masking compounds were identified in a natural white mold cheese. The oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography. The four fractions obtained were characterized by thin-layer chromatography and nuclear magnetic resonance spectroscopy. The fatty acid-containing fraction was found to have the highest bitterness-masking activity against quinine hydrochloride. Bitterness-masking activity was quantitated using a method based on subjective equivalents. At 0.5 mM, the fatty acid mixture, which had a composition similar to that of cheese, suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.0049–0.0060% and 0.5 mM oleic acid to that of 0.0032–0.0038% solution. The binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry. These results suggest that oleic acid masked bitterness by forming a complex with the bitter compounds.

Long-Chain Fatty Acids Elicit a Bitterness-Masking Effect on Quinine and Other Nitrogenous Bitter Substances by Formation of Insoluble Binary Complexes

We have previously found that fatty acids can mask the bitterness of certain nitrogenous substances through direct molecular interactions. Using isothermal titration calorimetry, we investigated the interactions between sodium oleate and 22 bitter substances. The hydrochloride salts of quinine, promethazine, and propranolol interacted strongly with fatty acids containing 12 or more carbon atoms. The (1)H NMR spectra of these substances, obtained in the presence of the sodium salts of the fatty acids in dimethyl sulfoxide, revealed the formation of hydrogen bonds between the nitrogen atoms of the bitter substances and the carboxyl groups of the fatty acids. When sodium laurate and the hydrochloride salt of quinine were mixed in water, an equimolar complex formed as insoluble heterogeneous needlelike crystals. These results suggested that fatty acids interact directly with bitter substances through hydrogen bonds and hydrophobic interactions to form insoluble binary complexes that mask bitterness.