Specificity of Enzyme System Producing C6-Aldehyde in Tea Chloroplasts

The substrate specificity of enzyme system producing C₆-aldehyde in Thea chloroplasts was clarified with an entire series of synthesized positional isomers, in which the position of cis-1, cis-4-pentadiene system varies from C-3 to C-13 in C₁₈ fatty acid and geometrical isomers of linoleic acid. The structural requirement for the substrate of enzyme system producing C₆-aldehyde is the presence of cis-1, cis-4-pentadiene system between ω-6 and ω-10.     

Ceramide in Rice Grain

A rice lamina inclination test that is simple and specific for brassinosteroids was used as a micro-quantitative bioassay for brassinolide 1 and its 6-keto congener, castasterone 2, in the concentration range of 5 x 10^–5 /ig/ml to 5 x 10^–3μg/ml, when uniform seedlings of the rice cultivars Arborio J-l and Nihonbare were selected. A phytohormone, indole-3-acetic acid (IAA), showed similar activity in this bioassay. Its lowest effective concentration, however, was 50 /ig/μl, about five orders of magnitude greater than that of brassinolide. Other phytohormones, abscisic acid (ABA) and the cytokinins kinetin and A⁶-benzyladenine, inhibited the lamina inclination of rice seedlings. The addition of a cytokinin reduced the promoting effect of brassinolide. Thus, the rice lamina inclination test can be used both as a micro-quantitative bioassay for brassinosteroids and as a method for detecting antibrassinolide compouds.     

Production and Characterization of a Pressure-induced Gel from Freeze-concentrated Milk

A process was developed to produce a characteristic milk gel. Raw and market milk samples were freeze-concentrated using bacterial ice nuclei. The concentrates were kept at 5°C and compressed at 300–600 MPa for 5 min. The combination of the freeze concentration and the pressurization gave a milk gel without adding any gelling agents. The addition of sugar at 10% to the concentrated milk improved its gel strength and viscoelasticity. The gel was characterized by a phase transition at about 62–75°C.     

Conversion of Quinate to 3-Dehydroshikimate by Ca-Alginate-Immobilized Membrane of Gluconobacter oxydans IFO 3244 and Subsequent Asymmetric Reduction of 3-Dehydroshikimate to Shikimate by Immobilized Cytoplasmic NADP-Shikimate Dehydrogenase

The membrane fraction of Gluconobacter oxydans IFO 3244, involving membrane-bound quinoprotein quinate dehydrogenase and 3-dehydroquinate dehydratase, was immobilized into Ca-alginate beads. The Ca-alginate-immobilized bacterial membrane catalyzed a sequential reaction of quinate oxidation to 3-dehydroquinate and its spontaneous conversion to 3-dehydroshikimate under neutral pH. An almost 100% conversion rate from quinate to 3-dehydroshikimate was observed. NADP-Dependent cytoplasmic enzymes from the same organism, shikimate dehydrogenase and D-glucose dehydrogenase, were immobilized together with different carriers as an asymmetric reduction system forming shikimate from 3-dehydroshikimate. Blue Dextran 2000, Blue Dextran-Sepharose-4B, DEAE-Sephadex A-50, DEAE-cellulose, and hydroxyapatite were effective carriers of the two cytoplasmic enzymes, and the 3-dehydroshikimate initially added was converted to shikimate at 100% yield. The two cytoplasmic enzymes showed strong affinity to Blue Dextran 2000 and formed a soluble form of immobilized catalyst having the same catalytic efficiency as that of the free enzymes. This paper may be the first one on successful immobilization of NAD(P)-dependent dehydrogenases.     

Production of 4-Keto-D-arabonate by Oxidative Fermentation with Newly Isolated Gluconacetobacter liquefaciens

Production of 4-keto-D-arabonate (4KAB) was confirmed in a culture medium of Gluconacetobacter liquefaciens strains, newly isolated from water kefir in Argentina. The strains rapidly oxidized D-glucose, D-gluconate (GA), and 2-keto-D-gluconate (2KGA), and accumulated 2,5-diketo-D-gluconate (25DKA) exclusively before reaching the stationary phase. 25DKA was in turn converted to 4KAB, and 4KAB remained stable in the culture medium. The occurrence of 4KAB was assumed by Ameyama and Kondo about 50 years ago in their study on the carbohydrate metabolism of acetic acid bacteria (Bull. Agr. Chem. Soc. Jpn., 22, 271–272, 380–386 (1958)). This is the first report confirming microbial production of 4KAB.     

Isolation and Analysis of Cinnamic Acid 4-Hydroxylase Homologous Genes from a Hybrid Aspen,Populus kitakamiensis

Cinnamic acid 4-hydroxylase (CA4H) is the second enzyme involved in phenylpropanoid biosynthesis and is a member of the cytochrome P-450 superfamily. Three CA4H homologous genes, cyp73a, cyp73b, and cyp73c, and a cDNA clone of cyp73a were isolated from a genomic library and a cDNA library of a hybrid aspen; Populus kitakamiensis, and were characterized. They might be interrupted by two introns each. cyp73a and cyp73b were very similar to each other not only in coding regions but also in non-coding regions. Southern blot analysis showed that four homologous genes for CA4H constructed a small gene family in the diploid genome of P. kitakamiensis. In the promoter regions, there were many common m-element-like sequences in phenylpropanoid biosynthesis genes.     

Glutamic Acid Independent Production of Poly(γ-glutamic acid) by Bacillus subtilis TAM-4

A bacterium that produced a large amount of poly(γ-glutamic acid) (PGA) when it was grown aerobically in a culture medium containing ammonium salt and sugar as sources of nitrogen and carbon, respectively, was isolated from soil. The bacterium, strain TAM-4, was classified as Bacillus subtilis. The maximum PGA production (22.1 mg/ml) was obtained when it was grown in a medium containing 1.8% ammonium chloride and 7.5% fructose at 30°C for 96 h with shaking. Some properties of the PGA obtained at different times of cultivation were investigated by gel permeation chromatography, SDS–PAGE, and measurement of viscosity, and calculation of the d/l ratio of glutamic acid constituting PGA. The results suggested that PGA was elongated with no changes in the diastereoisomer ratio in the molecule.     

Macamides and their synthetic analogs: Evaluation of in vitro FAAH inhibition

Maca (Lepidium meyenii), a traditional food crop of the Peruvian Andes is now widely touted as a dietary supplement. Among the various chemical constituents isolated from the plant are a unique series of non-polar, long-chain fatty acid N-benzylamides known as macamides. We have synthesized 11 of the 19 reported macamides and have tested each as potential inhibitors of the human enzyme, fatty acid amide hydrolase (FAAH). The five most potent macamides were FAAH inhibitors (IC₅₀ = 10–17 μM). These amides were derivatives of oleic, linoleic and linolenic acids and benzylamine or 3-methoxybenzylamine. Of the three compounds evaluated in a pre-incubation time study, two macamides were not irreversible inhibitors of FAAH. The third, a carbamate structurally related to macamides, was shown to be an irreversible inhibitor of FAAH (IC₅₀ = 0.153 μM).