Summary C‐Glycosides are characterized by their C–C bonds in which the anomeric carbon of the sugar moieties is directly bound to the carbon atom of aglycon. C‐Glycosides are remarkably stable, as their C–C bonds are resistant to glycosidase or acid hydrolysis. A variety of plant species are known to accumulate C‐glycosylflavonoids; however, the genes encoding for enzymes that catalyze C‐glycosylation of flavonoids have been identified only from Oryza sativa (rice) and Zea mays (maize), and have not been identified from dicot plants. In this study, we identified the C‐glucosyltransferase gene from the dicot plant Fagopyrum esculentum M. (buckwheat). We purified two isozymes from buckwheat seedlings that catalyze C‐glucosylation of 2‐hydroxyflavanones, which are expressed specifically in the cotyledon during seed germination. Following purification we isolated the cDNA corresponding to each isozyme [FeCGTa (UGT708C1) and FeCGTb (UGT708C2)]. When expressed in Escherichia coli, both proteins demonstrated C‐glucosylation activity towards 2‐hydroxyflavanones, dihydrochalcone, trihydroxyacetophenones and other related compounds with chemical structures similar to 2′,4′,6′‐trihydroxyacetophenone. Molecular phylogenetic analysis of plant glycosyltransferases shows that flavonoid C‐glycosyltransferases form a different clade with other functionally analyzed plant glycosyltransferases.

Citrus plants accumulate many kinds of flavonoids, including di-C-glucosyl flavonoids, which have attracted considerable attention due to their health benefits. However, the biosynthesis of di-C-glucosyl flavonoids has not been elucidated at the molecular level. Here, we identified the C-glycosyltransferases (CGTs) FcCGT (UGT708G1) and CuCGT (UGT708G2) as the primary enzymes involved in the biosynthesis of di-C-glucosyl flavonoids in the citrus plants kumquat (Fortunella crassifolia) and satsuma mandarin (Citrus unshiu), respectively. The amino acid sequences of these CGTs were 98% identical, indicating that CGT genes are highly conserved in the citrus family. The recombinant enzymes FcCGT and CuCGT utilized 2-hydroxyflavanones, dihydrochalcone, and their mono-C-glucosides as sugar acceptors and produced corresponding di-C-glucosides. The K and k values of FcCGT toward phloretin were <0.5 μm and 12.0 sec , and those toward nothofagin (3'-C-glucosylphloretin) were 14.4 μm and 5.3 sec , respectively; these values are comparable with those of other glycosyltransferases reported to date. Transcripts of both CGT genes were found to concentrate in various plant organs, and particularly in leaves. Our results suggest that di-C-glucosyl flavonoid biosynthesis proceeds via a single enzyme using either 2-hydroxyflavanones or phloretin as a substrate in citrus plants. In addition, Escherichia coli cells expressing CGT genes were found to be capable of producing di-C-glucosyl flavonoids, which is promising for commercial production of these valuable compounds.

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Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.Lactic acid bacteria (LAB) are widely used as starter cultures for dairy, meat, and vegetable fermentations (24, 38). Some LAB strains are known to produce various types of bacteriocins, which have bactericidal effects against gram-positive bacteria, including food-borne pathogens (17,22,23,32). This beneficial trait has led to utilization of bacteriocins as food additives (9, 30). For example, nisin, a bacteriocin produced by Lactococcus lactis, has been used for food preservation in a large number of countries (7,15).Fermented soybean paste (miso) is a traditional food seasoning in Japan. Miso has been assumed to contribute to the health and lifespan of Japanese due to its favorable physiological effects, such as its antioxidative activity (3), its antimutagenic effect (18,21,29,39), and its inhibitory effect on accumulation of cholesterol (1, 2). In the first step of the miso production process, called koji mold fermentation, a koji mold (Aspergillus oryzae) is grown on steam-cooked rice. During this process care must be taken to avoid contamination by sporeforming bacteria, such as Bacillus subtilis, since the conditions are favorable for growth of bacteria.We previously tried to use LAB strains with antibacterial activity in the koji mold fermentation process to suppress the growth of contaminating bacteria. However, a preliminary test revealed that most LAB strains that are used industrially do not grow well on cooked rice. This poor growth may be ascribed to a deficiency of nutrients in rice, which are required by the LAB strains tested. In addition, the fairly low water activity of cooked rice seems to be unfavorable for the growth of LAB strains. We also attempted to isolate LAB strains which grow well on cooked rice and have an inhibitory effect on the growth of vegetative cells of B. subtilis, which is known to often contaminate preparations because of its spore-forming capacity. A large number of lots of rice bran were collected and used for screening LAB strains, since isolated strains are expected to grow well on steam-cooked rice. We found that rice bran is a good source for isolation of LAB strains that produce bacteriocins. In this paper, we describe purification and characterization of a bacteriocin produced by one of the iso...

We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-chain gene segment showed a novel nucleotide substitution. This heterozygous mutation encodes both Cys (TGT) and Arg (CGT) at residue 153. To examine the basis for the fibrinogen deficiency, we prepared expression vectors containing mutant γ-chain DNAs encoding γ153R and γ153A for in vitro expression in Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay and immunoblot analysis of the culture media and cell lysates showed that CHO cells transfected with γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-chain was expressed. In cells expressing normal fibrinogen, assem- bly intermediates and intact fibrinogen were seen in cell lysates prepared after short (3 minutes) or long (1 hour) incubation with 35S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant γ-chains. These data suggest that γ-chains have an important early role in fibrinogen assembly. Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first step in assembly is the formation of γ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→Arg substitution does not allow fibrinogen assembly and secretion, and this is manifest in vivo as a fibrinogen deficiency. We designated this variant as fibrinogen Matsumoto IV.