Allergenic proteins of molecular mass 22 kDa, 36 kDa, 39-40 kDa and 70-72 kDa were identified from water -soluble fractions of common buckwheat cv. "Miyazaki -zairai". The identification was based on the reactivity of the protein with IgE antibodies from patients' allergic to buckwheat. The allergenic proteins fall within the $ and ' subunits of the globulin fraction indicating that the allergenic reactions are contributed by the major storage protein in buckwheat. The 22 kDa protein was the most consistent with IgE activity. N-terminal amino acid sequence of 22 kDa protein showed maximum homology with the seed globulins from both divot and monocot species. Based on the N-terminal amino acid sequence, two degenerate primers were selected to amplify the cDNA encoding the major allergenic protein. A single PCR product obtained was subsequently sequenced. The deduced amino acid sequence was identical in its N-terminal end. Homology search revealed 94 and 93 % homology to two buckwheat legumin like proteins.

Plants were regenerated from cotyledon tissue of greenhouse grown seedlings of common buckwheat (Fagopyrum esculentum Moench.). Maximum callus regeneration was induced on Murashige and Skoog (MS) medium containing 2,4-D (2.0 mg l 21 ) and kinetin (KIN) (0.2 mg l 21 ) and either 3 or 6% sucrose. Friable callus was transferred to MS media containing KIN and benzylaminopurine (BAP) at varied concentrations for embryogenic callus induction. The optimum medium for embryogenic callus induction was found to be MS medium supplemented with 0.2 mg l 21 KIN, 2.0 mg l 21 BAP and 3% (w/v) sucrose. Variation of sucrose from 3 to 6% did not show any significant effect on callus induction or embryogenesis. Regeneration of embryonic callus varied from 13 to 32%. Whole plants were obtained at high frequencies when the embryogenic calluses with somatic embryos and organized shoot primordia were transferred to half-strength MS media with 3% sucrose. Regenerated plants after acclimation were transferred to greenhouse conditions, and both vegetative and floral characteristics were observed for variation. This regeneration system may be valuable for genetic transformation and cell selection in common buckwheat.

The triglucoside of sesaminol, i.e., 2,6-O-di(β-D-glucopyranosyl)-β-D- glucopyranosylsesaminol (STG), occurs abundantly in sesame seeds and sesame oil cake and serves as an inexpensive source for the industrial production of sesaminol, an anti-oxidant that displays a number of bioactivities beneficial to human health. However, STG has been shown to be highly resistant to the action of β-glucosidases, in part due to its branched-chain glycon structure, and these circumstances hampered the efficient utilization of STG. We found that a strain (KB0549) of the genus Paenibacillus produced a novel enzyme capable of efficiently hydrolyzing STG. This enzyme, termed PSTG, was a tetrameric protein consisting of identical subunits with an approximate molecular mass of 80 kDa. The PSTG gene was cloned on the basis of the partial amino acid sequences of the purified enzyme. Sequence comparison showed that the enzyme belonged to the glycoside hydrolase family 3, with significant similarities to the Paenibacillus glucocerebrosidase (63% identity) and to Bgl3B of Thermotoga neapolitana (37% identity). The recombinant enzyme (rPSTG) was highly specific for β-glucosidic linkage, and k cat and k cat/K m values for the rPSTG-catalyzed hydrolysis of p-nitrophenyl-β-glucopyraniside at 37°C and pH 6.5 were 44 s−1 and 426 s−1 mM−1, respectively. The specificity analyses also revealed that the enzyme acted more efficiently on sophorose than on cellobiose and gentiobiose. Thus, rPSTG is the first example of a β-glucosidase with higher reactivity for β-1,2-glucosidic linkage than for β-1,4- and β-1,6-glucosidic linkages, as far as could be ascertained. This unique specificity is, at least in part, responsible for the enzyme’s ability to efficiently decompose STG.

Both common buckwheat (Fagopyrum esculentum) flour and meal cause an allergy in sensitive patients, and if unnoticed, it can be fatal. It has become a potential occupational hazard for some mill workers. The development of hypoallergenic buckwheat would be more efficient if natural mutants for allergenic protein are detected. A screening and selection method was developed using SDS-PAGE coupled with PCR techniques. SDS-PAGE analysis of 14 different species of buckwheat revealed that F. lineare and F. urophyllum lack the 22-kDa major allergenic protein. PCR-based screening with specific primers for sequences encoding the allergenic protein was also effective in distinguishing the allergen-deficient species.

A facultatively anaerobic, Gram-stain-positive, rod-shaped bacterium, designated strain KB0549 T , was isolated from sesame oil cake. Cells were motile, round-ended rods, and produced central or terminal spores. The cell wall peptidoglycan contained meso-diaminopimelic acid as the diamino acid. The major fatty acids were anteiso-C 15 : 0 and anteiso-C 17 : 0 . The DNA G+C content of strain KB0549 T was 51.9 mol%. On the basis of 16S rRNA gene sequence phylogeny, strain KB0549 T was affiliated with the genus Paenibacillus in the phylum Firmicutes and was most closely related to Paenibacillus cookii with 97.4 % sequence similarity. Strain KB0549 T was physiologically differentiated from P. cookii by the high content of anteiso-C 17 : 0 , inability to grow at 50 6C, spore position, and negative Voges-Proskauer reaction. Based on these unique physiological and phylogenetic characteristics, it is proposed that the isolate represents a novel species, Paenibacillus relictisesami sp. nov.; the type strain is KB0549 T (5JCM 18068 T 5DSM 25385 T ). Ash et al. (1993) proposed that 11 strains belonging to 'Group 3' within the genus Bacillus should be transferred to the genus Paenibacillus. The descriptions of Paenibacillus amylolyticus, Paenibacillus illinoisensis and Paenibacillus chibensis as species within the genus Paenibacillus were later amended by Shida et al. (1997). At the time of writing, the genus Paenibacillus contains 147 species and four subspecies (http://www.bacterio.net/p/paenibacillus.html).Members of the genus Paenibacillus are aerobic or facultatively anaerobic, rod-shaped, endospore-forming bacteria. Species of this genus have been isolated from various sources, including soil (Yoon et al., 2007), rhizosphere (Daane et al., 2002;Kuisiene et al., 2008), compost (Iida et al., 2005Ueda et al., 2013), warm springs (Saha et al., 2005) and human faeces (Hoyles et al., 2012). Several of the characterized strains have useful enzymic activities; for example, Paenibacillus sp. JDR-2 produces xylanase and is, therefore, able to utilize hemicellulosic polysaccharides (St John et al., 2006). Members of the genus Paenibacillus contain meso-diaminopimelic acid as the major diamino acid in peptidoglycan, and have anteiso-C 15 : 0 as the major cellular fatty acid. The DNA G+C content of species ranges from 36 to 59 mol% (Ash et al., 1993;Shida et al., 1997;De Vos et al., 2009). In this communication, we describe the polyphasic taxonomic characterization of a sesaminol-producing bacterium, strain KB0549 T , which was isolated from sesame oil cake (SOC) and was determined to be affiliated with the genus Paenibacillus.Sesaminol is one of several lignans found in sesame oil, having strong antioxidant activity as well as a variety of health benefits to humans (Fukuda et al., 1986;Kang et al., 1998;Kumazawa et al., 2003). Sesame seeds contain large amounts of sesaminol in glycosidic form. Sesaminolis the most abundant lignan in sesame seeds and is therefore a potentially important source of sesaminol. However, due to its bra...