Quantitative validation of nicotine production in tea (Camellia sinensis L.).

Endogenous nicotine was confirmed to be present in tea plants (Camellia sinensis L.) by liquid chromatography-tandem mass spectrometry of tea samples from tea-producing regions in six Asian countries. All samples contained nicotine (0.011-0.694 µg g-1 dry weight). Nicotine contents remained constant during manufacturing of green, oolong and black teas, implying that nicotine is stable against heating, drying, enzymatic oxidation and mechanical damage during processing. Flower buds and seeds of cultivar Yabukita also contained nicotine (0.030-0.041 µg g-1 dry weight). A comparison of two cultivars revealed that higher nicotine contents were found in the black tea cultivar Benifuki. All plant parts of hydroponic Yabukita contained nicotine (0.003-0.013 µg g-1 dry weight). Tea cells cultured in B5 medium as well as roots and stems of tea seedlings contained nicotine levels similar to those of new leaves from field-grown plants. Although the levels of endogenous nicotine in tea plants are extremely low and sample contamination cannot be discounted, these levels exceed the maximum acceptable limit in Japan (0.01 µg g-1 dry weight).

Pantoea theicola sp. nov., isolated from black tea.

A Gram-negative, facultatively anaerobic strain was isolated from black tea. On the basis of 16S rRNA gene sequence similarity comparisons, strain QC88-366T was grouped into the genus Pantoea, being related most closely to the type strains of Pantoea gaviniae (98.5 %) and Pantoea calida (98.3 %); sequence similarities were ≤ 97.0 % to the type strains of other species of the genus Pantoea. Multilocus sequence analysis based on partial sequences of the gyrB, rpoB, infB and atpD genes also revealed P. gaviniae and P. calida as the closest phylogenetic relatives. The fatty acid profile showed the major fatty acids of strain QC88-366T were C16 : 0, C16 : 1 and C18 : 1, the same as those of its closest related species. However, the ratio of C16 : 1, C17 : 0 cyclo, C18 : 1 and C18 : 2 differed slightly compared with those of the related neighbours. In addition, the results of physiological and biochemical tests also allowed the phenotypic differentiation of strain QC88-366T from its closest phylogenetic neighbours. The G+C content of the DNA was 57.2 mol%. Strain QC88-366T therefore represents a novel species of the genus Pantoea, for which the name Pantoea theicola sp. nov. is proposed. The type strain is QC88-366T ( = DSM 29212T = NBRC 110557T).

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli.

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13 degrees C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac alpha 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates.