Background: Carboxylesterases typically have an alkaline pH optima, which limits their industrial application. Results: An acidophilic carboxylesterase was crystallized and changed into an alkaliphilic enzyme via quadruple mutations. Conclusion:The extended hydrogen bonds in the active site were important for adaptation to a low pH. Significance: The first structure of an acidophilic carboxylesterase revealed a novel strategy for the low pH adaptation of carboxylesterase.

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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.

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Feeding development in infants is important not only for of the purpose of acquiring nutrition but also for developing the ability to intake liquids. Our previous study showed that the introduction of a straw was appropriate after an infant has acquired the ability to sip liquid from a spoon and/or cup. In this study, we investigated the effect of a bowlshaped spoon on liquid intake. The aim of this study was to determine the appropriate form of spoon for infant feeding development. Eleven healthy infants (3 girls and 8 boys, 10-18 months old, mean age: 13.3 months) were recruited with their guardians' consent. We made 3 types of prototype spoon: A, oval (a standard renge soup spoon); B, flared-out (with the margin of the bowl flared out); and C, hemispherical (with a hemispherical bottom, and smaller than type A or B). We observed infants taking liquid supported by their mothers and evaluated the following responses: 1) confusion with regard to lip position, 2) spillage and 3) choking. Type C showed statistically less confusion with regard to lip position than type A or B (pϽ0.01), and B showed less than type A (pϽ0.05). No statistically significant differences were observed in spillage or choking among the three types of spoon. The renge soup spoon is often used to smooth the transition from breast/ bottle to cup feeding. In this study, we demonstrated the appropriate spoon form for infant feeding development.