Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

Purification, gene cloning, and biochemical characterization of a ß-glucosidase capable of hydrolyzing sesaminol triglucoside from Paenibacillus sp. KB0549.

The triglucoside of sesaminol, i.e., 2,6-O-di(ß-D-glucopyranosyl)-ß-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 ß-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 ß-glucosidic linkage, and k cat and k cat/K m values for the rPSTG-catalyzed hydrolysis of p-nitrophenyl-ß-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 ß-glucosidase with higher reactivity for ß-1,2-glucosidic linkage than for ß-1,4- and ß-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.

Catalytic removal of acetaldehyde in saliva by a Gluconobacter strain.

Acetaldehyde (AA) accumulates in the oral cavity after alcohol intake and is responsible for an increased risk of alcohol-related upper aerodigestive tract (UDAT) cancer among aldehyde dehydrogenase 2-inactive heterozygotes in particular. Thus, the removal of AA from the saliva to a level below its mutagenic concentration (50 µM) after drinking is a potentially straightforward method for reducing the risk of alcohol-related UDAT cancer. Although microbial cells with AA-decomposing activity could potentially serve as a useful agent for the catalytic removal of AA from the saliva without the supplemental addition of cofactors, these cells generally exhibit strong AA-producing activity from ethanol, which is present in excess (50mM) over AA (100 µM) in the saliva after drinking. In this study, we observed that Gluconobacter kondonii (GK) cells efficiently decomposed salivary AA (100-390 µM) without the supplemental addition of cofactors irrespective of the type of alcoholic beverages consumed, even in the presence of an excess of ethanol (63 mM). Hydrogen peroxide, which is carcinogenic in animal experiments, was not produced because of the AA removal. The GK cells incubated at 45 °C and pH 3.5 for 15 h were killed, but they retained 80% of their original AA-decomposing activity. The treated cells were used as nonviable microcapsules that harbor a membrane-bound AA-decomposing activity.

Appropriate spoon form for feeding of liquids in infant feeding development.

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 (p<0.01), and B showed less than type A (p<0.05). 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.

Gene cloning and biochemical characterization of a catalase from Gluconobacter oxydans.

Gluconobacter oxydans has a large number of membrane-bound dehydrogenases linked to the respiratory chain that catalyze incomplete oxidation of a wide range of organic compounds by oxidative fermentation. Because the respiratory chain is a primary site of reactive oxygen species (ROS) production, the bacterium is expected to have a high capacity to detoxify nascent ROS. In the present study, a gene that encodes a catalase of G. oxydans, which might act as a potential scavenger of H(2)O(2), was cloned, and the expression product (termed rGoxCat) was characterized biochemically. rGoxCat is a heme b-containing tetrameric protein (molecular mass, 320 kDa) consisting of identical subunits. The recombinant enzyme displayed a strong catalase activity with a k(cat) of 6.28×10(4) s(-1) and a K(m) for H(2)O(2) of 61 mM; however, rGoxCat exhibited no peroxidase activity. These results, along with the phylogenetic position of the enzyme, provide conclusive evidence that rGoxCat is a monofunctional, large-subunit catalase. The enzyme was most stable in the pH range of 4-9, and greater than 60% of the original activity was retained after treatment at pH 3.0 and 40°C for 1h. Moreover, the enzyme exhibited excellent thermostability for a catalase from a mesophilic organism, retaining full activity after incubation for 30 min at 70°C. The observed catalytic properties of rGoxCat, as well as its stability in a slightly acidic environment, are consistent with its role in the elimination of nascent H(2)O(2) in a bacterium that produces a large amount of organic acid via oxidative fermentation.