Cell wall invertases (cwINVs), with a high affinity for the cell wall, are fundamental enzymes in the control of plant growth, development, and carbon partitioning. Most interestingly, defective cwINVs have been described in several plant species. Their highly attenuated sucrose (Suc)-hydrolyzing capacity is due to the absence of aspartate-239 (Asp-239) and tryptophan-47 (Trp-47) homologs, crucial players for stable binding in the active site and subsequent hydrolysis. However, so far, the precise roles of such defective cwINVs remain unclear. In this paper, we report on the functional characterization of tobacco (Nicotiana tabacum) Nin88, a presumed fully active cwINV playing a crucial role during pollen development. It is demonstrated here that Nin88, lacking both Asp-239 and Trp-47 homologs, has no invertase activity. This was further supported by modeling studies and site-directed mutagenesis experiments, introducing both Asp-239 and Trp-47 homologs, leading to an enzyme with a distinct Suc-hydrolyzing capacity. In vitro experiments suggest that the addition of Nin88 counteracts the unproductive and rather aspecific binding of tobacco cwINV1 to the wall, leading to higher activities in the presence of Suc and a more efficient interaction with its cell wall inhibitor. A working model is presented based on these findings, allowing speculation on the putative role of Nin88 in muro. The results presented in this work are an important first step toward unraveling the specific roles of plant defective cwINVs.

As steviol glycosides are now allowed as a food additive in the European market, it is important to assess the stability of these steviol glycosides after they have been added to different food matrices. We analyzed and tested the stability of steviol glycosides in semiskimmed milk, soy drink, fermented milk drink, ice cream, full-fat and skimmed set yogurt, dry biscuits, and jam. The fat was removed by centrifugation from the dairy and soy drink samples. Proteins were precipitated by the addition of acetonitrile and also removed by centrifugation. Samples of jam were extracted with water. Dry biscuits were extracted with ethanol. The resulting samples were concentrated with solid-phase extraction and analyzed by high-performance liquid chromatography on a C18 stationary phase and a gradient of acetonitrile/aqueous 25 mM phosphoric acid. The accuracy was checked using a standard addition on some samples. For assessing the stability of the steviol glycosides, samples were stored in conditions relevant to each food matrix and analyzed periodically. The results indicate that steviol glycosides can be analyzed with good precision and accuracy in these food categories. The recovery was between 96 and 103%. The method was also validated by standard addition, which showed excellent agreement with the external calibration curve. No sign of decomposition of steviol glycosides was found in any of the samples.

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t owards the heavily glycosylated a-1-acidg lycoprotein (AGP) is reported. Sodium dodecyl sulfate polyacrylamide gele lectrophoresis (SDS-PAGE) and high-performancea nion exchange chromatography with pulseda mperometric detection (HPAEC-PAD) show that after incubation of the protein with HPAs at 80 8Ca nd pH 2.8 complete hydrolysis of terminal glycosidic bond has been achieved,r esulting in the removal of sialic acids with no observed destruction of the protein core or the residual glycan chains. The 1 HNMR spectroscopy confirmed that the releaseds ialic acids preserve intact structure upon their excision from the protein,w hich makest he reported method suitable for the analysis of sialica cid modifications which play an important role in numerousb iological processes. The presence of other sugars was not detected by 1 HNMR and HPAEC-PAD, suggesting that HPAs hydrolyze only the terminal glycosidic bond in the glycoprotein, resulting in the selectiver elease of sialica cid from AGP.T he kineticr esults have shown that under equal temperature and pH conditions,t he hydrolysis of the terminal glucosidic bond occurredf aster in the presence of HPAs compared to conventionalm ineral acids. The observed rate constantsw erei n the range 6,7 10 À2 À11,9 10 À2 min À1 and the complete and selectivee xcision of sialic acids could be achieved within6 0min of incubation. The Trpf luorescencea nd CD spectroscopy show that non-covalent interaction between HPAa nd protein takes place in solution which could lead to stabilization of the sialosyl cation that is formed during the glycosidicbond hydrolysis by anionicH PA cluster. Figure 1. The two mostc ommonly occurringS IAs: N-acetylneuraminic acid (A) and N-glycolylneuraminic acid (B).