Thermotoga maritima invertase (␤-fructosidase) hydrolyzes sucrose to release fructose and glucose, which are major carbon and energy sources for both prokaryotes and eukaryotes. The name "invertase" was given to this enzyme over a century ago, because the 1:1 mixture of glucose and fructose that it produces was named "invert sugar." Despite its name, the enzyme operates with a mechanism leading to the retention of the anomeric configuration at the site of cleavage. The enzyme belongs to family GH32 of the sequence-based classification of glycosidases. The crystal structure, determined at 2-Å resolution, reveals two modules, namely a five-bladed ␤-propeller with structural similarity to the ␤-propeller structures of glycosidase from families GH43 and GH68 connected to a ␤-sandwich module. Three carboxylates at the bottom of a deep, negatively charged funnel-shaped depression of the ␤-propeller are essential for catalysis and function as nucleophile, general acid, and transition state stabilizer, respectively. The catalytic machinery of invertase is perfectly superimposable to that of the enzymes of families GH43 and GH68. The variation in the position of the furanose ring at the site of cleavage explains the different mechanisms evident in families GH32 and GH68 (retaining) and GH43 (inverting) furanosidases.

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.