Protein structure prediction: assembly of secondary structure elements by basin-hopping.

The prediction of protein tertiary structure from primary structure remains a challenging task. One possible approach to this problem is the application of basin-hopping global optimization combined with an all-atom force field. In this work, the efficiency of basin-hopping is improved by introducing an approach that derives tertiary structures from the secondary structure assignments of individual residues. This approach is termed secondary-to-tertiary basin-hopping and benchmarked for three miniproteins: trpzip, trp-cage and ER-10. For each of the three miniproteins, the secondary-to-tertiary basin-hopping approach successfully and reliably predicts their three-dimensional structure. When it is applied to larger proteins, correctly folded structures are obtained. It can be concluded that the assembly of secondary structure elements using basin-hopping is a promising tool for de novo protein structure prediction.

Characterization of free and immobilized (S)-aminotransferase for acetophenone production.

Enzyme immobilization often improves process economics, but changes in kinetic properties may also occur. The immobilization of a recombinant thermostable (S)-aminotransferase was made by entrapment on calcium alginate-3% (w/v)-and tested with (S)-(-)-(alpha)-methylbenzylamine for acetophenone production. The best immobilization results were obtained for beads of concentration of 10 mg of spray-dried cells (containing recombinant (S)-aminotransferase) per milliliter of sodium alginate bead. As a result of immobilization, the properties of immobilized spray-dried cells differed from the properties of free spray-dried cells. V (m) for the immobilized enzyme was between 0.08 and 0.09 mM/min, while the V (m) for free enzyme was 0.06-0.07 mM/min. K (m) values differed for immobilized and free spray-dried cells by a factor of between 3 and 5 for (S)-(-)-(alpha)-methylbenzylamine (6.05 mM for immobilized, 1.78 mM for free) and pyruvate (5.0 mM for immobilized, 1.01 mM for free) at 55 degrees C. Optimum pH values were 7.7 and 8.1 for the free spray-dried cells and the immobilized formulation, respectively. The maximum activity for free spray-dried cells was measured at 55 degrees C, whereas for immobilized ones, it was at 60 degrees C. Activation and deactivation energy values for free spray-dried cells were 15.13 and 41.73 kcal/mol, while those for immobilized spray-dried cells were 8.86 and 48.88 kcal/mol, respectively. Overall, as a result of immobilization, an increase in V (m) was measured for the (S)-aminotransferase by 28 to 33% with respect to free enzyme; K (m) increased by a factor of three- to fivefold and had a shift of 5 degrees C in optimum temperature, and the activation energy was 41% lower than the activation energy of free (S)-aminotransferase.