Previously, we calculated a consensus amino acid sequence from 13 homologous fungal phytases. A synthetic gene was constructed and recombinantly expressed. Surprisingly, consensus phytase-1 was 15-26 degrees C more thermostable than all parent phytases used in its design [Lehmann et al. (2000)Protein Eng., 13, 49-57]. In the present study, inclusion of six further phytase sequences in the amino acid sequence alignment resulted in the replacement of 38 amino acid residues in either one or both of the new consensus phytases-10 and -11. Since consensus phytase-10, again, was 7.4 degrees C more thermostable than consensus phytase-1, the thermostability effects of most of the 38 amino acid substitutions were tested by site-directed mutagenesis. Both stabilizing and destabilizing mutations were identified, but all affected the stability of the enzyme by <3 degrees C. The combination of all stabilizing amino acid exchanges in a multiple mutant of consensus phytase-1 increased the unfolding temperature from 78.0 to 88.5 degrees C. Likewise, back-mutation of four destabilizing amino acids and introduction of an additional stabilizing amino acid in consensus phytase-10 further increased the unfolding temperature from 85.4 to 90.4 degrees C. The thermostabilization achieved is the result of a combination of slight improvements from multiple amino acid exchanges rather than being the effect of a single or of just a few dominating mutations that have been introduced by chance. The present findings support the general validity of the consensus concept for thermostability engineering of proteins.

By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 8C more thermostable than the parent fungal phytases used in its design~Lehmann et al., 2000!. We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site~i.e., all the divergent residues! with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile. For the new protein termed consensus phytase-7, a major-although not complete-shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 8C decrease in unfolding temperature~T m ! as measured by differential scanning calorimetry, consensus phytase-7 still was Ͼ7 8C more thermostable than all wild-type ascomycete phytases known to date. Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that~most closely! matches the most favorable catalytic properties found among its family members.

The text describes the research Geopartitura executed by the team MidiaLab Computer art research laboratory, that raises the thought about social artists and urban space. As a work of art it can be considered activist action. As a system, it is composed by software, database, locative media and mobile devices. The work was created to be performed as urban interactive cyberintervention, in order to interact with passers-by, a bias of social inclusion, transforming the urban landscape and its noises, at a dimension of open space for discussion. As active and engaged art, presents the artist as social citizen.