From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase

Naturally-occurring phytases having the required level of thermostability f or application in animal feeding have not been found in nature thus far. We decided to de novo construct consensus phytases using primary protein sequ ence comparisons. A consensus enzyme based on 13 fungal phytase sequences h ad normal catalytic properties, but showed an unexpected 15-22 degrees C in crease in unfolding temperature compared with each of its parents. As a fir st step towards understanding the molecular basis of increased heat resista nce, the crystal structure of consensus phytase was determined and compared with that of Aspergillus niger phytase. Aspergillus niger phytase unfolds at much lower temperatures. In most cases, consensus residues were indeed e xpected, based on comparisons of both three-dimensional structures, to cont ribute more to phytase stabilization than non-consensus amino acids. For so me consensus amino acids, predicted by structural comparisons to destabiliz e the protein, mutational analysis was performed. Interestingly, these cons ensus residues in fact increased the unfolding temperature of the consensus phytase, In summary, for fungal phytases apparently an unexpected direct l ink between protein sequence conservation and protein stability exists.