Enhancement of activity and thermostability of Aspergillus niger ATCC 10864 phytase A through rational design.

Aspergillus niger ATCC 10864 phytase A was produced in Penicillium verruculosum. The enzyme was found to have two pH optima of 2.5 and 5.0, as well as a T-optimum of 50-55 °C. Two amino acid substitutions, A76M and S265P, were designed for improvement in thermostability, and two more, N300K and D363N, were designed for improvement in enzyme activity. The most thermostable variant, S265P, was characterized by a 3.8-fold increase in time of half-life at 55 °C and a 1.2-fold increase in residual activity at 90 °C compared to the wild-type. The most active variant, D363N, was 1.7-times more active at 40 °C and retained 1.3-times higher residual activity at 90 °C compared to the wild-type. The obtained results revealed the importance of substitutions with proline in α-helixes for the thermostability improvement of phytases. Also, the importance of sequence motif 361HDN363 was demonstrated with relevance to values of catalytic parameters.