Improved Thermal Stability of a Novel Acidophilic Phytase

Abstract

Phytase increases the availability of phosphate and trace elements by hydrolyzing the phosphomonoester bond in phytate present in animal feed. It is also an important enzyme from an environmental perspective because it not only promotes the growth of livestocks but also prevents phosphorus contamination released into the environment. Here we present a novel phytase derived from Turicimonas muris, TmPhy, which has distinctive structure and properties compared to other previously known phytases. TmPhy gene expressed in the Pichia system was confirmed to be 41 kDa in size and was used in purified form to evaluate optimal conditions for maximum activity. TmPhy has a dual optimum pH at pH3 and pH6.8 and exhibited the highest activity at 70°C. However, the heat tolerance of the wildtype was not satisfactory for feed application. Therefore, random mutation, disulfide bond introduction, and N-terminal mutation were performed to improve the thermostability of the TmPhy. Random mutation resulted in TmPhyM with about 45% improvement in stability at 60°C. Through further improvements, a total of three mutants were screened and their heat tolerance was evaluated. As a result, we obtained TmPhyMD1 with 46.5% residual activity, TmPhyMD2 with 74.1%, and TmPhyMD3 with 66.8% at 80°C heat treatment without significant loss of or with increased activity.

Keywords: Acidophilic; dual optimum pH; phytase; phytic acid; thermostable.

Fig. 1. Temperature profile (A) and pH profile (B) of TmPhy and mutants.

Fig. 2. Natural and engineered disulfide bonds in the TmPhyMD1 model structure.

Yellow shows the disulfide bonds of the wildtype, and green shows the additional disulfide bonds that were introduced to increase thermal stability.

Fig. 3. Thermostability of the TmPhy and mutants.

The bars in the graph are colored as follows. TmPhy is in purple, TmPhyM in blue, TmPhyMD1 in green, TmPhyMD2 in orange and TmPhyMD3 in red.