Thermostability Engineering of Fungal Phytases Using Low-Mr Additives and Chemical Crosslinking

With the ultimate goal to develop preparations of phytase (myo-inositol hexakisphosphate phosphohydrolase) with improved thermal resistance for inclusion in animal feed, several thermostabilization approaches were investigated with a set of fungal (Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, and Aspergillus niger phytase) and consensus phytases. Screening of different low-Mr additives revealed that polyethylene glycols increase the thermostability of all phytases in a molecular weight-dependent fashion. The polyols ribitol, xylitol (C5 sugars) and sorbitol (C6 sugar) also improved their thermostability, whereas polyols containing more or less carbon atoms, such as glycerol, erythritol and mannoheptulose, showed only minor effects. The stabilizing effects of PEGs and polyols were concentration dependent. In a second series of experiments, crosslinking of the carbohydrate chains of A. fumigatus and consensus phytase using sodium periodate and adipic acid dihydrazide resulted in the formation of oligomeric forms, which may explain the observed thermostability enhancement of 10-15d C.