Molecular Characterization and Expression of a Phytase Gene from the Thermophilic Fungus Thermomyces lanuginosus

Abstract

ABSTRACTThephyAgene encoding an extracellular phytase from the thermophilic fungusThermomyces lanuginosuswas cloned and heterologously expressed, and the recombinant gene product was biochemically characterized. ThephyAgene encodes a primary translation product (PhyA) of 475 amino acids (aa) which includes a putative signal peptide (23 aa) and propeptide (10 aa). The deduced amino acid sequence of PhyA has limited sequence identity (ca. 47%) withAspergillus nigerphytase. ThephyAgene was inserted into an expression vector under transcriptional control of theFusarium oxysporumtrypsin gene promoter and used to transform aFusarium venenatumrecipient strain. The secreted recombinant phytase protein was enzymatically active between pHs 3 and 7.5, with a specific activity of 110 μmol of inorganic phosphate released per min per mg of protein at pH 6 and 37°C. TheThermomycesphytase retained activity at assay temperatures up to 75°C and demonstrated superior catalytic efficiency to any known fungal phytase at 65°C (the temperature optimum). Comparison of this newThermomycescatalyst with the well-knownAspergillus nigerphytase reveals other favorable properties for the enzyme derived from the thermophilic gene donor, including catalytic activity over an expanded pH range.