Bioprocess for the production of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural and biochemical characteristics

- A ∼41-fold improvement in rStPhy production has been achieved under constitutive GAP promoter as compared to that of wild strain S. thermophile.
- Analysis of surface properties of rStPhy revealed the critical factors that determine thermostability of the protein.
- The catalytically important amino acids (Arg74, His75, Arg78, His368 and Asp369) were identified by docking and site directed mutagenesis studies.

Thermophilc mold Sporotrichum thermophile secretes an acidstable and thermostable phytase, which finds application as a food and feed additive because of its adequate thermostability, acid stability, protease insensitivity and broad substrate spectrum. Low extracellular phytase production by the mold is a major bottleneck for its application on a commercial scale. We have successfully overcome this problem by constitutive secretary expression of codon optimized rStPhy under glyceraldehyde phosphate dehydrogenase (GAP) promoter in Pichia pastoris. A ∼41-fold improvement in rStPhy production has been achieved. Circular Dichroism (CD) spectra revealed that rStPhy is composed of 26.65% α-helices, 5.26% β-sheets and 68.09% random coils at pH 5.0 and 60 °C, the optima for the enzyme activity. The melting temperature (Tm) of the enzyme is ∼73 °C. The 3D structure of rStPhy displayed characteristic signature sequences (RHGXRXP and HD) of HAP phytase. The catalytically important amino acids (Arg74, His75, Arg78, His368 and Asp369) were identified by docking and site directed mutagenesis. Fluorescence quenching by N-bromosuccinimide (NBS) and CsCl exposed tryptophan residues surrounded by negative charges, which play a key role in maintaining structural integrity of rStPhy.