title: Bioprocess for the production of recombinant HAP phytase of the thermophilic mold Sporotrichum thermophile and its structural and biochemical characteristics.
abstract: 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.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Anay Kumar
authors.initials: AK
authors.lastname: Maurya
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Deepak
authors.initials: D
authors.lastname: Parashar
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address: tsnarayana@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D010832:6-Phytase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D006358:Hot Temperature; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D010843:Pichia; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013175:Sporothrix / Q000201:enzymology
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D010832:6-Phytase
keywords: Circular dichroism; Fluorescence quenching; P. pastoris; Recombinant phytase; Site-directed mutagenesis; Sporotrichum thermophile