title: Recombinant HAP Phytase of the Thermophilic Mold Sporotrichum thermophile: Expression of the Codon-Optimized Phytase Gene in Pichia pastoris and Applications.
abstract: The codon-optimized phytase gene of the thermophilic mold Sporotrichum thermophile (St-Phy) was expressed in Pichia pastoris. The recombinant P. pastoris harboring the phytase gene (rSt-Phy) yielded a high titer of extracellular phytase (480 ± 23 U/mL) on induction with methanol. The recombinant phytase production was ~40-fold higher than that of the native fungal strain. The purified recombinant phytase (rSt-Phy) has the molecular mass of 70 kDa on SDS-PAGE, with K m and V max (calcium phytate), k cat and k cat/K m values of 0.147 mM and 183 nmol/mg s, 1.3 × 10(3)/s and 8.84 × 10(6)/M s, respectively. Mg(2+) and Ba(2+) display a slight stimulatory effect, while other cations tested exert inhibitory action on phytase. The enzyme is inhibited by chaotropic agents (guanidinium hydrochloride, potassium iodide, and urea), Woodward's reagent K and 2,3-bunatedione, but resistant to both pepsin and trypsin. The rSt-Phy is useful in the dephytinization of broiler feeds efficiently in simulated gut conditions of chick leading to the liberation of soluble inorganic phosphate with concomitant mitigation in antinutrient effects of phytates. The addition of vanadate makes it a potential candidate for generating haloperoxidase, which has several applications.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
authors.forename: Bibhuti
authors.initials: B
authors.lastname: Ranjan
authors.affiliation: Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India. tsnarayana@gmail.com.
authors.forename: T
authors.initials: T
authors.lastname: Satyanarayana
mesh_terms: D010832:6-Phytase / Q000235:genetics* / Q000378:metabolism*; D000821:Animal Feed; D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D003062:Codon; D004795:Enzyme Stability; D010833:Phytic Acid / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013175:Sporothrix / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D010833:Phytic Acid; D010832:6-Phytase
keywords: Broiler feeds; Dephytinization; Haloperoxidase; Pichia pastoris; Recombinant phytase; Thermophilic mold