Recombinant HAP Phytase of the Thermophilic Mold Sporotrichum thermophile: Expression of the Codon-Optimized Phytase Gene in Pichia pastoris and Applications

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• 作者：Bibhuti Ranjan ; T. Satyanarayana
• 关键词：Thermophilic mold ; Pichia pastoris ; Recombinant phytase ; Broiler feeds ; Dephytinization ; Haloperoxidase
• 刊名：Molecular Biotechnology
• 出版年：2016
• 出版时间：February 2016
• 年：2016
• 卷：58
• 期：2
• 页码：137-147
• 全文大小：616 KB
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• 作者单位：Bibhuti Ranjan (1)
  T. Satyanarayana (1)
  
  1. Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India
  
• 刊物主题：Biotechnology; Biochemistry, general; Cell Biology; Protein Science; Biological Techniques; Human Genetics;
• 出版者：Springer US
• ISSN：1559-0305

文摘

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 × 103/s and 8.84 × 106/M s, respectively. Mg2+ and Ba2+ 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. Keywords Thermophilic mold Pichia pastoris Recombinant phytase Broiler feeds Dephytinization Haloperoxidase