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<p>Phytases hydrolyze phytic acid (myo-inositol hexakisphosphate), releasing inorganic phosphorus and essential minerals, thereby increasing their bioavailability for animals and humans. However, low native production and the limited stability of wild-type enzymes hinder their industrial applications. In this study, the <i>PHY7227</i> gene from <i>Talaromyces pinophilus</i> was cloned and expressed in <i>Komagataella phaffii</i>, yielding a recombinant phytase with a specific activity of 371.29 U/mg. The identity of this phytase was confirmed by SDS-PAGE and LC-MS/MS. The recombinant phytase exhibited a molecular mass of ~ 75&#xa0;kDa, maximum activity at pH 5.5 and at 55&#xa0;°C and 60&#xa0;°C and showed higher specificity for sodium phytate, exhibiting <i>K</i><sub><i>m app</i></sub> and <i>V</i><sub><i>max app</i></sub> values of 0.947 mM and 7.67 µmol×s<sup>− 1</sup>, respectively, against this substrate. The enzyme showed significant thermostability at 50&#xa0;°C and it was not inhibited by EDTA, DTT, or β-mercaptoethanol. In order to immobilize the phytase using the cross-linked enzyme aggregate (CLEA), 70% (v/v) isopropanol provided the highest CLEA immobilization yield, 83%, and activity recovery of 70.8%. Compared to the free form, the immobilized phytase exhibited enhanced thermostability at 50&#xa0;°C and a broader pH activity range. The immobilized phytase maintained over 60% of its initial activity after ten cycles of reuse in sodium phytate hydrolysis. These results demonstrate the effectiveness of CLEA immobilization for the recombinant phytase and highlight its potential for industrial applications, especially in animal feed production.</p>

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Characterization and CLEA immobilization of Talaromyces pinophilus phytase produced in Komagataella phaffii

  • 期刊论文

  • 发表日期：

    2026年1月12日

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  • Rafaela Inês de Souza Ladeira Ázar,
  • Yan da Silva Clevelares,
  • Roberta Corsino Ferreira,
  • Riziane Ferreira Gomes,
  • Gabriela Piccolo Maitan-Alfenas,
  • Valéria Monteze Guimarães

摘要

Phytases hydrolyze phytic acid (myo-inositol hexakisphosphate), releasing inorganic phosphorus and essential minerals, thereby increasing their bioavailability for animals and humans. However, low native production and the limited stability of wild-type enzymes hinder their industrial applications. In this study, the PHY7227 gene from Talaromyces pinophilus was cloned and expressed in Komagataella phaffii, yielding a recombinant phytase with a specific activity of 371.29 U/mg. The identity of this phytase was confirmed by SDS-PAGE and LC-MS/MS. The recombinant phytase exhibited a molecular mass of ~ 75 kDa, maximum activity at pH 5.5 and at 55 °C and 60 °C and showed higher specificity for sodium phytate, exhibiting Km app and Vmax app values of 0.947 mM and 7.67 µmol×s− 1, respectively, against this substrate. The enzyme showed significant thermostability at 50 °C and it was not inhibited by EDTA, DTT, or β-mercaptoethanol. In order to immobilize the phytase using the cross-linked enzyme aggregate (CLEA), 70% (v/v) isopropanol provided the highest CLEA immobilization yield, 83%, and activity recovery of 70.8%. Compared to the free form, the immobilized phytase exhibited enhanced thermostability at 50 °C and a broader pH activity range. The immobilized phytase maintained over 60% of its initial activity after ten cycles of reuse in sodium phytate hydrolysis. These results demonstrate the effectiveness of CLEA immobilization for the recombinant phytase and highlight its potential for industrial applications, especially in animal feed production.