Recovery and purification of Aspergillus niger phytase from crude extract using AOT / isooctane reversed micelles

Alberto A Neira-Vielma; Anna Iliná; Georgina Michelena Álvarez; Cynthia O Nascimento; Cristóbal Noé Aguilar; José Luis Martínez-Hernández; Maria das Graças Carneiro-da-Cunha

doi:10.1016/j.btre.2020.e00471

Recovery and purification of Aspergillus niger phytase from crude extract using AOT / isooctane reversed micelles

Biotechnol Rep (Amst). 2020 May 20:26:e00471. doi: 10.1016/j.btre.2020.e00471. eCollection 2020 Jun.

Authors

Alberto A Neira-Vielma^{1

2}, Anna Iliná³, Georgina Michelena Álvarez⁴, Cynthia O Nascimento⁵, Cristóbal Noé Aguilar¹, José Luis Martínez-Hernández³, Maria das Graças Carneiro-da-Cunha²

Affiliations

¹ Food Research Department, Universidad Autónoma de Coahuila, México. Blvd. V. Carranza S/N. Col. República, CP 25280, Saltillo, Coahuila, México.
² Departamento de Bioquímica/Laboratório de Imunopatologia Keizo Asami, Universidade Federal de Pernambuco-UFPE, Av. Prof. Moraes Rego s/n, CEP 50.670-420, Recife, PE, Brazil.
³ Nanobiociences Group, Universidad Autónoma de Coahuila, Blvd. V. Carranza S/N. Col. República, CP 25280, Saltillo, Coahuila, México.
⁴ Instituto Cubano de Investigaciones de los Derivados de la Caña de Azúcar (ICIDCA), Vía Blanca #804 y Carretera Central, Zona postal 10, código 11 000, San Miguel del Padrón Ciudad de La Habana, Cuba.
⁵ Faculdade São Miguel, Rua João Fernandes Vieira 458/459, CEP 50.050-215, Recife, PE, Brazil.

Abstract

This work describes the successful extraction of Aspergillus niger phytase from a crude extract (CE) obtained from solid-state fermentation by reversed micelle system using anionic surfactant sodium bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane achieved in two simple steps: forward and backward extractions. The effects of potassium chloride (KCl) concentration, pH of the aqueous solution, and AOT concentration that affect the system were examined. The best result for the forward extraction was obtained with the CE solution at pH 4.0, 50 mM KCl, and 100 mM AOT, while for the backward extraction the best result was achieved with a stripping aqueous solution at pH 5.5 containing 200 mM KCl, achieving a purification factor of 4.03, 1.15 times higher than that reported for the conventional purification process. Phytase purity was demonstrated by SDS-PAGE (89 kDa) and its activity by zymogram, confirming the efficiency of the process with low time consumption (∼40 min).

Keywords: Liquid-liquid extraction; Phytase; Reversed micelles; Triticale.