A novel process for the recovery of superoxide dismutase from yeast exploiting electroextraction coupled to direct sorption

Abstract

BACKGROUND: This work presents a novel approach for the recovery and purification of superoxide dismutase (SOD; EC 1.15.1.1) produced in the yeast Kluyveromyces marxianus var. bulgaricus (NBIMCC 1984). Electroextraction was evaluated as an alternative to bead milling for SOD release. Seven anion exchange resins were studied in order to optimize SOD purification using ion exchange chromatography.

Potential application of aqueous two‐phase systems and three‐phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus

“…He and collaborators attained also 53% recovery using chloroform‐ethanol extraction, acetone precipitation, and ion exchange chromatography . Simental‐Martínez et al obtained a recovery of 84% using electroextraction and anionic adsorption . However, some of the aforementioned processes include a significant number of stages.…”

“…Total protein concentration was determined by the Bradford method using BSA (for complex ATPS) and commercial SOD (for model ATPS) as protein standards for the calibration curves . SOD activity determination was carried out using the SOD Assay Kit‐WST as detailed elsewhere . Briefly, the activity quantification was carried out in 96‐well microplates, adding per well 20 μL of sample, 20 μL of the kit enzyme (xanthine oxidase) and 250 μL of WST reagent (water‐soluble tetrazolium salt).…”

“…The Kluyveromyces marxianus strain was grown as described elsewhere. 17 Briefly, the yeast grew in 1 L flasks (20% working volume), using YPD medium (1% yeast extract, 2% peptone, 2% dextrose), using orbital shaking (220 rpm) at 30 C (MaxQ 6000, Thermo Scientific, MA) until the late exponential or stationary phases were reached. Growth was monitored turbidimetrically at 600 nm (Genesys 10S UV-VIS, Thermo Scientific, MA).…”

“…18 SOD activity determination was carried out using the SOD Assay Kit-WST as detailed elsewhere. 17 Briefly, the activity quantification was carried out in 96-well microplates, adding per well 20 lL of sample, 20 lL of the kit enzyme (xanthine oxidase) and 250 lL of WST reagent (water-soluble tetrazolium salt). After 20 min of incubation at 37 C, the absorbance was measured at 450 nm in a microplate reader.…”

“…8 Simental-Mart ınez et al obtained a recovery of 84% using electroextraction and anionic adsorption. 17 However, some of the aforementioned processes include a significant number of stages. Furthermore, some stages require the use of toxic solvents, expensive unit operations and reagents, and are difficult to scale up, limiting their industrial application.…”