TURK 2025

Effective downstream processing methods are needed for the large-scale purification of enzymes. In recent years, non-chromatographic methods like three-phase partitioning (TPP), which can be directly applied to crude extracts, are gaining popularity due to their fast, high efficiency, and cost-effectiveness. modified TPP variants are also being used and developed to enhance the extraction yield and purification fold of enzymes. In this study, the glutaminase enzyme produced from Pseudomonas iridis ZV-37 was partially purified for the first time using the Conventional three-phase partitioning (TPP) method and a new modification of TPP, alternating current magnetic field-assisted three phase partitioning (ACMF-TPP). Conventional TPP was optimized and used as a reference method, which yielded 131% of the enzyme with 4.48-fold purity with optimized experimental conditions such as (NH4)2SO4 saturation (50% w/v), crude enzyme solution to tert-butanol ratio (1:2 v/v), pH 5. A Box-Behnken design (BBD) of response surface methodology (RSM) was applied to optimize ACMF-TPP. With RSM optimized experimental conditions of ACMF-TPP such as magnetic field irradiation time (6 min), duty cycle (40%), and power (40 W), the maximum yield obtained was 188% with purity of 5.85 folds. Glutaminase purity and yield were enhanced within a considerably shorter processing time (6 min in ACMF-TPP as against 60 min in conventional TPP). The results show that ACMF-TPP is a more effective method for partially purifying glutaminase than conventional TPP.