Integrated Downstream Processing of Lactoperoxidase from Milk Whey Involving Aqueous Two-Phase Extraction and Ultrasound-Assisted Ultrafiltration

Abstract

The present work involves the adoption of an integrated approach for the purification of lactoperoxidase from milk whey by coupling aqueous two-phase extraction (ATPE) with ultrasound-assisted ultrafiltration. The effect of system parameters of ATPE such as type of phase system, polyethylene glycol (PEG) molecular mass, system pH, tie line length and phase volume ratio was evaluated so as to obtain differential partitioning of contaminant proteins and lactoperoxidase in top and bottom phases, respectively. PEG 6000-potassium phosphate system was found to be suitable for the maximum activity recovery of lactoperoxidase 150.70% leading to 2.31-fold purity. Further, concentration and purification of enzyme was attempted using ultrafiltration. The activity recovery and purification factor achieved after ultrafiltration were 149.85% and 3.53-fold, respectively. To optimise productivity and cost-effectiveness of integrated process, influence of ultrasound for the enhancement of permeate flux during ultrafiltration was also investigated. Intermittent use of ultrasound along with stirring (2 min acoustic and 2 min stirring) resulted in increased permeate flux from 0.94 to 2.18 l/m(2) h in comparison to the ultrafiltration without ultrasound. The use of ultrasound during ultrafiltration resulted in increase in flux, but there was no significant change in activity recovery and purification factor. The integrated approach involving ATPE and ultrafiltration may prove to be a feasible method for the downstream processing of lactoperoxidase from milk whey.

Partitioning of thermostable glucoamylase in polyethyleneglycol/salt aqueous two-phase system

“…This phenomenon enhanced the purification factor and yield at the bottom phase. The results are in accordance with reported literature (Nandini and Rastogi [19] and Naganagouda and Mulimani [22]). It can be visualized from Fig.…”

“…The decrease in pH makes the glucoamylase more positively charged and leads to stronger interaction between glucoamylase and polymer which migrated more enzymes to the PEG-rich phase. Similar results were observed by Nandini and Rastogi [19] and Ratanapongleka [20].…”

“…However, based on the purification factor, potassium phosphate resulted in a higher PF (1.46). Potassium phosphate system was recognized to be more effective for lactoperoxidase [19], laccase [20], polyphenol oxidase [21], and α-galactosidase [22]. Based on the preliminary results, PEG/potassium phosphate (K 2 HPO 4 and KH 2 PO 4 ) system was used for further studies.…”

“…Recent studies have employed the use of ATPS (polyethylene glycol (PEG)/potassium phosphate) for biomolecule extraction and primary purification, to a great extent. Nandini and Rastogi [19] dealt with the partitioning of lactoperoxidase from milk whey and studied the effect of phase-forming salt, PEG molecular weight, pH, TLL and V R , resulting in a purification-fold (PF) of 2.31. Ratanapongleka [20] studied the partitioning behavior of laccase from Lentinus polychrous Lev., to study the effect of PEG molecular weight and concentration, salt concentration, pH, and NaCl, leading to 99 % yield and PF of 3.…”

Extractive bioconversion of poly-ϵ-caprolactone by Burkholderia cepacia lipase in an aqueous two-phase system

“…Burkholderia lipase had an isoelectric point of 6.3 [22]. Therefore, at pH 7, it is slightly negatively charged and could interact strongly with PEG, which acts as a positively charged polymer [23]. Moreover, the high salt concentration in the lower phase led to the hydration of lipases, which exposed their hydrophobic parts and therefore, encouraged their hydrophobic interactions with the PEG [24].…”