Three Phase Partitioning System, an Emerging Non-Chromatographic Tool for Proteolytic Enzymes Recovery and Purification

Three phase partitioning, a scalable method for the purification and recovery of cucumisin, a milk-clotting enzyme, from the juice of Cucumis melo var . reticulatus

“…The findings showed also that during the TPP process, an enhancement of the activity of enzyme may be also possible. This is due to the protective phenomenon that the increased flexibility of enzyme can lead to higher catalytical activity in TPP process [5,25]. Accord-ingly, earlier studies reported an increase in the catalytic activity and yield of several proteases [4,28,[37][38][39].…”

“…Accord-ingly, earlier studies reported an increase in the catalytic activity and yield of several proteases [4,28,[37][38][39]. It was suggested that, the salting out of a protein in TPP system is depended to ionic strength effects, kosmotropy, osmotic stressor, exclusion crowding agent and binding of sulfate ion to cationic sites of a protein [5,25].…”

“…A major difference between conventional salting out procedure and TPP is the addition of C4 alcohols like t-butanol, which is a nonionic kosmotrope that is very soluble and acting as a differentiating solvent. t-Butanol has been selected in this study since it gives the highest recovery (Table 1) and due to its several advantages over other solvents as reported to deliver best results for separation [4,5,38,39]. t-Butanol is miscible in water, but after the addition of enough (NH 4 ) 2 SO 4 , the solution can make three-phase layers and remove lipids, phenolics, pigments and enzyme inhibitors efficiently.…”

“…These enzymes are all proteases, one of the most important enzymes used in food industry, contributing to about 60% of the total world enzyme market [3]. Increasing world cheese production and consumption together with the scarcity on calf rennet have stimulated MCE research from alternative new sources, which has been directed towards plant proteases sources [4,5]. Among plant proteases, we found serine proteases.…”

“…Three phase partitioning (TPP), an emerging non-chromatographic technique, may be considered a more attractive purification technology compared to the other processing methods since it offers major advantages in high throughput of desired product, easy to scale up and cost effective. Studies on the usage of TPP for proteases purification either as one-step purification methodology or being coupled with other techniques have been successfully reported [5,24]. This bioseparation technique is a three-stage batch method, which is a hybrid of salting out and alcohol precipitations for extracting, purifying and concentrating proteins for use in small or large manufacturing operations [25].…”