Abstract
Recombinant bromelain is a protease that was partially purified using aqueous two-phase system (ATPS). The process variables (pH, PEG 6000 and potassium phosphate concentration) were optimized on enzyme activity and partition coefficient using response surface methodology (RSM) based on a face-centered central composite design (FCCCD) model. The optimum conditions for purification were at 18.47% [w/w] PEG6000 and 13% [w/w] potassium phosphate, pH 7.0 with enzyme activity was obtained as 0.272±0.0036 unit m/L, and partition coefficient as 1.394±0.093. The recombinant bromelain was preferentially partitioned into the top phase and the band was reduced in contrast to crude sample on SDS-PAGE gel.
Highlights
Bromelain is the major protease enzyme present prominently in pineapple (Ananas comosus) fruit and wastes. [1] The increase demand of bromelain in various industrial applications such as food, beverage, tenderization, cosmetic, pharmaceutical and textile [2] has drawn attention among researchers to clone the bromelain gene in various hosts including E.coli BL21-A1,[3] E.coli BL21 DE3pLysS, [4] Pichia pastoris, [5] and Brassica rapa. [6] the recovery of the recombinant bromelain released to the medium is not simple
The plots exhibited a pronounced increase of enzyme activity and KE of recombinant bromelain as the potassium phosphate and PEG concentration reached at 18% [w/w] and 19% [w/w], respectively
Due to the salting-out effect, the ability of salts to capture water molecules is improved and the ionic strength of salts in the aqueous two-phase system (ATPS) medium increased as the potassium phosphate concentration increased.[35]The plots of enzyme activity and KE against pH and potassium phosphate concentration were shown in Figures 1 (a) and 1 (c)
Summary
Bromelain is the major protease enzyme present prominently in pineapple (Ananas comosus) fruit and wastes (peel, core, stem and crown). [1] The increase demand of bromelain in various industrial applications such as food, beverage, tenderization, cosmetic, pharmaceutical and textile [2] has drawn attention among researchers to clone the bromelain gene in various hosts including E.coli BL21-A1,[3] E.coli BL21 DE3pLysS, [4] Pichia pastoris, [5] and Brassica rapa. [6] the recovery of the recombinant bromelain released to the medium is not simple. Current finding by Arshad et al [7] demonstrated a mechanical disruption technique using ultrasonication with an extractant buffer containing sodium phosphate, cysteine and EDTA is found to be effective to release the recombinant bromelain. While such disruption method effectively liberates the recombinant bromelain to the medium, the purification step becomes more challenging because the host cell proteins and nucleic acids are released, thereby contaminating the recombinant bromelain with undesired cellular proteins. Proteins from the E. coli host sometimes naturally binds to the nickel ligand and are co-eluted with the recombinant bromelain during the purification process. Multiple steps in the chromatographic method associates with the huge amount of manufacturing cost for the large-scale process. [11]
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