Bromelain is a protease that has multiple applications in food industry. This study focused on the partial purification of bromelain from pineapple crown using the three‐phase partitioning (TPP) method. Different parameters of TPP (ratio of crude extract to t‐butanol (v/v), percentage of (NH4)2SO4, and pH) were optimized for the concentration and purification of bromelain. The optimized parameters of the TPP system, that is, 1.0:0.5 crude extract to t‐butanol ratio at pH 7.0 with 70% (NH4)2SO4 saturation, gave significantly high purification fold of 3.4 (p‐value ≤.000) with a significant increase in percent enzyme activity recovery of 244% (p‐value ≤.000). The purified enzyme was predominantly partitioned at the interfacial phase and the molecular weight of bromelain was found to be around 26 kDa. Characterization of bromelain demonstrated the highest relative activity at pH 7.0 and 35°C. This study showed the effective and economical process development of bromelain purification from pineapple waste at laboratory scale using the TPP method. Novelty impact statement The current study reported the purification of industrially important enzyme Bromelain using three‐phase partitioning (TPP) method, which has not been previously studied. TPP method was effectively used as a single, scalable, non‐chromatographic method for bromelain purification from crown waste of pineapple. The optimized parameters (70% (NH4)2SO4 saturation with 1.0:0.5 ratio of crude extract to t‐butanol at pH 7.0), resulted in the purification fold of 3.4 with a recovery of 244%.

Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% HIGHLIGHTS• Bromelain was extracted from crown, core, and peel waste of pineapple.• Ammonium sulphate (NH4)2SO4 precipitation method was used as a simple and cost-effective approach for partial purification of bromelain.• A purification of 4.34, 2.75 and 2.59-fold was achieved for crown, core and peel bromelain samples respectively.• Enzyme activity recovery of 170.41 %, 192.37 % and 337.59 % was achieved for crown, core, and peel bromelain, respectively. 2 Gul, A., et al.