The pineapple waste from the pineapple industry has contributed to an increase in waste in Malaysia and worldwide every year. A major type of endopeptidase enzymes found in pineapple is fruit bromelain, stem bromelain, ananain, and comasain. This study aims to extract and purify protease from the crown of MD2 pineapple. Protease was extracted and purified using anion exchange chromatography, gel filtration, and desalting before being identified using liquid chromatography-mass spectrometry (LC-MS). Proteolytic activity was determined using the well diffusion method and Casein Digestion Unit. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/ml, a specific activity of 3937.50 U/mg. In the present study, the proteolytic assay showed that 1 kg crown of MD2 cultivar produced an activity of 126.0 ± 3.86 U/mL, a specific activity of 3937.50 U/mg and the total activity of 3.94 × 109 U. The molecular weight of the purified enzyme was in the range of 25 to 35 kDa under the optimum condition of pH 7 and 37°C. Purification of the extract yielded a band at the molecular weight of 20–25 kDa at the optimum pH of 3 and 9 at 60°C. From LC-MS analysis, the purified enzyme from the crown extract was similar to ananain under accession number A0A199VSS3 (according to Uniprot). It had five unique peptides and covered 97/356 amino acids (44.9% coverage). The ananain (EC 3.4.22.31) is classified in the subfamilies of cysteine protease C1A (clan CA, family C1), a peptidase family related to papain. In conclusion, protease was extracted and identified as an ananain-like protease from the crown.

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