Purification of pineapple bromelain by IMAC chromatography using chlorophyll-activated macroporous matrices

•A polymeric affinity matrix was developed immobilized chlorophyll in its structure.•The matrix was able to adsorb proteases extracted from the pineapple fruit.•The new adsorbent shows promise in protein purification.•A product with a purity about 6 times greater than the crude extract was obtained.•Chlorophyll shows promise as a chelating agent in chromatographic processes. This study investigated the purification of bromelain obtained from pineapple fruit using a new adsorbent for immobilized metal ion affinity chromatography (IMAC), with chlorophyll obtained from plant leaves as a chelating agent. The purification of bromelain was evaluated in batches from the crude extract of pineapple pulp (EXT), and the extract precipitated with 50 % ammonium sulfate (EXT.PR), the imidazole buffer (200 mM, pH 7.2) being analyzed and sodium acetate buffer, pH 5.0 + 1.0 NaCl as elution solutions. All methods tested could separate forms of bromelain with molecular weights between ±21 to 25 kDa. Although the technique using EXT.PR stood out in terms of purity, presenting a purification factor of around 3.09 ± 0.31 for elution with imidazole and 4.23 ± 0.12 for acetate buffer solution. In contrast, the EXT methods obtained values between 2.44 ± 0.23 and 3.21 ± 0.74 for elution with imidazole and acetate buffer, respectively, for purification from EXT.PR has lower yield values (around 5 %) than EXT (around 15 %). The number of steps tends to reduce yield and increase process costs, so the purification process in a monolithic bed coupled to the chromatographic system using the crude extract was evaluated. The final product obtained had a purification factor of 6, with a specific enzymatic activity of 59.61 ± 0.00 U·mg−1 and a yield of around 39 %, with only one band observed in the SDS-PAGE electrophoresis analysis, indicating that the matrix produced can separate specific proteins from the total fraction in the raw material. The IMAC matrix immobilized with chlorophyll proved promising and viable for application in protease purification processes.