Expression, purification, and characterization of a recombinant stem bromelain from Ananas comosus

Abstract

Commercially available bromelain is prepared by performing a tedious and costly purification method that yields bromelain at different degrees of purity. In the current study, a gene encoding stem bromelain from Ananas comosus was amplified using polymerase chain reaction. This bromelain gene was initially cloned into the pENTR/TEV/D-TOPO vector before being sub-cloned into the pDEST17 expression vector. DNA sequencing of the amplified products exhibited a high level of homology to the corresponding gene from the NCBI public database. Protein expression was conducted in the BL21-AI Escherichia coli strain. The recombinant bromelain was then purified in a single step using immobilized metal affinity chromatography, specifically a Ni-NTA spin column. The purified recombinant bromelain was detected by Western blotting. In addition, the purified enzyme exhibited hydrolytic activity towards gelatin and a synthetic substrate, LNPE. The purified recombinant bromelain exhibited optimum activity at pH 4.6 and 45°C.