Recombinant bromelain production in Escherichia coli: process optimization in shake flask culture by response surface methodology

The use of highly purified proteins for therapeutic purposes has been in existence for many decades (Paul, 2004). Enzymes, mostly proteases, constitute the largest portion of these purified proteins for industrial and therapeutic applications. Proteases are enzymes that catalyze the hydrolysis of peptide linkages in proteins. They have wide applications in food, pharmaceutical and detergent industries. In fact, these enzymes constitute about 60% of all commercial enzymes in the world (Lucia and Tomas, 2010). Recently, microbial enzymes have been substituting those from other sources and might now account for almost 90% of the total market (Illanes, 2008). This is due to the fact that microbial cells are excellent systems for enzyme production. Thus, there is a great stimulation for extensive research works on recombinant proteins (Illanes, 2008).Bromelain is a general name given to the family of sulfhydryl proteolytic enzymes (cysteine proteases) obtained from the pineapple plant, Ananas comosus. Depending on the source, it is usually classified as either fruit bromelain or stem bromelain (Kelly 1996). The sulfhydryl proteolytic fraction is the primary component of bromelain. The pineapple enzyme also contains several protease inhibitors, a peroxidase, acid phosphatase, and organically bound calcium (Kelly, 1996).A member of papain family, stem bromelain (E.C.3.4.22.32) contains 212 amino acid residues including seven cysteines, one of which is involved in catalysis (Bitange et al., 2008). Pure stem bromelain is stable when stored at -20°C and has an optimum pH range of 6-8.5 for most of its substrates (casein, gelatin, synthetic peptides, etc.). The optimum temperature range for the enzyme is 50-60°C. It is mostly activated by cysteine while hydrogen sulfide and sodium cyanide are less effective (Bencucci et al. 2011). However, heavy metals such as mercury and silver, and L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane [also known as E-64] deactivate the enz