Purification and proteolytic activity of Caseinolytic protease (Clp) from Lactobacillus plantarum IIA-1AS

A gene encoding caseinolytic protease (Clp) was isolated from Lactobacillus plantarum IIA-1A5 and overexpressed in Escherichia coli. This enzyme belongs to the serine protease and plays essential role in cellular protein homeostasis. This study investigated the purification and proteolytic activity of the protease produced by Lactobacillus plantarum IIA-1A5 isolated from Peranakan Ongole (PO) beef meat. The protein was purified by his-tag affinity column chromatography. The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 26 kDa. Enzyme activity was increased up to 1.9 fold after his-tag affinity chromatography with yield 83.06% and the specific activity of this protein is 606,36 AU mg−1. The optimum pH and temperature for the purified enzyme were found around pH 9.0 and 60°C respectively. Different metal ions were tested for their effect on Clp activity. The enzyme was strongly inhibited by Fe2+, Zn2+, Mg2+ and Cu2+ while Mn2+, and Ca2+ were as activators. The Clp was stable in the presence of various organic solvents especially ethanol and methanol at 10% (v/v) concentration. Also, the proteolytic activity of this protein was higher in casein, bovine serum albumin (BSA), collagen and skim milk compared with sarcoplasmic and myofibril protein from meat. In summary, this enzyme is more suitable for applications in milk protein than meat protein.