Purification and identification of different lipases contained in PPL commercial extracts: A minor contaminant is the main responsible of most esterasic activity

Abstract

Porcine pancreatic lipase extract has been used as a biocatalyst for many years, however it is a rather complex mixture with various active enzymes. In this work, we have developed a protocol to purify three different enzymes with esterase activity and lipase properties: the actual Porcine pancreatic lipase; a previously reported 33 kDa lipase and a third one with a molecular weight of 25 kDa. They can be adsorbed on hydrophobic supports at low ionic strength, in some cases exhibiting a certain hyper-activation and recognizing a wide range of substrates. Moreover, there was some selectivity in the adsorption of the different lipases on different hydrophobic supports, but it is not enough to have fully pure enzymes. Using ionic exchangers, combined with adsorption in hydrophobic supports at low ionic strength, we have been able to have fully purified lipases. The 25 kDa enzyme was, in fact, the most interesting one because it was responsible for a great percentage of the esterase activity (including against glycidyl butyrate), even though it only accounts for less than 1% of the total protein. It presented a p I of 5.5 and the amino terminal sequence was identical to π-chymotrypsine B.