Purification and characterization of a phospholipid-hydrolyzing phosphoesterase produced by Pediococcus acidilactici isolated from Gouda cheese.

Lipolysis occurs during ripening of dairy products as a result of esterase or lipase activity. Lactic acid bacteria (LAB) are considered to be weakly lipolytic bacteria compared with other species. In cheeses with extended ripening periods, lipolytic LAB may have several advantages. Pediococcus acidilactici is a LAB frequently found in fermented dairy products, but no previous reports exist on their production of esterases or lipases. Our interest in the relationship of LAB and enzymatic characterization is due to the multiple reports of the benefits of LAB in the gut microbiome, particularly at the intestinal membrane. Pediococci have been characterized as probiotic and especially active in membrane interactions. The aim of this project was to purify, characterize, and identify the phosphoesterase produced by P. acidilactici originally isolated from Gouda cheese and determine its phospholipid (PL) hydrolysis profile, with a focus on increased absorption of these compounds in the human gut. Native zymograms were performed to identify a protein with lipolytic activity in the intracellular fraction of P. acidilactici. The enzyme was purified via size-exclusion HPLC, concentrated via ultrafiltration, and identified using sequence analysis in liquid chromatography (LC)-MS/MS. The purified fraction was subjected to biochemical characterization as a function of pH, temperature, ion concentration, hydrolysis of different substrates, and PL. A single protein with a molecular weight of 86 kDa and esterase activity was detected by zymography. Analysis of the LC-MS/MS results identified a putative metallophosphoesterase with a calculated molecular weight of 45.5 kDa, suggesting that this protein is active as a homodimer. The pure protein showed an optimal activity between pH 8.0 to 9.0. The optimal temperature for activity was 37°C, and the enzyme lost 15% of activity after incubation at 90°C for 1 h. This enzyme showed activity on short-chain fatty acids and exhibited high hydrolysis of phosphatidylinositol. It also hydrolyzed phosphatidylserine, phosphatidylcholine, and sphingomyelin. Phosphatidylethanolamine was hydrolyzed but with less efficiency. The characteristics and lipolytic actions exerted by this protein obtained from LAB hold promise for a potential strain of esterase or lipase that may exert human health benefits through increased digestibility and absorption of nutrients found in dairy products.