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

摘要

Lipolysis occurs during ripening of dairy products asa result of esterase or lipase activity. Lactic acid bacteria(LAB) are considered to be weakly lipolytic bacteriacompared with other species. In cheeses with extendedripening periods, lipolytic LAB may have several advantages.Pediococcus acidilactici is a LAB frequentlyfound in fermented dairy products, but no previousreports exist on their production of esterases or lipases.Our interest in the relationship of LAB and enzymaticcharacterization is due to the multiple reports of thebenefits of LAB in the gut microbiome, particularly atthe intestinal membrane. Pediococci have been characterizedas probiotic and especially active in membraneinteractions. The aim of this project was to purify,characterize, and identify the phosphoesterase producedby P. acidilactici originally isolated from Goudacheese and determine its phospholipid (PL) hydrolysisprofile, with a focus on increased absorption of thesecompounds in the human gut. Native zymograms wereperformed to identify a protein with lipolytic activity inthe intracellular fraction of P. acidilactici. The enzymewas purified via size-exclusion HPLC, concentrated viaultrafiltration, and identified using sequence analysisin liquid chromatography (LC)-MS/MS. The purifiedfraction was subjected to biochemical characterizationas a function of pH, temperature, ion concentration,hydrolysis of different substrates, and PL. A singleprotein with a molecular weight of 86 kDa and esteraseactivity was detected by zymography. Analysis ofthe LC-MS/MS results identified a putative metallophosphoesterasewith a calculated molecular weight of45.5 kDa, suggesting that this protein is active as ahomodimer. The pure protein showed an optimal activitybetween pH 8.0 to 9.0. The optimal temperature foractivity was 37°C, and the enzyme lost 15% of activityafter incubation at 90°C for 1 h. This enzyme showedactivity on short-chain fatty acids and exhibited highhydrolysis of phosphatidylinositol. It also hydrolyzedphosphatidylserine, phosphatidylcholine, and sphingomyelin.Phosphatidylethanolamine was hydrolyzed butwith less efficiency. The characteristics and lipolytic actionsexerted by this protein obtained from LAB holdpromise for a potential strain of esterase or lipase thatmay exert human health benefits through increaseddigestibility and absorption of nutrients found in dairyproducts.