Identification and characterization of a polyurethanase with lipase activity from Serratia liquefaciens isolated from cold raw cow's milk

Abstract

Lipases are associated with food spoilage and are also used in various biotechnological applications. In this study, we sought to purify, identify, and characterize a lipase from S. liquefaciens isolated from cold raw cow's milk. The lipase partially purified by ultrafiltration and gel filtration showed a specific activity of 2793 U/mg. By zymography, the enzyme presented approximately 65 kDa, and LC-MS/MS allowed the identification of a polyurethanase with a conserved domain of family I.3 lipase. The modeled and validated structure of polyurethanase was able to bind to different fatty acids and urethane by molecular docking. The polyurethanase showed optimum activity at pH 8.0 and 30 °C. In the presence of ions, activity was decreased, except for Ca²⁺, Mg²⁺, and Ba²⁺. Reducing agents did not alter the activity, while amino acid modifiers reduced enzyme activity. It is concluded that polyurethanase with lipase activity represents a potential enzyme for the deterioration of milk and dairy products, as well as a candidate for industrial applications.

Keywords: Biotechnological applications; Deterioration; Diethyl pyrocarbonate (DEPC) – PubChem CID: 3051; Dithiothreitol (DTT) – PubChem CID: 446094; Ethylenediaminetetraacetic acid (EDTA) – PubChem CID: 6049; Family I.3 lipase; Methylumbelliferyl butyrate (MUB) – PubChem CID: 87247; Phenylmethylsulfonyl fluoride (PMSF) – PubChem CID: 4784; Polyurethanase; Tosyl-l-phenylalanine chloromethyl ketone (TPCK) – PubChem CID: 4339647; p-nitrophenol (p-NP) – PubChem CID: 980; p-nitrophenyl palmitate (p-NPP) – PubChem CID: 73891; β-mercaptoethanol (BME) – PubChem CID: 1567.