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Immobilized Sphingolipid Ceramide N-Deacylase As A Tool For Simple Semisynthesis Of C17:0 Sphingolipid Isoforms

Autoři: Čadková Michaela | Korecká Lucie | Kuchař Ladislav | Černá Veronika | Bílková Zuzana

Rok: 2012

Druh publikace: ostatní - přednáška nebo poster

Strana od-do: nestránkováno

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Jazyk	Název	Abstrakt	Klíčová slova
eng	Immobilized Sphingolipid Ceramide N-Deacylase As A Tool For Simple Semisynthesis Of C17:0 Sphingolipid Isoforms	Sphingolipid ceramide N-deacylase (SCDase; EC 3.5.1.69) is an enzyme hydrolizing N-acyl linkage between fatty acids and sphingosine bases in ceramides of various sphingolipids as well as in sphingomyelin to form their lyso-form and appropriate fatty acid. This enzyme could also catalyze reversible hydrolytic reaction of lyso-sphingolipids and fatty acids to produce sphingolipids 1,2,3. In this contribution the preparation and characterization of magnetic carrier with covalently bond enzyme SCDase for sphingolipid modification is presented. The main advantage of immobilized enzyme compared to its soluble form is the possibility of easy removing from the reaction mixture, repeated use as well as it enables to gain the pure forms of sphingolipids at low cost. For preparation of effective enzyme reactor magnetic macroporous bead cellulose with hydroxyl function groups was elected for the immobilization of SCDase isolated from Pseudomonas sp. TK4.. Catalytic activity of both forms of enzyme (soluble and immobilized) is examined by use of specific substrate GM1 Asialo for hydrolytic reaction and lyso-Gb3Cer with heptadecanoic acid (C17:0) for reversible hydrolytic reaction. Formed products are detected by high performance thin layer chromatography (HPTLC) and verified by mass spectrometry ESI-MS. The prepared carrier with immobilized enzyme was characterized by determination of operational stability for possible reuse and storage stability for long term storage. Subsequently, this system can be used for acquirement of pure forms of sphingolipids and lyso-sphingolipids, which could serve as internal standards for sphingolipids studies.	sfingolipid ceramide N-deacylase; immobilized enzyme; magnetic microspheres;

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