Abstract
Backgroundα-l-Fucosidases are enzymes involved in metabolism of α-l-fucosylated molecules, compounds with a fundamental role in different life essential processes including immune response, fertilization and development, but also in some serious pathological events. According to the CAZy database, these enzymes belong to families 29 and 95. Some of them are also reported to be able to catalyze transglycosylation reactions, during which α-l-fucosylated molecules, representing compounds of interest especially for pharmaceutical industry, are formed.MethodsActivity-based screening of a genomic library was used to isolate the gene encoding a novel α-L-fucosidase. The enzyme was expressed in E.coli and affinity chromatography was used for purification of His-tagged α-L-fucosidase. Standard activity assay was used for enzyme characterization. Thin layer chromatography and mass spectrometry were used for transglycosylation reactions evaluation.ResultsUsing a genomic library of Paenibacillus thiaminolyticus, constructed in E.coli DH5α cells, nucleotide sequence of a new α-l-fucosidase isoenzyme was determined and submitted to the EMBL database (HE654122). However, no similarity with enzymes from CAZy database families 29 and 95 was detected. This enzyme was produced in form of histidine-tagged protein in E.coli BL21 (DE3) cells and purified by metaloaffinity chromatography. Hydrolytic and transglycosylation abilities of α-l-fucosidase iso2 were tested using different acceptor molecules.ConclusionsIn this study, new enzyme α-l-fucosidase iso2 originating from Paenibacillus thiaminolyticus was described and prepared in recombinant form and its hydrolytic and transglycosylation properties were characterized. As a very low amino acid sequence similarity with known α-l-fucosidases was found, following study could be important for different biochemical disciplines involving molecular modelling.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0160-x) contains supplementary material, which is available to authorized users.
Highlights
Saccharides and different types of glycoconjugates constitute a diverse group of molecules, function of which range from both energy and building elements supply to facilitation of specific interactions and to modifications of properties and functions of many biologically active molecules [1, 2]
According to Carbohydrate-Active Enzymes database (CAZy database), they belong to families 29 and 95, which differ among each other in mechanism used for the Benešová et al BMC Biotechnology (2015) 15:36 hydrolytic reaction catalysis [7]
While testing the possibility to purify an enzyme with α-L-fucosidase activity by chromatographic methods, it was accidentally discovered that P. thiaminolyticus produces two isoenzymes of α-Lfucosidase and that it is possible to separate them using chromatography with hydrophobic interactions on the column HiTrap Butyl FF (1 mL)
Summary
Saccharides and different types of glycoconjugates constitute a diverse group of molecules, function of which range from both energy and building elements supply to facilitation of specific interactions and to modifications of properties and functions of many biologically active molecules [1, 2]. First of them, coordinated by Nomenclature Committee α-L-Fucosidases (3.2.1.51) are glycosidases that are able to cleave terminal α-L-fucosyl moiety from different types of oligosaccharides and glycoconjugates. 29 enzymes use a two-step double-displacement mechanism for the catalysis, (retaining enzymes i.e. the anomeric configuration of the released product is the same as in the originally cleaved molecule), while glycosidases from family 95 are among so called inverting enzymes catalyzing the hydrolytic process by direct displacement mechanism. Many retaining enzymes are able to catalyze hydrolytic reactions and transglycosylation; i.e. the reaction, in which the final acceptor of cleaved glycosidic residue is a hydroxyl group-possessing molecule that differs from water [7, 8, 9, 10]
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