Benešová Eva, Lipovová Petra, Krejzová Jana, Kovaľová Terezia, Buchtová Patricie, Spiwok Vojtěch, Králová Blanka
Department of Biochemistry and Microbiology, UCT Prague, Prague, 166 28, Czech Republic.
BMC Biotechnol. 2015 May 27;15:36. doi: 10.1186/s12896-015-0160-x.
α-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.
Activity-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.
Using 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.
In 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.
α-L-岩藻糖苷酶是参与α-L-岩藻糖基化分子代谢的酶，这些化合物在包括免疫反应、受精和发育等不同生命必需过程中具有重要作用，在一些严重病理事件中也发挥作用。根据CAZy数据库，这些酶属于第29和95家族。据报道，其中一些酶还能够催化转糖基化反应，在此过程中会形成α-L-岩藻糖基化分子，这些分子尤其受到制药行业的关注。
基于活性的基因组文库筛选用于分离编码新型α-L-岩藻糖苷酶的基因。该酶在大肠杆菌中表达，亲和色谱法用于纯化带有His标签的α-L-岩藻糖苷酶。标准活性测定用于酶的表征。薄层色谱法和质谱法用于评估转糖基化反应。
利用在大肠杆菌DH5α细胞中构建的解硫胺芽孢杆菌基因组文库，确定了一种新的α-L-岩藻糖苷酶同工酶的核苷酸序列，并提交至EMBL数据库（HE654122）。然而，未检测到与CAZy数据库第29和95家族的酶有相似性。该酶在大肠杆菌BL21(DE3)细胞中以带有组氨酸标签的蛋白质形式产生，并通过金属亲和色谱法纯化。使用不同的受体分子测试了α-L-岩藻糖苷酶同工酶2的水解和转糖基化能力。
在本研究中，描述并制备了源自解硫胺芽孢杆菌的新型酶α-L-岩藻糖苷酶同工酶2的重组形式，并对其水解和转糖基化特性进行了表征。由于发现与已知α-L-岩藻糖苷酶的氨基酸序列相似性非常低，后续研究对于涉及分子建模的不同生物化学学科可能具有重要意义。
