title: Purification, expression and characterization of a novel α-l-fucosidase from a marine bacteria Wenyingzhuangia fucanilytica.
abstract: α-l-Fucosyl residues are frequently found in oligosaccharides, polysaccharides and glycoconjugates which play fundamental roles in various biological processes. α-l-Fucosidases, glycoside hydrolases for catalyzing the removal of α-l-fucose, can serve as desirable tools in the study and the modification of fucose-containing biomolecules. In this study, an α-l-fucosidase named as Alf1_Wf was purified from a marine bacterium Wenyingzhuangia fucanilytica by using a combination of chromatographic procedures. The sequence of Alf1_Wf was identified via proteomics analysis against the predicted proteome of the bacterium. Recombinant Alf1_Wf with 6×His tag was expressed in E. coli and showed α-l-fucosidase activity. Sequence annotation revealed that Alf1_Wf contained a combination of GH29 catalytic domain and CBM35 accessory domain. Alf1_Wf was confirmed as a member of GH29-A subfamily based on the phylogenetic analysis. Furthermore, biochemical properties and kinetic characteristics of the enzyme were also determined. Substrate specificity determination showed that Alf1_Wf was capable in hydrolyzing α1,4-fucosidic linkage and synthetic substrate pNP-fucose. Besides, Alf1_Wf could act on partially degraded fucoidan. This study successfully purified, identified, cloned, expressed and characterized a novel α-l-fucosidase, and meanwhile revealed a new multidomain structure of glycoside hydrolase. The knowledge gained from this study should facilitate the further research and application of α-l-fucosidases.
authors: College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China.
authors: Shujun
authors: S
authors: Dong
authors: College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China. Electronic address: changyg@ouc.edu.cn.
authors: Yaoguang
authors: Y
authors: Chang
authors: College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China.
authors: Jingjing
authors: J
authors: Shen
authors: College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China.
authors: Changhu
authors: C
authors: Xue
authors: College of Food Science and Engineering, Ocean University of China, Qingdao, 266003, China.
authors: Feng
authors: F
authors: Chen
mesh_terms: D059001:Aquatic Organisms / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D041965:Flavobacteriaceae / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005644:alpha-L-Fucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005644:alpha-L-Fucosidase
keywords: Alf1_Wf; Fucoidan; Fucosidase; GH29; Marine bacterium