• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to cleave a fluorogenic substrate 4-methylumbelliferyl-alpha -L-fucopyranoside. The specific activity is >1,600 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Ile2-Glu449, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met

• Predicted Molecular Mass

  53 kDa

• SDS-PAGE

  43-50 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 50 mM MES, 200 mM NaCl, pH 5.5

• Recombinant T. maritima alpha ‑L‑Fucosidase (rT. maritima alpha -L-Fucosidase) (Catalog # 6556-GH)

• Substrate: 4-Methylumbelliferyl-alpha -L-fucopyranoside (Research Products International Corp, Catalog # M65200), 50 mM stock in DMSO

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rT. maritima alpha -L-Fucosidase to 2 ng/μL in Assay Buffer.

2. Dilute Substrate to 400 μM in Assay Buffer.

3. Load into a plate 50 μL of 2 ng/μL rT. maritima alpha -L-Fucosidase, and start the reaction by adding 50 μL of 400 μM Substrate. For Substrate Blanks, load 50 μL of Assay Buffer and 50 μL of 400 μM Substrate.

4. Read plate at excitation and emission wavelengths of 365 nm and 445 nm, respectively, in kinetic mode for 5 minutes.

5. Calculate specific activity:

     *Adjusted for Substrate Blank
     **Derived using calibration standard 4-Methylumbelliferone (Sigma, Catalog # M1381).

Per Well:

• rT. maritima alpha -L-Fucosidase: 0.100 μg

• Substrate: 200 μM

Background: alpha-L-Fucosidase

Fucosylated glycoconjugates play numerous roles in biological events, including development and apoptosis (1, 2), and are involved in the pathology of inflammation, cancer, and cystic fibrosis (3, 4). Fucosidases are generally used for studying fucosylated glycans. With a K  _cat/K  _m value of 160,000 M  ^-1s  ^-1, the alpha -L-fucosidases of   Thermotoga maritima efficiently hydrolyzes 4-nitrophenyl fucoside (5). The enzyme is the closest bacterial relative of mammalian fucosidase with 38% identity to its human homologue. It is thought to remove alpha ‑1,2‑ and alpha -1,4-linked fucosyl side chains from algal fucoidan (5), while the activity on alpha -1,3- and alpha ‑1,6‑ linked fucose has not been tested. The enzyme assembles as a hexamer and displays a two-domain fold, with Asp  ²²⁴ and Glu  ²⁶⁶ being critical for enzyme activity (6).

• References:

1. Hiraishi, K. et al. (1993) Glycobiology 3:381.

2. Solter, D. and Knowles, B.B. (1978) Proc. Natl. Acad. Sci. USA 75:5565.

3. Shah, M. et al. (2008) cancer 113:338.

4. Becker, D.J. and Lowe, J.B. (2003) Glycobiology 13:41R.

5. Tarling, C.A. et al. (2003) J. Biol. Chem. 278:47394.

6. Sulzenbacher, G. et al. (2004) J. Biol. Chem. 279:13119.

• Entrez Gene IDs:

  897238 (T. maritima)

• Alternate Names:

  alphaLFucosidase; alpha-L-Fucosidase