BfFuc29B

Ex-Fuc0245

(EC 3.2.1.51) exo-α-Fucosidase

CAZy Family: GH29

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~48kDa)

Fig.1 Electrophoresis analysis of BfFuc29B. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate Bbefore IPTG induction; lane 2, culture lysate after IPTG induction; lane 3, BfFuc29B purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

3.35 U/mg protein (on pNP-α-fuc) at pH 7.0 and 37°C.

One Unit of pNP-α-fuc activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenyl per minute from pNP-α-fuc (5 mM) insodium phosphate buffer (20 mM) pH7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BfFuc29B on different substrates^a.

^aReactions were performed with 5 mM substrate, pH 7.0, at 37°C for 5 min.

^bAbsorption caused by released p-nitrophenol was measured at 405 nm. The relative activity on pNPαFuc was taken as 100%.

^cThe data are reported as means±standard errors from the mean for three independent experiments.

Figure2. Time course of BfFuc29B-catalyzed defucosylation of Rituxan.The deglycosylated Rituxan was used for the defucosylation activity evaluation of BfFuc29B. Mixtures of BfFuc29B and deglycosylated Rituxan were incubated at different temperatures and aliquots were taken atdifferent times to evaluate the defucosylation activity by AAL lectin blotting. The staining intensity is highly relevant to the content of core fucose.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:7.5

Temperature Optima:60°C

Figure3.Biochemical properties and specificity of BfFuc29B.(a) pH profile of BfFuc29B(b) Profile of BfFuc29B at different temperatures

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in sodium phosphate buffer (20 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Tsai T I , Li S T , Liu C P , et al. An Effective Bacterial Fucosidase for Glycoprotein Remodeling[J]. Acs Chemical Biology, 2017, 12(1):63.