BbFuc95A

Exo-Gal0202

(EC.3.2.1)exo-α-1,2-Fucosidase

CAZy Family: GH95

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~100 kDa)

Figure 1. Electrophoresis analysis of BbFuc95A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before IPTG induction; lane 2, culture lysate after IPTG induction; lane 3, BbFuc95A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

1.64 U/mg protein (on pNP-α-Fuc) at pH 7.0 and 37°C

One Unit of pNP-α-Fucactivity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenyl per minute from pNP-α-Fuc (5 mM) in phosphate buffer (20 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BbFuc95A 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.

Figure 2.HPAEC was used to detect the enzymatic hydrolysis of BbFuc95A to WGRP-N-B

Table 2.Comparison of monosaccharide composition before and after enzymatic hydrolysis of WGRP-N-B by BbFuc95A.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.5

Temperature Optima:37°C

5.STORAGE CONDITIONS

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

6. REFERENCES

[1] Katayama T, Sakuma A, Kimura T, et al. Molecular cloning and characterization of Bifidobacterium bifidum 1,2-alpha-L-fucosidase (AfcA), a novel inverting glycosidase (glycoside hydrolase family 95). J Bacteriol. 2004 Aug;186(15):4885-93.