BtGal42A

Exo-Gal0070

(EC.3.2.1.23)exo-β-Galactosidase

CAZy Family: GH42

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~78 kDa)

Figure 1. Electrophoresis analysis of BtGal42A. 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, BtGal42A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

19.35 U/mg protein (on pNP-β-gal) at pH 7.0 and 37°C.

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

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BtGal42A on different substrates^a.

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

^bAbsorption caused by releasedp-nitrophenol was measured at 405nm. The relative activity on pNPβGal (19.35mol/min/mg) was taken as 100%.

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

Table 2. Hydrolysis kinetic parameters of BtGal42A from B. animalis subsp. lactis Bl-04.

a. As judged by thin-layer chromatography.

There was no activity towards substrates with N-acetylhexosamine at the penultimate position: lacto-N-biose I (LNB; Galβ1-3GlcNAc), galacto-N-biose (GNB; Galβ1-3GalNAc), lacto-N-tetraose (LNT ; Galβ1-3GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose II (Galβ1-3[Fucα1–4]GlcNAcβ1-3Galβ1-4Glc), Lewis A (Galβ1-3[Fucα1–4]GlcNAc), N-Acetyl-D-lactosamine (LacNAc; Galβ1-4GlcNAc), lacto-N-neotetraose (LNnT ; Galβ1-4GlcNAcβ1-3Galβ1-4Glc), Lewis X (Galβ1-4[Fucα1–3]GlcNAc), and lacto-N-fucopentaose III (Galβ1-4[Fucα1–3]GlcNAcβ1-3Galβ1-4Glc).n.d., not detected.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 5.5

Temperature Optima:45°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] Viborg AH, Fredslund F, Katayama T, et al. A β1-6/β1-3 galactosidase from Bifidobacterium animalis subsp. lactis Bl-04 gives insight into sub-specificities of β-galactoside catabolism within Bifidobacterium. Mol Microbiol. 2014 Oct 7.