BtGal36E

Ex-Gal0041

(EC.3.2.1.22)exo-α-Galactosidase

CAZy Family: GH36

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~73kDa)

Figure 1. Electrophoresis analysis of BtGal36E. 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, BtGal36E purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.1 U/mg protein (on pNP-α-gal) at pH 7.0 and 30°C.

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

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BtGal36E on different substrates^a.

^aReactions were performed with 5mM substrate, pH7.0, at 30°C for 30 min.

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

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

Figure 2. TLC analysis of BtGal36E catalyzed hydrolysis of polysaccharides. Gum arabic from acacia (left two lanes) or arabinogalactan from larch wood (right two lanes) was incubated with (+) or without (-)BtGal36Efor 1 h at 35°C in 40 mM sodium acetate buffer, pH 5.5, containing 10 mM CaCl₂ and 0.002 mg/mLBSA. The sugars on the TLC were developed twice using a solvent system of acetonitrile:water (4:1, v/v). Standard arabinose and galactose are in the leftmost lane.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima:5.5

pH Stability:4.0-12.0

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1]Kikuchi A, Okuyama M , Kato K , et al. A novel glycoside hydrolase family 97 enzyme: Bifunctional β-l-arabinopyranosidase/α-galactosidase from Bacteroides thetaiotaomicron[J]. Biochimie, 2017,142:41-50.