BtGal2A

Ex-Gal0062

(EC.3.2.1.23)exo-β-1,4-Galactosidase

CAZy Family: GH2

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~92kDa)

Figure 1. Electrophoresis analysis of BtGal2A. 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, BtGal2A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

1.11 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 BtGal2A on different substrates^a.

^aReactions were performed with 5 mM substrate, pH 7.0, at 37 ◦C for 10 min.

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

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

Figure 3. Signal molecule protection. (a), Each panel shows the catalytic efficiency of key enzymes implicated in signal molecule degradationfor galactan. Data taken from technical replicates, n= 3. (b), Models for degradation of galactan.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.5

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] Luis AS, Briggs J, Zhang X, et al. Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides. Nat Microbiol. 2018 Feb;3(2):210-219.