BtGal43A

Endo-Gal0053

(EC. 3.2.1.181) endo-β-1,3-Galactanase

CAZy Family: GH43

PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~65 kDa)

Figure 1. Electrophoresis analysis of BtGal43A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate of E. coli BL21-pET28a-BtGal43A before IPTG induction; lane 2, culture lysate of E. coli BL21-pET28a-BtGal43A after IPTG induction; lane 3, BtGal43A purified from Ni sepharose fastflow column.

2. SPECIFIC ACTIVITY

0.39 U/mg protein (on pNP-β-gal) at pH7.0 and 37°C

One Unit of pNP-β-gal activity is defined as the amount of enzyme required to release one μmole 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 BtGal43A on different substrates.

Figure 2 TLC of BtGal43A hydrolyzed lactose. The concentration of lactose was 2 mg/mL in all reactions. The reactions were incubated in 20 mM sodium phosphate buffer (pH 7.0) for 12 hours.

Figure 3. HPAEC analysis of the activity of BtGal43A. The AGPs were at 25 mg/ml for all reactions, the β-1,3- galactan backbone was at 1.5 mg/ml. Enzyme concentration was 1 µM. Reactions were incubated for 16 h in 20 mM sodium phosphate buffer pH 7.0 containing 150 mM NaCl buffer. The right figure shows a time course of BtGal43A acting on β-1,3-galactan. Peaks containing a defined galactooligosaccharide are identified by a yellow circle with the degree of polymerization shown in subscript.

4. PHYSICOCHEMICAL PROPERTIES

pH Optima: suggested use 7.0

Temperature Optima: suggested use 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] Cartmell A, Muñoz-Muñoz J, Briggs JA, et al. A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation. Nat Microbiol. 2018, 3(11): 1314-1326.