LpGal2A（LpGal2）

Exo-Gal0068

(EC.3.2.23)exo-β-1,6-Galactosidase

CAZy Family: GH2

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~35.197 kDa)

Figure 1. Electrophoresis analysis of LpGal2A. 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, LpGal2A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.36 U/mg protein (on pNP-β-gal) at pH 7.0 and 50°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 LpGal2A on different substrates^a.

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

^bAbsorption caused by released p-nitrophenol was measured at 405nm. 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 2. Hydrolysis of LpGal2A on galactise detected by HPAEC.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.0

pH Stability: 6.0-9.0

Temperature Optima: 50°C

Temperature Stability: < 45°C

Figure 3.Effect of pH on the activity and stability of recombinant LpGal2A. (A) Optimum pH. Assays were carried out at 37°C for 10 min in buffers ranging in pH from 2.0 to 11.0. The activity at optimum pH was defined as 100%. (B) pH stability. Assays were carried out after the incubation of the enzyme in buffers ranging from pH 2.0-11.0 at 4°C.

Figure 3.Effect of temperature on the activity and stability of recombinant LpGal2A. (A) Optimum temperature. Activity was measured between 20°C and 90°C at pH 7.0 for 10 min. The activity at optimum temperature was defined as 100%. (B)Thermostability. Assays were carried out in20 mM phosphate buffer (pH 7.0) at 65°C for 10 min after the incubation of the enzyme at 35-60°C. The activity without heat treatment was defined as 100%.

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] Zhang XY, Yuan Y, et al. Glycoside hydrolase family2 exo-β-1,6-galactosidase LpGal2 from Lactobacillus plantarum: Cloning, expression, and enzymatic characterization. Process biochemistry, 2021, 102: 269-274.