PpGal42A (PpBGal42A)

Exo-Gal0251

(EC.3.2.1.23)exo-β-Galactosidase

CAZy Family: GH42

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~77.16 kDa)

Figure 1. Electrophoresis analysis of PpGal42A. 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, PpGal42A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

1.24 U/mg protein (on pNP-β-gal) at pH 6.0 and 30°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) inacetate buffer (20 mM) pH 6.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of PpGal42C on different substrates^a.

^aReactions were performed with 5 mM substrate, pH 6.0, at 30°C for 5 min.

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

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

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.0

Temperature Optima: 30°C

Figure 2. Effect of pH and temperature on activity and stability of PpBGal42A. (A) Using pNPG as substrate. (A-a) Optimal pH. (A-b) pH stability. (A-c) Optimaltemperature.(A-d) Temperature stability. (B) Using pNPαArap as substrate. (B-a) Optimal pH. (B-b) pH stability. (B-c) Optimal temperature. (B-d) Temperature stability. Relative activity was calculated using the maximum activity as 100%. Results are presented as the mean ± standard deviation (n=3)

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in acetate buffer (20 mM) pH 6.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Cui J, Yuan Y, et al. Cloning, Expression, Purification, and Characterization of a Novel β-Galactosidase/α-L-Arabinopyranosidase from Paenibacillus polymyxa KF-1. Molecules. 2023, 28, 7464.