PoGal35A

Ex-Gal00342

(EC.3.2.1.23)  exo-β-1,3/4/6-Galactosidase

CAZy Family: GH35

PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~111 kDa)

Figure 1. Electrophoresis analysis of PoGal35A. M, molecular weight marker (PageRuler Prestained Protein Ladder, Thermo Scientific); lane 1, culture lysate before Methanol induction; lane 2-4, culture lysat after Methanol induction; lane 5, PoGal35A purified from Ni sepharose fastflow column.

2. SPECIFIC ACTIVITY

191 U/mg protein (on pNP-β-gal) at pH 5.0 and 70°C

One Unit of pNP-β-gal activity is defined as the amount of enzyme required to release one μmole of galactose per minute from pNP-β-gal (5 mM)in Na-Acetate buffer (20 mM) pH 5.0.

3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figure 2. HPAEC-PAD analysis of hydrolysis products of galactobiose generated by PoGal35A.

Figure 3. HPAEC-PAD analysis of the products of the degradation of Galactan by PoGal35A.

4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 5.0

pH Stability: 4.0-7.0

Temperature Optima: 70°C

Temperature Stability: 40°C-50°C

Figure 4. Effect of pH on activity (a) and stability (b) of PoGal35A using potato galactan as substrate. The optimal pH (a) was determined at different pH from 2 to 11. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 3 h at different pH. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(n = 3).

Figure 5. Effect of temperature on activity (a) and stability (b) of PoGal35A using potato galactan as substrate.  The optimal temperature (a) was determined at different temperatures from 20 to 90℃. The maximum activity obtained was defined as 100% activity. Thermal stability was determined by incubating the enzyme for 3 h at different temperatures. The activity of the enzyme before incubation was defined as 100%. Results are presented as means ± standard deviations(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 4.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Zhou AD, Yi HT et al. Cloning, Expression, Purification and Characterization of the β-galactosidase PoβGal35A from Penicillium oxalicum. Molecular biotechnology, 2023, 65(7):1140-1150.

[2] 衣昊庭. 可降解RG-I果胶分子中半乳聚糖链的水解酶制备及其功能研究[D]. 东北师范大学, 2023.