PoGal35B（exo-β-1,3/4/6-Galactosidase）

PoGal35B

Ex-Gal00343

(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 PoGal35B. 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, PoGal35B purified from Ni sepharose fastflow column.


2. SPECIFIC ACTIVITY

1.2 U/mg protein (on pNP-β-gal) at pH 6.0 and 60°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 6.0.


3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figure 2. HPAEC-PAD analysis of hydrolysis products of galactobiose generated by PoGal35B.

Figure 3. HPAEC-PAD analysis of the products of the degradation of Galactan by PoGal35B.


4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.0

pH Stability: 4.0-7.0

Temperature Optima: 60°C

Temperature Stability: 40°C-50°C

Figure 4. Effect of pH on activity (a) and stability (b) of PoGal35AB using pNP-β-gal 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 PoGal35B using pNP-β-gal 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. REFERENCE

[1] 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.

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PoGal35B（exo-β-1,3/4/6-Galactosidase）

PoGal35B

Ex-Gal00343

(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 PoGal35B. 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, PoGal35B purified from Ni sepharose fastflow column.


2. SPECIFIC ACTIVITY

1.2 U/mg protein (on pNP-β-gal) at pH 6.0 and 60°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 6.0.


3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figure 2. HPAEC-PAD analysis of hydrolysis products of galactobiose generated by PoGal35B.

Figure 3. HPAEC-PAD analysis of the products of the degradation of Galactan by PoGal35B.


4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 6.0

pH Stability: 4.0-7.0

Temperature Optima: 60°C

Temperature Stability: 40°C-50°C

Figure 4. Effect of pH on activity (a) and stability (b) of PoGal35AB using pNP-β-gal 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 PoGal35B using pNP-β-gal 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. REFERENCE

[1] 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.