BtGal89A

endo-Gal0273

(EC 3.2.1)endo-α-1,6-Galactanase

CAZy Family: GH89

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~85 kDa)

Figure 1. Electrophoresis analysis of BtGal89A. 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, BtGal89A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.14 U/mg protein (on α-1,6-galactan) at pH 7.0 and 37^oC.

One Unit of α-1,6-galactanase activity is defined as the amount of enzyme required to release 1 μmol of galactose per minute from α-1,6-galactan (1 mg/ml) in PBS buffer pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of BtGal89A on different substrates^a.

^aReactions were performed with 5 mM (p-nitrophenyl glycosides) or 1 mg/ml (polysaccharides), pH 7.0, at 37°C for 10 min.

^bAbsorption caused by released p-nitrophenol or reducing sugars was measured at 405 or 540 nm. The relative activity on α-1,6-galactan  was taken as 100%.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.0

pH Stability: 6.0-8.0

Temperature Optima: 37°C

Temperature Stability: < 45°C

Figure2.pH profile of BtGal89 Aactivity.pH Optima: use buffers with different pH(2-11), the substrate is 1 mg/ml, add enzyme (enzyme:substrate=1:1), react at 37°C for 30 minutes, then stop the reaction at 100°C for 5 minutes.pH Stability: add the enzyme in buffers with different pH (2-11), and incubate at 4°C for 12 h; add the substrate (final concentration of substrate is 1 mg/ml, enzyme:substrate=1:1) at 37°C reaction for 30min, then stop the reaction at 100°C for 5 minutes.

Figure3.Temperature profile ofBtGal89Aactivity.Temperature Optima:use PBS as Buffer, the substrate is 1 mg/ml, add enzyme (enzyme:substrate=1:1), React at different temperatures(20-80°C) for 30min, then stop the reaction at 100°C for 5 minutes.Temperature Stability: the enzymes were incubated in a 30-60 °C water bath for 2 h, and the subsequent operations were carried out according to the optimal temperature experiment. The changes in enzyme activity after incubation at different temperature conditions were compared, and the temperature was plotted.

5.STORAGE CONDITIONS

The enzyme is supplied in PBS buffer and should be stored at -20°C. For assay, this enzyme should be diluted in PBS buffer pH 7.0. Swirl to mix the enzyme immediately prior to use.