PoAra54A

Ex-Ara00349

(EC.3.2.1.55)  exo-α-1,2/3/5-Arabinofuranosidase

CAZy Family: GH54

PROPERTIES

1. ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~53 kDa)

Figure 1. Electrophoresis analysis of PoAra54A. 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, PoAra54A purified from Ni sepharose fastflow column.

2. SPECIFIC ACTIVITY

16.9 U/mg protein (on pNPαAraf) at pH 4.0 and 40°C;

0.12 U/mg protein (on Sugar Beet Linear Arabinan) at pH 4.0 and 40°C;

6.80 U/mg protein (on Sugar Beet Arabinan) at pH 4.0 and 40°C;

0.21 U/mg protein (on Arabinoxylan) at pH 4.0 and 40°C;

One Unit of pNPαAraf activity is defined as the amount of enzyme required to release one μmole of galactose per minute from pNPαAraf (5 mM)in Na-Acetate buffer (20 mM) pH 4.0.

3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figure 2. Release of reducing sugar from arabinan substrates by PoAra54A.

Figure 3. HPAEC-PAD analysis of hydrolysis products of arabino-oligosaccharides (AOS: AA, AAA, AAAA, AA³A, AAA³A, AA²⁺³A), arabinoxylan-oligosaccharides (AXOS: A³X, A²XX, A²⁺³XX, XA²XX, XA³XX) generated by PoAra54A.

4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 4.0

pH Stability: 2.0-5.0

Temperature Optima: 40°C

Temperature Stability: 30°C-50°C

Figure 4. Effect of pH on activity (a) and stability (b) of PoAra54A using pNPαAraf 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 PoAra54A using pNPαAraf 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 5.0. Swirl to mix the enzyme immediately prior to use.