PoAra62A

Ex-Ara0130

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

CAZy Family: GH62

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~32 kDa)

Figure 1. Electrophoresis analysis of PoAra62A. 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, PoAra62A purified from Ni sepharose fastflow column.

2. SPECIFIC ACTIVITY

0.45 U/mg protein (on pNP-α-Araf) at pH 5.0 and 37 °C.

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

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

One Unit of pNP-α-Araf activity is defined as the amount of enzyme required to release one μmole of p-nitrophenyl per minute from pNP-α-Araf (5 mM) in sodium acetate buffer (20 mM) pH 5.0.

3. RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of PoAra62A on different substrates ^a.

^a Reactions were performed with 5 mM substrate, pH 7.0, at 37°C for 5 min.

^b Absorption caused by released p-nitrophenol was measured at 405 nm. The relative activity on pNPαAraf (4.31 μmol/min/mg) was taken as 100%.

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

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 PoAra62A.

4. PHYSICOCHEMICAL PROPERTIES

pH Optima: 4.5

pH Stability: 4.0-5.0

Temperature Optima: 35°C

Temperature Stability: <35°C

Figure 4. pH and temperature profiles of the purified recombinant enzymes (PoAra62A) The effect of pH on activity (a) and stability (b) of PoAra62A used pNPαAraf as substrate; and the effect of temperature on activity (c) and stability (d) of PoAra62A also used pNPαAraf as substrate. 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 sodium acetate (20 mM) pH 5.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Hu Y, Yan X, Zhang H, et al. Cloning and expression of a novel α-1, 3-arabinofuranosidase from Penicillium oxalicum sp. 68. AMB Express, 2018, 8(1): 1-10.