PcAra51A

Ex-Ara00149

(EC.3.2.1.55 ) exo-α-Arabinofuranosidase

CAZy Family: GH51

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~68 kDa)

Figure 1. Electrophoresis analysis of PcAra51A. 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, PcAra51A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

2.79 U/mg protein (on pNP-α-araf) at pH 6.0 and 37°C.

One Unit of pNP-α-araf activity is defined as the amount of enzyme required to release 1 μmol of pNP per minute from pNP-α-araf(5 mM) in Sodium acetate buffer (20 mM) pH 6.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of PcAra51A on different substrates^a.

^aReactions were performed with 5 mM substrate, pH 6.0, at 37°C for 5 min.

^bAbsorption caused by released p-nitrophenol was measured at 405 nm. The relative activity on pNPαAraf was taken as 100%,.

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

Figure 2. Hydrolysis of PcAra51A on substratesdetected by HPAEC.Reactions were performed with 10 μg substrate, pH 6.0, at 37◦C for 24 h.The green five pointed star represents arabinose and the orange five pointed star represents xylose.The results show that PcAra51A can hydrolyze any kind of bond linked α-ara.

Table 2. Enzyme activity of PcAra51A towards various substrates

Enzyme activity was tested by incubating 18 mUpNP-α-arafof the enzyme with 500 μL of substrate (0.02% forpNP α-L-arabinofuranoside and 0.1% for polysaccharides) in 20 mM Na–acetate buffer (pH 5.0) at 37 °C for indicated times. The experiments were performed in duplicate. ND, not determined

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 5.0

Temperature Optima: 50°C

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Sakamoto T , Inui M , Kana Yasui, et al.Substrate specificity and gene expression of twoPenicillium chrysogenumα-l-arabinofuranosidases belonging to glycoside hydrolase families 51 and 54[J]. Applied Microbiology & Biotechnology, 2013, 97(3):1121-1130.