BsAra51A

Ex-Ara0133

(EC.3.2.1.55) exo-α-arabinofuranosidase

CAZy Family: GH51

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~56 kDa)

Figure 1. Electrophoresis analysis ofBsAra51A. 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,BsAra51Apurified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

2.28 U/mg protein (on pNP-α-Araf) at pH 7.0 and 37°C.

One Unit of pNP-α-Araf activity is defined as the amount of enzyme required to release 1 μmol of p-nitrophenyl per minute from pNP-α-Araf (5 mM) in NaH₂PO₄-Na₂HPO₄ buffer (20 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity ofBsAra51Aon different substrates^a.

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

^bAbsorption caused by releasedp-nitrophenol was measured at 405nm. The relative activity onpNPαAraf(2.28μmol/min/mg) was taken as 100%.

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

Figure 3. The non-redundant roles of AbfA and BsAra51Ain arabino-oligosaccharide degradation in B. subtilis. Arabinooligosaccharides resulting from the action of extracellular enzymes in different hemicellulosic homopolysaccharides (branched and debranched arabinans) and heteropolysaccharides (arabinoxylans, arabinogalactans) are transported into the cell by specific transport systems. Arabinose oligomers enter the cell most probably via AraNPQ, an ABC-type transporter. Mixed oligomers, arabinoxylo-oligosaccharides and arabinogalacto-oligosaccharides may be transported into the cell also via AraNPQ or by other unidentified transport systems (?). The transport of the monosaccharides, arabinose, xylose and galactose, is accomplished by the same transport system, the AraE permease. Once inside the cell, arabino-oligosaccharides with a-1,5, a-1,2, and a-1,3 linkages are degraded by the concerted action of the two GH51, AbfA and BsAra51. L-Arabinose is then converted, by the action of the products of the araA, araB and araD genes, to D-xylulose 5-phosphate, which is further catabolized through the pentose phosphate pathway (PPP).

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 8.0

Temperature Optima: 60°C

5.STORAGE CONDITIONS

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

6. REFERENCES

[1] Inacio J M, Correia I L, de Sa-Nogueira I. Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis[J]. Microbiology, 2008, 154(9): 2719-2729.