BsAra51B (exo-α-Arabinofuranosidase,BsAra5A)
BsAra51B (BsAra5A)
Ex-Ara0135
(EC.3.2.1.55)exo-α-arabinofuranosidase
CAZy Family: GH51
PROPERTIES
1.ELECTROPHORETIC PURITY
-Single band on SDS-gel electrophoresis (MW ~58 kDa)
Figure 1. Electrophoresis analysis ofBsAra51B. 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,BsAra51Bpurified from Ni sepharose fastflow column.
2.SPECIFIC ACTIVITY
25.1 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 phosphate buffer (20 mM) pH 7.0.
3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES
Table 1. Relative activity ofBsAra51Bon different substratesa.
Substrateb | Relative activity (%)c |
pNPβGlu | _ |
pNPβGal | _ |
pNPβMan | _ |
pNPβXyl | _ |
pNPαGlc | _ |
pNPαGal | _ |
pNPαMan | _ |
pNPαAraf | 100±0.0 |
pNPαArap | _ |
aReactions were performed with 5 mM substrate, pH 7.0, 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.
Table2.Catalytic activity of BsAra51B
Enzyme | Substrate | kcat (s−1) | K m (mM) | kcat/Km (s−1 mM−1) |
BsAra51B | pNPAf | 306±14 | 0.49±0.07 | 614 |
BsAra51B | Sugar beet arabinan (branched) | 8.2±1.0 | 4.4±0.6 | 1.8 |
BsAra51B | Linear α-1,5-L-arabinan | 12±0.4 | 0.36±0.007 | 33 |
BsAra51B | Arabinobiose | 32±1.3 | 0.78±0.11 | 41 |
BsAra51B | Arabinotriose | 88±5.7 | 1.1±0.08 | 80 |
BsAra51B | Wheat arabinoxylan | nd | nd | nd |
BsAra51B | Larch wood arabinogalactan | nd | nd | nd |
nd, Although enzyme activity was present it was not possible to measure the individual kinetic constants.
4.PHYSICOCHEMICAL PROPERTIES
pH Optima: 8.0
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] Inacio J M, Correia I L, de Sa-Nogueira I. Two distinct arabinofuranosidases contribute to arabino-oligosaccharide degradation in Bacillus subtilis. Microbiology, 2008, 154(9): 2719-2729.
