MeAra2A

Ex-Ara0143

(EC.3.2.1)exo-α-Arabinofuranosidase

CAZy Family: GH2

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~87 kDa)

Figure 1. Electrophoresis analysis of MeAra2A.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, MeAra2A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

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

One Unit of pNP-α-Arafactivity is defined as the amount of enzyme required to release 1 μmole of p-nitrophenyl per minute frompNP-α-Araf (5 mM) in phosphate buffer (20 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of MeAra2A on 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(1.61μmol/min/mg) was taken as 100%.

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

Figure 2. HPLC analysis of the time course of the transformation of ginsenoside Rb2 byMeAra2A. S ginsenoside standards, Rd (1) metabolite 1, 20(S)-Rg3 (2) metabolite 2.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.0

Temperature Optima: 40°C

Figure2.Effect of pH and temperature on the stability and activity of recombinant MeAra2A, which was determined using pNP-a-L-arabinopyranoside as a substrate. 

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in sodiumphosphate (20 mM) pH 6.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1]Quan L H, Wang C, Jin Y, et al. Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20 (S)-Rg3[J]. Antonie Van Leeuwenhoek, 2013, 104(1): 129-137.