PcAra93A

Ex-Ara0144

(EC.3.2.1)endo-α-1,5-Arabinanase

CAZy Family: GH93

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~42kDa)

Figure 1. Electrophoresis analysis of PcAra93A.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, PcAra93A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

65 U/mg protein (on debranched arabinan) at pH 5.0 and 37^oC.

One Unit of activity is defined as the amount of enzyme required to release 1 mmol of arabinobiose per minute from debranched arabinan (5 mM) in Na-acetate buffer (20 mM) pH5.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Kinetic parameters of the recombinantPcAra93Atowards arabino-oligosaccharides with different DPs.

In a typical assay, 0.168 μg of the enzyme was incubated with various concentrations of arabino-oligosaccharide as the substrate. Content of arabino-oligosaccharides used for the substrates was determined by the phenol/sulfuric acid method. The data were analyzed by Lineweaver^Burk plots using a leastsquares linear regression.

Figure 2. Analysis of the enzymatic products ofα-1,5-L-arabinoheptaose with the recombinantPcAra93A. A reaction mixture containing 7μg of the enzyme and 100μl of 5 mM arabinoheptaose in 20 mM Na-acetate buffer, pH5.0, was incubated at 37^oC for the times shown. Aliquots were taken at intervals and inactivated by boiling for 5 min, followed by analysis of the reaction products with HPAEC. Ara1 to Ara8 represent arabinose to arabinooctaose, respectively.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 4.0

pH Stability:4.0-8.0

Temperature Optima: 40°C

Temperature Stability:<50°C

Figure3.Effects of temperature (a) and pH(b) on activity (closedcircles) and stability (open circles) of the recombinant MBP fusionPcAra93A.

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, Ihara H, Shibano A, et al. Molecular characterization of a Penicillium chrysogenum exo-1, 5-α-L-arabinanase that is structurally distinct from other arabinan-degrading enzymes[J]. FEBS letters, 2004, 560(1-3): 199-204