SpMan38A

Ex-Man0093

(EC.3.2.1.-)exo-α-1,3-Mannosidase

CAZy Family: GH38

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~103 kDa)

Figure 1. Electrophoresis analysis of SpMan38A. 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, SpMan38A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.01 U/mg protein (on pNP-α-man) at pH 7.0 and 30°C.

One Unit of pNP-α-Man activity is defined as the amount of enzyme required to release 1 μmol of mannose per minute from pNP-α-man(5 mM) in phosphate buffer (50 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity of SpMan38A on different substrates^a.

^aReactions were performed with 1 mM (p-nitrophenyl glycosides) substrate, pH 7.0, at 30°C for 30 min.

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

Figure 2. Figure 3. SpMan38A-catalysed hydrolysis of Man9(GlcNAc)2 glycans. (A) Action of SpMan38A, alone, on Man9(GlcNAc)2. The glycan remains unmodified. (B) Action of SpMan38A in combination with a specific α-1,2 mannosidase theBacteroides thetaiotaomicronBt3990. Following α-1,2 mannoside removal, SpMan38A is able to further degrade the unmasked glycans, with the action pattern most indicative of α-1,3 mannosidase activity.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.0

Temperature Optima: 35°C

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (50 mM) pH 7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1] Michael D. L. Suits, Yanping Zhu, Edward J. Taylor, et al. Structure and Kinetic Investigation ofStreptococcus pyogenesFamily GH38 a-Mannosidase Plos One, 2010, 5(2): e9006.