SpMan125A

Ex-man0096

(EC.3.2.1.163) exo-α-1,6-Mannosidase

CAZy Family: GH125

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~49 kDa)

Figure 1. Electrophoresis analysis of SpMan125A. 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, SpMan125A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.08 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 pNP 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 SpMan125A on different substrates^a.

^areactions were performed with 2 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.CE traces of Man9 treated with SpMan125A(i), CpGH125 (ii), Man5 treated with SpMan125A(iii), CpGH125 (iv), and Man3a treated with SpMan125A(v), CpGH125 (vi),α(1,2)(1,3)-mannosidase (vii),SpMan125A+α(1,2)(1,3)-mannosidase (viii), and CpGH125 +α(1,2)(1,3)-mannosidase (ix). The structures of the N-glycans are shown above the traces. The Man2a product indicated with an asterisk is inferred. The identities of the peaks were determined from the mobilities of standards [1].

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] Gregg K J, Zandberg W F, Hehemann J H, et al. Analysis of a New Family of Widely Distributed Metal-independent α-Mannosidases Provides Unique Insight into the Processing of N-Linked Glycans[J]. Journal of Biological Chemistry, 2011, 286(17): 15586-15596