BtMan38A

Ex-Man0080

(EC.3.2.1.113)exo-α-1,2-Mannosidase

CAZy Family: GH38

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~134kDa)

Figure 1. Electrophoresis analysis of BtMan38A. 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, BtMan38A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

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

One Unit of pNP-α-Man activity is defined as the amount of enzyme required to release 1 nmol of p-nitrophenyl per minute from  pNP-α-Man (5 mM) in PBS buffer (20 mM) pH 7.0.

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Table 1. Relative activity ofBtMan38A on different substrates^a.

Reactions were performed with 1 mM (p-nitrophenyl glycosides and disaccharides) or 5 mg/ml (polysaccharides) substrate, pH 7.0, at 30°C for 10 min.

^bNot detected.

Figure 2.Phosphorylated high mannose N-glycan MNN4 and RNAase B incubated with BT2629 (GH92α-mannosidase), BT3783 (phosphatase) or BtMan38A (GH38α-mannosidase) and the products analysed by capillary electrophoresis. Man5 to Man9 (subscript numbers refer to the number of Man units in the N-glycans) lack phosphate and mannose-1-phosphate groups. P* indicates the N-glycan contains a single phosphate, which is in the following structure: Man-α-1-phosphate-α-6-Man-α-1,2-Man.

4.PHYSICOCHEMICAL PROPERTIES

pH Optima: 7.0

Temperature Optima: 37°C

5.STORAGE CONDITIONS

The enzyme should be stored at -20°C. For assay, this enzyme should be diluted in phosphate buffer (20 mM) pH7.0. Swirl to mix the enzyme immediately prior to use.

6. REFERENCES

[1]Cuskin F, Lowe E C, Temple M J, et al. Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism. Nature, 2015, 517(7533): 165-169.