BtMan92A

Ex-Man0082

(EC.3.2.1.113)exo-α-1,2-Mannosidase

CAZy Family: GH92

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band onSDS-gel electrophoresis (MW ~86kDa)

Figure 1. Electrophoresis analysis of BtMan92A. 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, BtMan92A purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.39 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 one nmole 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 of BtMan92A on different substrates^a.

^aReactions were performed with 5 mM substrate, pH 7.0, at 37°C for 10 min.

^bAbsorption caused by released p-nitrophenol was measured at 405 nm. The relative activity on pNPαMan was taken as 100%.

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

Figure.2 GH92α-mannosidases at high concentrations (50 mM)were incubated with yeast mannan for 5 h in the absence (labelled GH92)or in the presence (GH92/GH76) of the endo-α-1,6-mannanase BT3782.The GH92α-mannosidases in this example were BtMan92A (1), BT2130(2) and BT3773 (3). The GH76 endo-α-1,6-mannanase only releases mannooligosaccharides in the presence of BtMan92A.

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[J]. Nature, 2015, 517(7533): 165-169.