BtMan92E

Ex-Man0091

(EC.3.2.1.113)exo-α-1,2-Mannosidase

CAZy Family: GH92

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~87 kDa)

Figure 1. Electrophoresis analysis of BtMan92E. 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, BtMan92D purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

0.87 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 of BtMan92E on different substrates^a.

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

^bAbsorption caused by releasedp-nitrophenol was measured at 405 nm. The relative activity onpNPαMan (0.87μmol/min/mg) was taken as 100%.

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

Table 2.Catalytic activity ofBtMan92E

^aMannose release indicated the enzyme was active against the substrate (+), while no release of the sugar is indicative of no activity (–). The score for each protein is based on the HPLC signal for mannose after 16 h incubations with 1 µM of enzyme: HPLC signals > 500 nanocoulomb (nc), +++; 200–500 nc, ++; <200 nc, +;cNA: no activity was detected.

^bSubstrate: the substrate used was the most active for the enzyme determined from the screen.

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]Zhu Y, Suits M D L, Thompson A J, et al. Mechanistic insights into a Ca2+-dependent family of α-mannosidases in a human gut symbiont[J]. Nature chemical biology, 2010, 6(2): 125-132.