BtMan76D

Endo-Man0102

(EC.3.2.1.101)endo-α-1,6-Mannanase

CAZy Family: GH76

PROPERTIES

1.ELECTROPHORETIC PURITY

-Single band on SDS-gel electrophoresis (MW ~60 kDa)

Figure 1. Electrophoresis analysis ofBtMan76D. 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, BtMan76D purified from Ni sepharose fastflow column.

2.SPECIFIC ACTIVITY

3.RELATIVE RATES OF HYDROLYSIS OF SUBSTRATES

Figure2.BtMan76D is a retaining glycoside hydrolase. Enzymatic hydrolysis of 4-nitrophenyl α-D-mannopyranosyl-1,6-α-D-mannopyranoside (S) was monitored by 1H-NMR spectroscopy (500 MHz).The stacked spectra show the reaction progress over time. SH1α is the anomeric proton of the reducing end mannopyranoside of the substrate, and SH19a is the anomeric proton of the non-reducing end mannopyranoside. The reaction proceeds with the initial formation of the product, the a-anomer of α-1,6-mannobiose (P-α, peaks PH_1αand P_H1'α), which slowly mutarotates to theβ-anomer (P-β, peaks PH_1βand PH_1'β)

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.