High-level expression of a novel thermostable and mannose-tolerant β-mannosidase from Thermotoga thermarum DSM 5069 in Escherichia coli

Table 1 Purification of the recombinant Tth Man5 β-mannosidase

Purification step	Total volume (mL)	Total activity (μmol min^-1)	Total protein (mg)	Specific activity (μmol mg^-1min^-1)	Recovery (%)	Purification (fold)
Crude extract^a	10	2160	135	16	100	1
Heat treatment^b	10	1922	31	62	89.0	3.9
Ni affinity chromatography^c	1	1734	17	102	80.3	6.4

^aThe recombinant strain was grown in LB medium (200 ml) with 100 μg ampicillin/ml at 37°C to OD₆₀₀ 0.4-0.5 and was incubated further with isopropyl-β-thiogalactopyranoside (IPTG) for 5 h. The cells were harvested by centrifugation at 10,000 g for 15 min at 4°C and resuspended in 10 ml imidazole buffer (10 mL of 5 mM imidazole, 0.5 mM NaCl, and 20 mM Tris–HCl buffer, pH 7.9), followed by sonication.
^bThe cell extracts after sonication were heat treated at 70°C for 30 min, and then cooled in an ice bath, centrifuged at 15,000 g for 20 min at 4°C and the supernatant was kept.
^cThe obtained supernatants were loaded on to an immobilized metal affinity column (Novagen, USA), and eluted with 0.4 M imidazole, 0.5 M NaCl, and 20 mM Tris–HCl buffer (pH 7.9).

ISSN: 1472-6750