Preliminary characterization of a novel β-agarase from Thalassospira profundimonas

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Table 1 Purification of agarase-fst from T. profundimaris fst-13007

Purification step	Total protein (mg)	Total activity (U)	Specific activity (U mg⁻¹)	Purification fold	Yield (%)
Crude enzyme solution	55.31	4134	74.74	1	100
(NH₄)₂SO₄ precipitation	32.23	3903	121.08	1.62	94.4
DEAE FF	2.10	674	321	4.29	16.3
Sephacryl S-100	0.40	573	1418	18.97	13.87

After 20 min centrifugation at 12,000g and filtration through a 0.45 μm membrane, crude agarase-fst from the culture broth was precipitated by 80 % (w/v) ammonium sulfate saturation and left to stand overnight. After 20 min of centrifugation at 12,000 g, the precipitate was resuspended in 20 mM Tris/HCl buffer (pH 7.5, buffer B) and dialyzed against the same buffer overnight. The dialyzed section was ultrafiltrated through an ultrafilter (5000–10,000 kDa, transmembrane pressure of 0.5 bars). The active fractions were collected and applied onto a DEAE Sepharose Fast Flow (1.6 cm × 10 cm; GE Healthcare) equilibrated with buffer B. The bound proteins were eluted with gradient of 0–1 M NaCl in the same buffer. The flow rate was maintained at 1 ml min ⁻¹. Active fractions were loaded onto Sephacryl S-100 gel column (1.6 cm × 60 cm; GE Healthcare) that had previously been equilibrated with buffer B. The enzyme was eluted with same buffer, at a flow rate of 1 ml min ⁻¹. Unless stated, all purification procedures were performed at 4 °C