Fig 1.
Domain arrangements and structures of representative GH3 enzymes.
(A) Comparison of domain arrangements in HvExoI [25], TnBgl3B [28], GlyA1 [11], and BrGH3A. (B) The structural model of BrGH3A. The (β/α)8 barrel domains (magenta), the (α/β)6 sandwich domains (cyan), the FnIII domains (green), and the linker regions (brown) are indicated. The additional C-terminal domain in GlyA1 is shown in orange. (C) Superimposition of the structural model of BrGH3A (cyan) and the structures of TnBgl3B (PDB code 2X41, magenta) and GlyA1 (PDB code 5K6L, green).
Fig 2.
Comparison of the active site architectures of BrGH3A (cyan) and GlyA1 (PDB code 5K6L, green).
Fig 3.
10% SDS‒PAGE analysis (left) and Western blot analysis (right) of the purified BrGH3A. Lane M is the protein size marker.
Fig 4.
The effects of (A) temperature and (B) pH on the activity of BrGH3A. The optimal conditions are shown as filled symbols and solid lines, and the stability conditions are shown as open symbols and dotted lines. The effects of pH were tested in 0.1 M sodium citrate, pH 2.0–6.5 (circles), and 0.1 M McIlvaine buffer, pH 3.0–9.0 (triangles).
Table 1.
Relative hydrolytic activities of BrGH3A toward glucooligosaccharides.
Fig 5.
Time-course reactions of BrGH3A with laminaritetraose (A) and pNP-Glc (B). (A) The reaction consisted of 2 μg of BrGH3A and 10 mM laminaritetraose in 20 mM sodium citrate, pH 5.0, at 40°C for 1 h. The STD contained 20 nmol of each glucose (G), laminaribiose (L2), laminaritriose (L3), and laminaritetraose (L4). The incubation times are indicated below the lanes. (B) The reactions contained 20 μg of BrGH3A and 5–40 mM pNP-Glc in 20 mM sodium citrate, pH 5.0, at 40°C for 6 h. Lane A contains 60 nmol of pNP, 40 nmol of pNP-Glc, and 20 nmol of pNP-Cel. Lane B contains 40 nmol of each glucose (G) and cellobiose (C2). The incubation times and the concentrations of pNP-Glc are indicated below the lanes. The unknown product is labeled pX.
Table 2.
Kinetic analysis of BrGH3A toward various pNP-glycosides and oligosaccharides.