Characterization of two novel alpha-glucosidases from Bifidobacterium breve UCC2003

Abstract

Two alpha-glucosidase-encoding genes (agl1 and agl2) from Bifidobacterium breve UCC2003 were identified and characterized. Based on their similarity to characterized carbohydrate hydrolases, the Agl1 and Agl2 enzymes are both assigned to a subgroup of the glycosyl hydrolase family 13, the alpha-1,6-glucosidases (EC 3.2.1.10). Recombinant Agl1 and Agl2 into which a His(12) sequence was incorporated (Agl1(His) and Agl2(His), respectively) exhibited hydrolytic activity towards panose, isomaltose, isomaltotriose, and four sucrose isomers--palatinose, trehalulose, turanose, and maltulose--while also degrading trehalose and, to a lesser extent, nigerose. The preferred substrates for both enzymes were panose, isomaltose, and trehalulose. Furthermore, the pH and temperature optima for both enzymes were determined, showing that Agl1(His) exhibits higher thermo and pH optima than Agl2(His). The two purified alpha-1,6-glucosidases were also shown to have transglycosylation activity, synthesizing oligosaccharides from palatinose, trehalulose, trehalose, panose, and isomaltotriose.

FIG. 1.

Effects of temperature (A) and pH (B) on the activity of Agl1_His (open squares) and Agl2_His (open triangles). The optimum pH and temperature conditions for catalytic activity, using palatinose as a substrate, were determined to be pH 5.5 and 30°C and 37°C for Agl1_His and Agl2_His, respectively.

FIG. 2.

Substrate specificities of Agl1_His and Agl2_His as determined by HPTLC. Reaction conditions were as described in Materials and Methods. Lane 1, carbohydrate standards listed on the left of panels A and C. Lanes 2 to 10 contained hydrolysis products obtained from (24 h [A]; 5 min [B]) the incubation of purified proteins (Agl1_His [A] or Agl2_His [B]) with palatinose (lane 2), sucrose (lane 3), trehalose (lane 4), turanose (lane 5), maltulose (lane 6), melezitose (lane 7), stachyose (lane 8), glycogen (lane 9), and maltodextrins (lane 10). (C) Lanes 2 to 9 contained hydrolysis products obtained from the incubation of Agl1_His (lanes 2 to 5) and Agl2_His (lanes 6 to 9) with trehalulose (lanes 2 and 6), isomaltose (lanes 3 and 7), panose (lanes 4 and 8), and isomaltotriose (lanes 5 and 9).

FIG. 3.

Growth profile analysis of B. breve UCC2003 in modified Rogosa broth supplemented with different carbohydrates.

FIG. 4.

HPTLC identification of the transferase activities of Agl1_His (A) and Agl2_His (B). Lane 1, carbohydrate standards listed on the left of both images. Lanes 2 to 7 contained hydrolysis and transglycosylation products obtained from the incubation of purified proteins (Agl1_His [A] or Agl2_His [B]) with the following 150 mM substrates: palatinose (lane 2), trehalulose (lane 3), isomaltose (lane 4), trehalose (lane 5), panose (lane 6), and isomaltotriose (lane 7). (C) HPAEC analysis of reaction products formed after the transglycosylation of panose by Agl1_His and Agl2_His. Open diamond, glucose; open circle, maltose; filled circle, panose (as identified by standards St2 and St1, respectively). Black arrows point to the newly synthesized oligosaccharides.