Purification and evaluation of the enzymatic properties of a novel fructosyltransferase from Aspergillus oryzae: a potential biocatalyst for the synthesis of sucrose 6-acetate

Abstract

A novel fructosyltransferase (AoFT) capable of synthesizing sucrose 6-acetate (S6A) from sucrose and glucose 6-acetate has been purified to homogeneity from Aspergillus oryzae ZZ-01. Its molecular mass was ~50 kDa by SDS-PAGE; optimal activity was at 45 °C and it was stable from pH 4.5 to 7.5 with an optimum pH of 6. Mg(2+), K(+) (5 mM), propanol, toluene (50%, v/v), Tween 20 or Triton X-100 (1%, w/v) increased the transfructosylation activity by 20, 17, 17, 10, 25 and 20%, respectively. An overall conversion of 32% was achieved under optimal conditions over 24 h. This is the first report that the purified and characterized the fructosyltransferase from Aspergillus capable of synthesis of S6A from sucrose and glucose 6-acetate.

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 β‐fructofuranosidase with transfructosylating activity

“…Guo et al reported the same optimum temperature for transfructosylating activity of recombinant FTase from Aspergillus oryzae overexpressed in Pichia pastoris . The optimum pH was 6.0 for both activities (Figure b), a value coincident with that reported for an A. oryzae FTase . However, the hydrolytic activity was slightly more active under acidic conditions (4.0 ≤ pH ≤ 5.0), whereas the transfructosylating one under neutral to alkaline conditions (7.0 ≤ pH ≤ 9.0).…”

“…The optimum pH was 6.0 for both activities (Figure 1b), a value coincident with that reported for an A. oryzae FTase. 31 such as proteases, 32 cellulases, 33 pectin lyases, 34 and FFase. 35 From the matrix of independent variables and responses used in CCRD (Table 1), one can see that the highest hydrolytic (159.81-160.63 U mL −1 ) and transfructosylating (63.16 U mL −1 ) activities were obtained in the central points (runs 9 and 10 carried out at 50 C and pH 6.00) and in run 6 (50 C and pH 7.41), respectively, conditions under which the response surfaces showed optimum regions for both ( Figure 2).…”

Cloning, Expression and Characterization of a Novel Fructosyltransferase from Aspergillus oryzae ZZ-01 for the Synthesis of Sucrose 6-Acetate

“…The resulting mixtures were then incubated at 55˝C for 24 h with constant shaking at 200 rpm. The resulting solutions were analyzed by high-performance liquid chromatography using an Xbridge Amide column (4.6ˆ250 mm, 3.5 µm, Waters, USA) , as described previously [9,10]. Samples were eluted isocratically with a 3:1 (v/v) mixture of acetonitrile and water at a flow rate of 1 mL/min.…”

“…2.4.1.9) can be used to catalyze the cleavage of the β-(1,2)-glycosidic linkages between glucose and fructose units, as well as the transfer of the resulting fructosyl group from one sucrose unit to another [8][9][10]. FTase coding genes have been isolated from several species, including plants [11][12][13], fungi [1,8,14,15] and bacteria [16][17][18], where they encode for extra-or intracellular FTases representing the eight highly conserved motifs in the glycoside hydrolases belonging to glycoside hydrolase families 32 (GH32) and 68 (GH68) [1,19].…”

“…Only one fructosyltransferase from Aureobasidium pullulans has been reported to date to catalyze the synthesis of Suc6A from glucose 6-acetate and sucrose [30]. We previously reported the use of Aspergillus oryzae ZZ-01 fructosyltransferase (AoFT) to synthesize Suc6A using glucose 6-acetate (Glu6A) and sucrose as substrates [9,10]. However, the industrial application of AoFT from wild-type A. oryzae ZZ-01 has been limited by low yields, and it is therefore necessary to isolate and express the AoFT gene in a bacterial or fungal expression system to improve its productivity.…”