Effective Enzymatic Synthesis of Lactosucrose and Its Analogues by β-d-Galactosidase from Bacillus circulans

Abstract

In the present study, beta-d-galactosidase from Bacillus circulans was proved to be a suitable biocatalyst for the production of lactosucrose (beta-d-Galp-(1-->4)-alpha-d-Glcp-(1-->2)-beta-d-Fruf, I) and its analogues from lactose and sucrose. During the hydrolysis of lactose, the formation of four transfer products was followed by high performance liquid chromatography with refraction index detector. In addition, the transfer products were isolated from the reaction mixture and identified to be I, beta-d-Galp-(1-->3)-alpha-d-Glcp-(1-->2)-beta-d-Fruf (II), beta-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-alpha,beta-d-Glcp (III), and beta-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-alpha-d-Glcp-(1-->2)-beta-d-Fruf (IV) by mass spectrometry with an electrospray ionization source and nuclear magnetic resonance spectroscopy. The order for the production of the transfer products was III > I > IV > II in the initial stage of the reaction, and the same relationship was also observed for the hydrolytic rates of transfer products. Furthermore, the effects of synthetic conditions including reaction temperature, reaction time, concentration of substrate, molar ratio of donor/acceptor, and enzyme concentration on the formation of transfer products were examined. We found that the optimal synthetic conditions were different for the production of I and II. Under the optimal conditions, the amount of total transfer products kept increasing during the early 4 h incubation, and a maximum yield of 146 g/L for total transfer products was obtained at 4 h of reaction.

Enzymatic synthesis of galacto-oligosaccharides and other lactose derivatives (hetero-oligosaccharides) from lactose

“…Lactosucrose yields obtained by transfructosylation of lactose are comparable with those observed after transgalactosylation of sucrose (Han et al, 2009;Li et al, 2009). A mixed enzyme system coupling transfructosylation of lactose to glucose removal by glucose oxidase and catalase increased the yield of lactosucrose from 29% to 43% (Han et al, 2009).…”

“…High donor (lactose) concentrations favour formation of GOS as byproducts, while a large excess of acceptor results in untransformed substrate. The yield of lactulose and lactosucrose, produced with fructose and sucrose as galactosyl acceptors, respectively, was the highest at a molar donor to acceptor ratio in the range of 1:1e1:2 (Lee, Kim, & Oh, 2004;Li et al, 2009;Mayer et al, 2004;Schuster-Wolff-Bührig et al, 2010). Likewise, transgalactosylation of fucose, N-acetylglucosamine, N-acetylgalactosamine, and mannose was achieved with high yield at a donor : acceptor ratio of 1:1 or an excess of galactosyl-acceptor (Li, Sun, Ye, & Zeng, 2010;Miyasato & Ajisaka, 2004;Schwab, Lee, Sørensen, & Gänzle, 2011).…”

Recent novel applications of levansucrases

“…The highest production of lactosucrose, of 285 g L −1 , is achieved using Pseudomonas aurantiaca levansucrase from 205 g L −1 sucrose and 410 g L −1 lactose (Han et al 2007), while the majority of lactosucrose-producing microorganisms generate 100-200 g L −1 of lactosucrose via transfructoylation from various concentrations of sucrose and lactose. In addition, lactosucrose may also be produced through the transgalactosylation activity of Bacillus circulans β-D-galactosidase, employing lactose as a donor and sucrose as an acceptor (Li et al 2009). However, transgalactosylation generates several oligosaccharide compounds and only produces 56 g L −1 of lactosucrose from 30 % (w/v) sucrose and 30 % (w/v) lactose, with a conversion ratio of less than 10 %.…”

Biosynthesis of lactosylfructoside by an intracellular levansucrase from Bacillus methylotrophicus SK 21.002

“…23,24 In addition, by transferring the galactosyl moiety from the donor lactose to the acceptor sucrose, the transgalactosylation reaction may synthesize lactosylfructoside by b-galactosidase (EC 3.2.1.23) from Bacillus circulans. 25,26 However, the capabilities of the three types of enzymes to produce lactosylfructoside are greatly different, even though the reactions are catalyzed by the same type of enzyme derived from different microorganisms.…”