Kinetics and products of Thermotoga maritima β-glucosidase with lactose and cellobiose

Abstract

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother's milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose. KEY POINTS: • β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield. • Thermotoga maritima β-glucosidase has high activity and high thermostability. • Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages.

Keywords: Galacto-oligosaccharides; Galactosidase; Gluco-oligosaccharides; Glucosidase; Thermostable.

Fig. 1

HPAEC-PAD profile of (A) Vivinal GOS and (B) GOS synthesized by rTmBglA from 57%wt lactose at 75 °C, using 3.75 U/g lactose for 24 h. Annotated peaks were identified based on references from previous work (Fig. 5) and NMR spectroscopy (Table 3; Fig. 4)

Fig. 2

Release of Glc and Gal determined by quantitative HPAEC-PAD profiling in 24-h incubations using 3.75 U/g lactose rTmBglA incubated with 57%_wt lactose at 75 °C

Fig. 3

MALDI-TOF–MS spectra of oligosaccharides synthesized by incubation of rTmBglA with (A) lactose and (B) cellobiose. Oligosaccharide peaks are labeled with mass and hexose composition

Fig. 4

One-dimensional 500 MHz.¹H NMR spectrum of GOS synthesized by rTmBglA from 57%wt lactose at 75 °C, using 3.75 U/g lactose for 24 h. Structural-reporter-group signals are annotated (Table 3; Fig. 5) and were used in identification of HPAEC-PAD peaks (Fig. 1)

Fig. 5

Graphical representation of structures identified in A GOS and B GlcOS; synthesized by rTmBglA from lactose and cellobiose, respectively. Structure numbers match with peaks in Figs. 1 and 6. The structure numbers of GOS used in this figure are the same as in previous publications (van Leeuwen et al. ; Yin et al. , ; Kittibunchakul et al. 2020)

Fig. 6

HPAEC-PAD profile of GlcOS synthesized by rTmBglA from 20%wt cellobiose (C2). Structures are identified based on reference standards, C3: cellotriose [β-(1 → 4)], C4: cellotetraose [β-(1 → 4)], G2: gentiobiose [β-(1 → 6)], L2: laminaribiose [β-(1 → 3)] and L3: laminaritriose [β-(1 → 3)], matching with identified structures shown in Fig. 5