Optimisation of enzymatic reactions for galactooligosaccharides synthesis using beta-galactanase from Geobacillus mahadii Geo-05

Abstract

Galacto-oligosaccharide (GOS) is one of the prominent prebiotics used in infant formula milk. Endo-beta-1,4-galactanase hydrolyses galactan and produces GOS with different degree of polymerisation (DP). DP of the GOS could vary depending on the enzyme source. Besides, reaction parameters such as enzyme loading, substrate loading, incubation temperature, pH and reaction time also contribute to various DP of GOS synthesis. In this study, beta-galactanase from Geobacillus mahadii Geo-05 was synthesised with a codon optimised sequence (named BgcGC) and biochemically characterised for its optimum function. The BgcGC reaction was optimised to obtain the highest yield of the targeted GOS (galactotriose, G3 and galactotetraose, G4) from the potato galactan. Then, the optimised conditions were applied on pectin extracted from sweet potato peels (SPP) to produce the GOS. BgcGC could actively hydrolyse potato galactan at 60°C in McIlvaine buffer pH 6 and stable to a wide range of pH between 4 to 10. According to the screening by the factorial design, three reaction parameters significantly contributed to the highest yield of GOS. Due to curvature prediction, the central composite design was further applied to optimise BgcGC reaction to produce GOS. The optimum reaction parameters for the GOS synthesis were identified at 0.15 U/ml BgcGC loading, 1.5 h incubation time and 50°C reaction temperature yielding a 1.5-fold increment of the GOS synthesis. The reaction of BgcGC towards SPP pectin resulted in a 0.081 mg/g yield of G3. These findings showed that GOS synthesised by BgcGC could be enhanced through the manipulation of reaction parameters with a notable increment of GOS yield. Moreover, BgcGC exhibited the potential to be used as an enzyme cocktail for the depolymerisation of pectin polysaccharides.