Improving the Secretion Yield of the β-Galactosidase Bgal1-3 in Pichia pastoris for Use as a Potential Catalyst in the Production of Prebiotic-Enriched Milk

Abstract

In this study, three kinds of milk were treated with the β-galactosidase Bgal1-3 (4 U/mL), resulting in 7.2–9.5 g/L galactooligosaccharides (GOS) at a lactose conversion of 90–95%. Then, Bgal1-3 was secreted from Pichia pastoris X33 under the direction of an α-factor signal peptide. After cultivation for 144 h in a flask culture with shaking, the extracellular activity of Bgal1-3 was 4.4 U/mL. Five more signal peptides (HFBI, apre, INU1A, MF4I, and W1) were employed to direct the secretion, giving rise to a more efficient signal peptide, W1 (11.2 U/mL). To further improve the secretion yield, recombinant strains harboring two copies of the bgal1-3 gene were constructed, improving the extracellular activity to 22.6 U/mL (about 440 mg/L). This study successfully constructed an engineered strain for the production of the β-galactosidase Bgal1-3, which is a promising catalyst in the preparation of prebiotic-enriched milk.

Improving Galactooligosaccharide Synthesis Efficiency of β-Galactosidase Bgal1-3 by Reshaping the Active Site with an Intelligent Hydrophobic Amino Acid Scanning

“…The enzyme activity was determined according to the method described in previous works. , In brief, 50 μL of diluted enzyme solution was added into 450 μL of lactose solution (5%, w/v) to initiate the reaction. After being incubated for 10 min at 40 °C in 50 mM potassium phosphate buffer (pH 7.0), the mixtures were boiled for 10 min to stop the reactions.…”

Cloning and Characterization of a New β-Galactosidase from Alteromonas sp. QD01 and Its Potential in Synthesis of Galacto-Oligosaccharides

“…Usually, the β-galactosidases from eukaryotes mainly belong to GH35 [ 24 , 25 ], while bacteria-derived β-galactosidases are mainly in GH families 42 and 2 [ 25 , 26 , 27 , 28 ]. The microorganisms used for the commercial production of β-galactosidase mainly include Bifidobacterium longum , Bacillus subtilis , Lactobacillus , Aspergillus niger , and Kluyveromyces lactis [ 29 , 30 , 31 ]. The properties and structures of β-galactosidase from different sources are quite different, so the scope of application also varies.…”

Whey valorization using transgalactosylation activity of immobilized β‐galactosidase

“…Until recently, the majority of the GOS synthesis related research was focused on using pure lactose solutions as a transgalactosylation substrate. However, with arousing awareness on lactose abundance in dairy effluents, cheaper and more versatile substrates, such as whey (Fischer & Kleinschmidt, 2015;Geiger et al, 2016;Sen et al, 2016) and whey permeate (Eskandarloo & Abbaspourrad, 2018;Mano et al, 2018), or even milk (Cao et al, 2017;Zhu et al, 2018), have been taken into account. Also, despite the fact that the various methods for the immobilization of b-galactosidases were proposed so far (Giacomini et al, 1998;Albayrak & Yang, 2002;Torres et al, 2003;Gaur et al, 2006;Grosov a et al, 2008;Neri et al, 2009;Guidini et al, 2010;Klein et al, 2012;Urrutia et al, 2013;Carevi c et al, 2016b;de Albuquerque et al, 2018;Levin et al, 2018;Guerrero et al, 2019;Kessi & Arias, 2019), the examples of immobilized enzyme preparations utilized in these reactions are rather scarce (Jovanovic-Malinovska et al, 2012;de Albuquerque et al, 2018;Eskandarloo & Abbaspourrad, 2018).…”