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. 2015 Apr 30;20(5):7874-89.
doi: 10.3390/molecules20057874.

Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization

Rocio Benavente  1 , Benevides C Pessela  2 , Jose Antonio Curiel  3 , Blanca de las Rivas  4 , Rosario Muñoz  5 , Jose Manuel Guisán  6 , Jose M Mancheño  7 , Alejandra Cardelle-Cobas  8 , Ana I Ruiz-Matute  9 , Nieves Corzo  10
Affiliations

Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization

Rocio Benavente et al. Molecules. .

Abstract

A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SDS-PAGE analysis of (A) LPG purification by adsorption on agarose-IDA-Ni support. Lane 1: Low molecular weight marker, Lane 2: Crude protein extract. Lane 3: Purified LPG and (B) different immobilized LPG preparations. Lane 1: Low molecular weight marker; Lane 2: Crude protein extract; Lane 3: Crude protein extract desorbed from CNBr support; Lane 4: Proteins desorbed from glyoxyl support.
Figure 2
Figure 2
Inhibition of β-galactosidase from Lactobacillus plantarum (LPG) by different sugars. The reaction was carried out at pH 7, as described in the Experimental Section. Glucose (triangles), galactose (squares), lactose (circles).
Figure 3
Figure 3
Influence of pH (A) and temperature (B) on residual activity of different preparations of β-galactosidase from Lactobacillus plantarum (LPG); Glyoxyl-LPG (rhombus), CNBr-LPG (one-point immobilized) (circles); and thermostability (C) at pH 7.0 and 50 °C of different preparations of LPG: Glyoxyl-LPG (rhombus), CNBr-LPG (one-point immobilized) (circles), soluble enzyme (triangles).
Figure 4
Figure 4
HPAEC-PAD profile of oligosaccharides from synthesis mixtures (A) and carbohydrate yields (%) (B) obtained during lactose hydrolysis by glyoxyl-immobilized β-galactosidase from Lactobacillus plantarum (LPG) at the two pH assayed (5 and 7). Compounds: (1) galactose + glucose; (2) 6-galactobiose; (3) lactose; (4) 6'-galactosyl-lactose; (5) 3'-galactosyl-lactose; (*) other oligosaccharides (GOS). Total GOS includes 6-galactobiose, 6'-galactosyl-lactose, 3'-galactosyl-lactose and other GOS. Squares: pH 7; rhombus: pH 5.
Figure 5
Figure 5
HPAEC-PAD profile of oligosaccharides from synthesis mixtures (A) and carbohydrate yields (%) (B) Obtained during lactulose hydrolysis by glyoxyl-immobilized β-galactosidase from Lactobacillus platarum (LPG) at the pHs assayed (5 and 7). Compounds: (1) galactose; (2) fructose; (3) 6-galactobiose; (4) lactulose; (5) 6'-galactosyl-lactulose; (6) 1-galactosyl-lactulose; (7) 3'-galactosyl-lactulose; (*) other oligosaccharides derived from lactulose (OsLu). Total OsLu include 6-galactobiose, 6'-galactosyl-lactulose, 1-galactosyl-lactulose, 3'-galactosyl-lactulose and other OsLu. Squares: pH 7; rhombus: pH 5.
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