Release of Soybean Isoflavones by Using a β-Glucosidase from Alicyclobacillus herbarius

Abstract

β-Glucosidases are used in the food industry to hydrolyse glycosidic bonds in complex sugars, with enzymes sourced from extremophiles better able to tolerate the process conditions. In this work, a novel β-glycosidase from the acidophilic organism Alicyclobacillus herbarius was cloned and heterologously expressed in Escherichia coli BL21(DE3). AheGH1 was stable over a broad range of pH values (5-11) and temperatures (4-55 °C). The enzyme exhibited excellent tolerance to fructose and good tolerance to glucose, retaining 65 % activity in the presence of 10 % (w/v) glucose. It also tolerated organic solvents, some of which appeared to have a stimulating effect, in particular ethanol with a 1.7-fold increase in activity at 10 % (v/v). The enzyme was then applied for the cleavage of isoflavone from isoflavone glucosides in an ethanolic extract of soy flour, to produce soy isoflavones, which constitute a valuable food supplement, full conversion was achieved within 15 min at 30 °C.

Keywords: biocatalysis; extremophiles; hydrolases; isoflavones; soy.

Figure 1

Structure of the main isoflavone glucosides (glycitin, genistin, and daidzin) found in soybean. The isoflavone moiety (glycitein, genistein, and daidzein) is highlighted in red. The sugar may be further acetylated or malonylated.

Figure 2

3D structure of AheGH1. A) The AheGH1 tetramer present in the asymmetric unit. B) Top and side views of the AheGH1 monomer, with ribbons coloured according to secondary structure (β‐strands in orange, α‐helices in green, 3‐, 4‐ and 5‐turns in yellow, unstructured regions in grey). N‐ and C‐terminal regions are labelled.

Figure 3

The AheGH1 active site. A) The AheGH1 putative active site showing active‐site residues delineating the pocket (sticks). Proposed catalytic resides E356 and E167 are indicated. B) Structural superposition of the active sites of AheGH1 (blue) and the homologous protein BglA (gold; PDB ID: 1E4I) complexed with 2‐deoxy‐2‐fluoro‐α‐d‐glucopyranose catalytic intermediate (yellow sticks) and C) superposition of AheGH1 (ice blue) with BglA (yellow) and BglB (pink) in complex with glucose (PDB ID: 2O9T) and a detailed view of the glucose binding site. Glucose is shown as black sticks. BglB residues interacting with the glucose molecule, and the corresponding AheGH1 residues, are depicted as sticks and coloured accordingly. AheGH1 residues conserved in BglA are coloured in purple. AheGH1 W329, which is conserved in all the three homologues is coloured in orange. Residue numbering in all panels is for AheGH1. This figure was made with CCP4mg. ^[30]

Figure 4

A) Specific activity with various pNP‐glucosides. B) Substrate inhibition kinetics with pNPG. Effects of C) glucose and fructose and D) co‐solvents on activity. Represented in the figures are the mean and the standard deviation. The activity was tested in a standard activity assay at 25 °C. Error bars represent standard deviations (n=3).

Figure 5

Effect of incubation in different co‐solvents on AheGH1 stability. The activity was tested in a standard activity assay at 25 °C. Activity expressed relative to the activity at t=0. Error bars represent standard deviations (n=3).

Figure 6

Enzymatic hydrolysis of 3 glucosides: daidzin, glycitin and genistin at t=0 and after 15 min of reaction at 30 °C. Error bars represent standard deviations (n=3).

Figure 7

Hydrolysis reaction over soybean isoflavones glucosides at t=0 and after 15 min, 30 min, 1 h, 3 h, 24 h and 48 h at 30 °C when AheGH1 is present in the reaction. Error bars represent standard deviations (n=3).