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.


Supporting information
The activity was tested using the standard activity assay at 25 ºC. Activity expressed relative to the activity at t=0. Error bars represent standard deviations (n = 3).    detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. Figure S11. UV spectra of genistin (top) and malonyl-daidzin (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. The peaks for genistin and malonyl-daidzin are not well separated in the soy-flour extract. Figure S12. UV spectra of malonyl-genistin (top) and daidzein (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. Figure S13. UV spectra of glycitein (top) and genistein (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. The peaks for daidzin and glycitein are not well separated in the soy-flour extract.
Supporting tables: Table S0: Table for size exclusion chromatography in the conditions referred. Ve makes reference to the elution volume expressed in minutes. MW is the molecular weight of each standard in kDa. Ve/Vo is the elution volume divided by the void volume of the column. Table S2: Data collection and refinement statistics. Data collection and refinement statistics for X-ray diffraction data collected on a single crystal of AheGH1. Values in parenthesis correspond to the high-resolution shell. For cross-validation, 5% experimental reflections were randomly selected to calculate the Rfree.

Sequence alignment
Sequence alignment based on the structural alignment of AheGH1 chain B (Ahe) and BglB (PDB: 2O9P), created using pymol.  Red shading indicates sequence conservation, with the darker colouring representing the highest sequence conservation. Structure conservation is indicated by sausage thickness with the least conserved regions indicated by increased thickness. The catalytic glutamate residues are indicated, as are the conserved residues shared in the loops of BglA and BglB that form the entrance to the active site tunnel. The gap in the AheGH1, due to lack of electron density is indicated. Figure S4: Structural superposition of the active sites of AheGH1 (ice blue) and the homologous proteins BglA (yellow, pdb code 1E4I) and BglB (pink, pdb code 2O9P). The residues delineating the pocket are shown as sticks, labeled and colored accordingly. The catalytic glutamate residues are depicted with thicker sticks. The AheGH1-E318 side-chain is missing. This figure was generated using CCP4mg. Figure S5: Effect of the incubation in a different range of pH on AheGH1 stability. The activity was tested using the standard activity assay at 25 ºC. Activity expressed relative to the activity at t=0. Error bars represent standard deviations (n = 3). Figure S6: Effect of the incubation in a different range of temperatures on AacGH1 stability. The activity was tested using the standard activity assay at 25 ºC. Activity expressed relative to the activity at t=0. Error bars represent standard deviations (n = 3). Figure S7: Thermofluorometric melting profile of AheGH1, over a temperature gradient of 15-99 °C, increasing at a rate of 2 °C/min.  Figure S10: UV spectra of daidzin (top) and glycitin (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. Figure S11. UV spectra of genistin (top) and malonyl-daidzin (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. The peaks for genistin and malonyl-daidzin are not well separated in the soy-flour extract. Figure S12. UV spectra of malonyl-genistin (top) and daidzein (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. Figure S13. UV spectra of glycitein (top) and genistein (bottom), obtained with the diode array detector. Left: authentic standard; right: corresponding peak in soy-flour-extract chromatogram. Black: spectrum at peak, green: spectrum at 50% peak height leading the peak, pink: spectrum at 50% peak height tailing the peak. The peaks for daidzin and glycitein are not well separated in the soy-flour extract.
Supporting Tables   Table S1: Table for size exclusion chromatography in the conditions referred. Ve makes reference to the elution volume expressed in minutes. MW is the molecular weight of each standard in kDa. Ve/Vo is the elution volume divided by the void volume of the column.  Table S2: Data collection and refinement statistics. Data collection and refinement statistics for X-ray diffraction data collected on a single crystal of AheGH1. Values in parenthesis correspond to the high-resolution shell. For cross-validation, 5% experimental reflections were randomly selected to calculate the Rfree. † Redundancy-independent merging R factor Rmeas estimated by multiplying the conventional Rmerge value by the factor [N/(N − 1)] 1/2 , where N is the data multiplicity. + CC1/2 is the correlation coefficient of the mean intensities between two random half-sets of data.

Crystal
Space group