Characterization of a novel cold-adapted GH1 β-glucosidase from Psychrobacillus glaciei and its application in the hydrolysis of soybean isoflavone glycosides

Abstract

The novel β-glucosidase gene (pgbgl1) of glycoside hydrolase (GH) family 1 from the psychrotrophic bacterium Psychrobacillus glaciei sp. PB01 was successfully expressed in Escherichia coli BL21 (DE3). The deduced PgBgl1 contained 447 amino acid residues with a calculated molecular mass of 51.4 kDa. PgBgl1 showed its maximum activity at pH 7.0 and 40 °C, and still retained over 10% activity at 0 °C, suggesting that the recombinant PgBgl1 is a cold-adapted enzyme. The substrate specificity, K_m, V_max, and K_cat/K_m for the p-Nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate were 1063.89 U/mg, 0.36 mM, 1208.31 U/mg and 3871.92/s, respectively. Furthermore, PgBgl1 demonstrated remarkable stimulation of monosaccharides such as glucose, xylose, and galactose, as well as NaCl. PgBgl1 also demonstrated a high capacity to convert the primary soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their respective aglycones. Overall, PgBgl1 exhibited high catalytic activity towards aryl glycosides, suggesting promising application prospects in the food, animal feed, and pharmaceutical industries.

Keywords: Catalytic efficiency; Cold adaptation; Isoflavone glycosides; Psychrobacillus glaciei; Sugar tolerance; β-Glucosidase.

Graphical abstract

Fig. 1

Multiple sequence alignment of PgBgl1 with available protein sequences from GenBank. The aligned sequences originated from the following organisms: WP_211895071.1, Psychrobacillus sp. INOP01; BcBgl1A, Evansella cellulosilytica DSM 2522; 1QOX, Niallia circulans subsp. alkalophilus; 6QWI, Paenibacillus polymyxa; EaBgl1A, Exiguobacterium antarcticum; The alignment was performed by the Clustal W method and annotated by GeneDoc. *: The catalytic site of the enzyme. Identical amino acids are shown on a black background, similar amino acids are shown on a gray background, and different amino acids are shown on a white background.

Fig. 2

The homology modeling of the recombinant protein PgBgl1. The catalytic sites of PgBgl1 have been labeled as E166 and E353. The 3D structure of PgBgl1 was predicted using the Swiss-Model server (https://swissmodel.expasy.org/on 6 June 2023).

Fig. 3

SDS-PAGE of recombinant protein PgBgl1. M: marker; lane1: IPTG; lane2: +IPTG; lane 3: crude enzyme; lane4: purified protein.

Fig. 4

Characterization of recombinant PgBgl1. (A) Optimum temperature. (B) Optimum pH. (C) Thermal stability (30 °C, 40 °C and 50 °C). (D) pH stability. The optimum activity was set as 100%. Data represent the means of triplicate measurements and error bars represent standard deviation.

Fig. 5

Different concentrations of methanol, ethanol, NaCl, and sugars on the activity of PgBgl1. (A) Methanol and ethanol tolerance of recombinant PgBgl1. Activity was measured in the presence of 0–20% ethanol and 0–30% methanol. Activity with 0% ethanol or methanol was set as 100%. (B) NaCl and various sugars (glucose, cellobiose, D-xylose, D-galactose and D-sucrose) tolerance of recombinant PgBgl1. Activity was measured in the presence of 0–2 M NaCl or sugars. Activity with 0 M NaCl or sugars was set as 100%. Data represent the means of triplicate measurements and error bars represent standard deviation.

Fig. 6

HPLC of soybean isoflavone standards and PgBgl1 hydrolyzed soybean isoflavone glycosides. (A) HPLC profiles of daidzin, glycitin, genistin, daidzein, glycitein, and genistein. (B) HPLC detection of soybean isoflavone glycoside daidzin hydrolyzed by PsBgl1. (C) HPLC detection of soybean isoflavone glycoside glycitin hydrolyzed by PsBgl1. (D) HPLC detection of soybean isoflavone glycoside genistin hydrolyzed by PsBgl1. Notes: 1, daidzin (9.781 min); 2, glycitin (10.490 min); 3, genistin (14.298 min); 4, daidzein (20.531 min); 5, glycitein (20.821 min); 6, genistein (25.311 min).