P nitrophenyl β d glucopyranoside

α-Glucosidase (Saccharomyces cerevisiae, EC3.2.1.20, 20 U/mg) and the substrate, p-nitrophenyl-β-d-glucopyranoside (p-NPG) were purchased from Sigma-Aldrich, and the assay was performed exactly according to our previous report^{34 (link),35 (link)}.

The β-glucosidase activity was measured by determining the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma) using the initial rate of accumulation of coloured reaction product as described by^{9 (link)}. One hundred eighty microliter of 5 mM pNPG substrate was diluted in 50 mM Tris HCl buffer (pH 8.5) and mixed with a 20 μl aliquot of the acquired enzyme. Then, the mixture was incubated at 50 °C for 10 min, and the reaction was terminated by adding 100 μl of ice-cold 0.5 M Na₂CO₃. The release of p-nitrophenol (pNP) via enzymatic hydrolysis was indicated by the appearance of a yellow colour and the absorbance was measured with a UV/Vis microplate spectrophotometer (Multiskan GO, Thermo Scientific) at 405 nm. One unit (U) of β-glucosidase activity was defined as the amount of enzyme that released 1 μmol of pNP per minute from the substrate. All experiments were performed in three technical replicates. Statistical significance between treatments and a control group was assessed using post-hoc Tukey's Honestly Significant Difference (HSD) test. Differences were considered statistically significant at a p-value of < 0.05.
Relative activity is as follows: $$\mathrm{Relative}\mathrm{Activity}(\%)=(\mathrm{Activity}\mathrm{of}\mathrm{sample}(\text{U})/\mathrm{Maximum}\mathrm{enzyme}\mathrm{activity}(\text{U}))\ast 100$$

The recombinant pGEX-4T-1 vector containing S. griseus β-glucosidase gene (GST-tagged) in E. coli BL21 (DE3) was developed in a previous study [14 (link)]. Ampicillin, glycerol, Isopropyl-β-D-thiogalactopyranoside (IPTG), LB broth, p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenol (pNP), cellobiose, fructose sucrose, tryptone, yeast extract, beef extract, CaCl₂, DTT, KOH, MgCl₂, (NH₄)₂S₄, ZnSO₄, Triton X-100, M PMSF, Lysozyme, Bradford reagent, and Glutathione Sepharose 4B resin were purchased from Sigma Aldrich (Ireland).

Mannobiose (M₂), mannotriose (M₃) and mannotetraose (M₄) standards were purchased from Megazyme (Bray, Ireland). Locust bean gum (LBG), solka floc, glucose, mannose, guar gum, p-nitrophenyl-α-d-galactopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-mannopyranoside, p-nitrophenol (pNP) and other chemicals were sourced from Sigma-Aldrich, USA. Copra meal was obtained from Parker Biotech Private Ltd., Tamil Nadu, Chennai, India. Food-grade konjac gum (glucomannan) was obtained from New Foods, Bloomingdale, Illinois, USA. Fenugreek seed (Trigonella foenum-graecum) meal, Aloevera pulp, rice husk, wheat straw and wheat bran were purchased from local markets.

Monacolin K, citrinin, liquiritin, liquiritigenin, trifluoroacetic acid, acetic acid, and p-nitrophenyl-β-D-glucopyranoside were purchased from Sigma-Aldrich Co. (USA), isoliquiritin and isoliquiritigenin from ChemFaces (China), and liquiritin apioside and isoliquiritin apioside from Chengdu Biopurify Phytochemicals Ltd. (China). All HPLC-grade solvents were purchased from Fisher Scientific Korea Ltd. (Korea). Extraction solution and methanol were purchased from Samchun Pure Chemical (Korea). Potato dextrose agar (PDA) was purchased from Acumedia Inc.(USA), and glucose from Duksan Pure Chemicals (Korea). Yeast extract-peptone-dextrose and yeast nitrogen base media were purchased from Sigma-Aldrich Co. G. uralensis base media were composed of 2.0 5.0% G. uralensis extracts and 2% glucose. Licorice (G. uralensis) was purchased from a regional market in Chungcheongbuk-do, Korea. Licorice extracts were prepared by adding 5 L of distilled water to 1.2 kg of licorice, followed by boiling at 95oC for 5 h. The extraction supernatant recovered by filtration with a Whatman filter paper No. 1 (UK) was concentrated to a solid content of 15% using a vacuum evaporator.