4 nitrophenyl β d glucopyranoside pnpg

Saccharomyces cerevisiae α-glucosidase (E.C 3.2.1.20), acarbose, and 4-nitrophenyl β-D-glucopyranoside (pNPG) were obtained from Sigma Aldrich Co (Saint Louis, MO, USA). The alpha-glucosidase (0.2 U/mL) and substrate (5.0 mM pNPG) were dissolved in 100 mM pH 6.9 sodium phosphate buffer [19 (link)]. The inhibitor (50 µL) was preincubated with alpha-glucosidase at 37 °C for 20 min, and the substrate (40 µL) was subsequently added to the reaction mixture. The enzymatic reaction was conducted at 37 °C for 20 min and ended by adding 0.2 M Na₂CO₃ (130 μL). Enzymatic activity was quantitatively measured at an absorbance of 405 nm (CLARIOstar plus, BMG LABTECH, Ortenberg, Germany). All samples were analyzed in triplicate at five different concentrations around the IC₅₀ values, and the mean values were retained. The following equation was used to calculate the inhibition percentage (%): Inhibition (%) = [1 − (A_sample/A_control)] × 100.

Wogonoside, wogonin, and 4-Nitrophenyl β-d-glucopyranoside (pNPG) (purity > 98) were purchased from Sigma Aldrich (St. Louis, MO, USA). Monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), and sodium chloride (NaCl) were purchased from VWR International (Radnor, PA, USA). KPI buffer was made using KH2PO4 and K2HPO4 and the pH was adjusted using sodium hydroxide and hydrochloride. Other chemicals (typically analytical grade or better) were used as received. C57BL6 mice (male, 8 weeks) were obtained from Jackson lab (Bar Harbor, ME, USA) and F344 rats (male, 8 weeks) were bought from Charles River (Wilmington, MA, USA).

C. morifolium flowers were purchased from Tongxiang Shine Herb Health products Co., Ltd., (Jiaxing, China). The plant material was powdered and stored in a sealed bag until use. 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), trifluoroacetic acid (TFA), 1-phenyl-3-methyl-5-pyrazolone (PMP), trichloroacetic acid (TCA), butyl hydroxyanisole (BHA), 4-nitrophenyl-β-D-glucopyranoside (pNPG), and α-glucosidase (biological reagent, 50 U/mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acarbose, 1,10-phenanthroline, bovine serum albumin (BSA), and monosaccharide standards (glucose, fructose, rhamnose, galactose, xylose, arabinose) were purchased from J & K Scientific Co., Ltd. (Beijing, China). All other chemicals used were of analytical grade.

pNPX, 4-nitrophenyl-α-l-arabinofuranoside (pNPA) (Sigma, Copenhagen, DK) and 4-nitrophenyl-β-d-glucopyranoside (pNPG) (Sigma, Copenhagen, DK), azurine-cross-linked (AZCL) arabinoxylan, AZCL xylan oat, AZCL debrancharabinan, AZCL HE-cellulose, AZCL xyloglucan, and AZCL barley-glucan (Megazyme, Bray, IE) were used as substrates to test the enzyme-specific activity. When using the pNPX, pNPA, and pNPG as substrates, the reaction and unit calculation was performed as described above (“Enzyme activity analysis” section). The assay using the AZCL substrates was performed as described by Huang et al. (2015 (link)). The enzyme activity was indicated by the diameter of blue haloes measured as millimeters.

The candidate lactic acid bacterial-starter strains isolated from fermented herbs, food, and rice wine were screened for their β-glucosidase enzymatic activity. The bacteria were grown in Lactobacilli MRS broth (Difco Laboratories, Detroit, MI, United States) at 37°C for 24 h. The bacterial suspensions were then centrifuged at 12,000 g for 3 min, and the supernatants were discarded. The bacterial pellets were washed three times with PBS (pH 7.4), and the cells were re-suspended in PBS. A portion of this suspension was then mixed with 3 mM 4-nitrophenyl β-D-glucopyranoside (p-NPG, Sigma Aldrich, St. Louis, MO, United States) at a 1:1 (v/v) ratio, and the mixture was incubated at 37°C for 10 min. The β-glucosidase enzymatic activity of the samples was measured colorimetrically at 420 nm using a microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, CA, United States).

Okra fruits were harvested at a commercial orchard in Chengdu, Sichuan, China. The samples were washed, hot-air-dried (75 °C and 12 h), smashed, screened, and stored at −20 °C. Acarbose, α-amylase (1000 U/mg), α-glucosidase (10 U/mg), arabinose (Ara), galactose (Gal), galacturonic acid (GalA), glucose (Glc), glucuronic acid (GlcA), mannose (Man), rhamnose (Rha), xylose (Xyl), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 1-phenyl-3-methyl-5-pyrazolone (PMP), and 4-nitrophenyl β-d-glucopyranoside (pNPG) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). A free cholesterol assay kit and DEAE Cellulose-52 were purchased from Solarbio (Beijing, China). Lactobacillus acidophilus CICC 6089, Lactobacillus rhamnosus CICC 6133, and Lactobacillus rhamnosus CICC 6151 were purchased from China Center of Industrial Culture Collection. All other chemicals and reagents used were of analytical grade.

Recombinant plasmid pPIC9 harboring the coding sequence bgl3A (gene bank accession KU363626), constructed in our previous work [14 (link)], was used as the template for site-saturation mutagenesis. Pichia pastoris strain GS115 (Invitrogen, Carlsbad, CA, USA) was employed as a heterologous expression host for protein preparation. The substrates 4-nitrophenyl β-d-glucopyranoside (pNPG), cellobiose, gentibiose, and salicin were purchased from Sigma–Aldrich (St. Louis, MO, USA). LA Taq DNA polymerase, restriction endonucleases, and DNA purification kit were purchased from TaKaRa (Otsu, Japan). DNA ligase and total RNA isolation system kit were purchased from Promega (Madison, WI, USA). All other chemicals were of analytic grade and commercially available.