β glucosidase

Manufactured by Merck Group

Sourced in United States, Germany, China, Japan

β-glucosidase is an enzyme that catalyzes the hydrolysis of β-1,4-glycosidic bonds in cellulose and other glucosides. It plays a crucial role in the breakdown of plant cell walls and the conversion of cellulose to glucose.

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Lab products found in correlation

64 protocols using β glucosidase

Kinetic Analysis of Glucoside Hydrolysis

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Stock
solutions of 1 mM Glc-o-MPN and Glc-p-MPN were prepared in distilled water
and used within a few hours. A 1 mg/mL stock solution of commercial
β-glucosidase (purchased from Sigma who derived it from almonds)
was prepared in 1X PBS buffer, pH 5.33, and stored at 2–8 °C.
An aliquot of Glc-o-MPN (50 μM)
and Glc-p-MPN was added in a cuvette
containing 1× PBS buffer, pH 5.33, followed by the addition of
200 μg/mL β-glucosidase. The final 1 mL reaction mixture
was mixed by inversion five times before monitoring the change in
the absorption spectra over an hour. Enzyme inhibition studies were
also performed using castanospermine (CAT) (10 μg/mL), a plant
alkaloid inhibitor of β-glucosidase (purchased from Sigma).
The order of addition was Glc-o-MPN or Glc-p-MPN (50 μM), CAT (10 μg/mL),
and β-glucosidase (200 μg/mL). The Michaelis–Menten
kinetic parameters were determined in 1× PBS buffer, pH 5.33,
at room temperature by varying the concentration of Glc-o-MPN (10–500 μM) or Glc-p-MPN (5–200 μM) in the reaction solutions.
β-glucosidase (100 μg/mL) was added to each reaction,
and the change in absorption at 300 nm for the appearance of o-MPN and 312 nm for the appearance of p-MPN was measured at 5 min intervals over
an hour. A Lineweaver–Burk plot was created, and the Michaelis–Menten
constant (K_m) and maximum rate (V_max) were calculated.

Morsby J.J., Thimes R.L., Olson J.E., McGarraugh H.H., Payne J.N., Camden J.P, & Smith B.D. (2022). Enzyme Sensing Using 2-Mercaptopyridine-Carbonitrile Reporters and Surface-Enhanced Raman Scattering. ACS Omega, 7(7), 6419-6426.

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Enzymatic Production of Betanidin

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Betanidin was obtained by deglycosylation of the betanin in beet root juice. 80 µL of β-glucosidase from almonds (Sigma–Aldrich) and 420 µL of 10 mM Na- ascorbate (pH 5.5) were added to 500 µL of sterile filtered beet root juice (ca. 200 mg/L betanin concentration) and incubated for 2 h at 37 °C. The Na-ascorbate ensures the stability of the formed betanidin. To verify the successful deglycosylation of betanin and formation on betanidin, the samples were quantified in HPLC, confirming that 94% of the (iso-)betanin has been deglycosylated to (iso-)betanidin. Betanidin concentration was determined by Beer-Lambert equation assuming a molar extinction coefficient ε = 5.4 × 10⁴ M⁻¹ cm⁻¹. The β-glucosidase was removed from the sample by size-exclusion, using a 30 kDa centrifugal filter unit (Merck Millipore, USA). The reaction mix for the glycosylation assay contained 140 mmol/L KPO₄ buffer (pH 6), 3.2 mmol/L UDP-glucose (Sigma–Aldrich), ca. 20 µmol/L betanidin and ca. 50 µg (5 µL) purified UGT73A36 in a total volume of 50 µL. The reaction was started by addition of the enzyme and incubated at 30 °C for 16 h. For the control, H₂O was added instead of purified UGT73A36. The reaction was stopped by keeping the mix at max. 4 °C before analysis by HPLC.

Babaei M., Thomsen P.T., Dyekjær J.D., Glitz C.U., Pastor M.C., Gockel P., Körner J.D., Rago D, & Borodina I. (2023). Combinatorial engineering of betalain biosynthesis pathway in yeast Saccharomyces cerevisiae. Biotechnology for Biofuels and Bioproducts, 16, 128.

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Phytochemical Profiling and Antioxidant Analysis

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Methyl jasmonate (MeJA), coronatine (COR), the Folin–Ciocalteu reagent, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), triphenyl tetrazolium chloride (TTC), Murashige and Skoog Basal Medium (MS), sucrose, gallic acid, quercetin, podophyllotoxin, β-glucosidase, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and salts were purchased from Merck (Darmstadt, Germany); the solvents were from Honeywell (Milan, Italy); the pre-coated TLC-plates SIL G-100/UV254 were from Macherey-Nagel (Dueren, Germany); water (purified water) was obtained from MilliQ (Millipore, Darmstadt, Germany).

Alfieri M., Mascheretti I., Dougué Kentsop R.A., Consonni R., Locatelli F., Mattana M, & Ottolina G. (2021). Enhanced Aryltetralin Lignans Production in Linum Adventi-Tious Root Cultures. Molecules, 26(17), 5189.

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Quantifying Salicylic Acid in Plants

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The shoot from 10–20 plants (80–160 mg) per sample was used for SA quantification. The samples were homogenized in 1 ml of 90% methanol and extracted using 1 ml of 100% methanol. The extracts were mixed and dried at 40°C, and the residues were dissolved in 4 ml of water at 80°C for 15 min. One milliliter of each extract was used for quantifying free and total SA. For total SA quantification, 1 ml of β-glucosidase (Merck, Darmstadt, Germany) solution (3 units/ml) was prepared with 0.1 M sodium acetate buffer, added to 1 ml of extract and incubated at 37°C for 4–6 h. For free SA quantification, 1 ml of 0.1 M sodium acetate buffer was added to 1 ml of extract. Subsequently, 2.5 ml of ethyl acetate–cyclohexane (1:1) and 50 µl of concentrated HCl were added and mixed intensely. The upper layer was dried at 35°C and dissolved in 1 ml of 20% methanol in 20 mM sodium acetate buffer. This solution was used for HPLC analysis (JASCO FP-1520S) using the CAPCELL PAK C18 MG column (5 µm, 150 mm × 2 mm) with 20% methanol in 20 mM sodium acetate buffer at a flow rate of 1 ml/min. Detection was performed at an excitation wavelength of 295 nm and an emission wavelength of 370 nm.

Hashimoto S., Shikanai Y., Kusajima M., Nakamura H., Fujiwara T, & Kamiya T. (2023). Inhibition of NPR1 Leads to Shoot Growth Improvement under Low-Calcium Conditions in Arabidopsis. Plant and Cell Physiology, 64(12), 1579-1589.

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Phloroglucinol-HCl Staining and HPLC Analysis

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Histochemical stains (Phloroglucinol, HCl, Fluorol Yellow 088) and ethanol were purchased from Merck (Darmstadt, Germany), while FineFIX working solution was obtained from Milestone SRL (Sorisole, Bergamo, Italy). Picric acid solution 1.3% H₂O (saturated), sodium carbonate, LC/MS-ELSD grade prunasin (purity ≥ 90%), β-glucosidase, phosphate buffer, sodium acetate, sodium hydroxide, chloridric acid, chloramine T, barbituric acid, pyridine, and HPLC-grade solvents and reagents (acetonitrile and formic acid) were purchased from Merck (Darmstadt, Germany).

Malaspina P., Betuzzi F., Ingegneri M., Smeriglio A., Cornara L, & Trombetta D. (2022). Risk of Poisoning from Garden Plants: Misidentification between Laurel and Cherry Laurel. Toxins, 14(11), 726.

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Quantifying Beta-Glucosidase Inhibition in Plant Extracts

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β-glucosidase inhibitory activity of plant extracts was carried out according to a previous study [28 (link)] and adapted in a 96-well plate. Briefly, 20 µL of substrate (p-nitrophenyl-β-D-glucopyranoside, Sigma Chemical Co., 1 mg/mL), 10 µL of varying concentrations of samples (1, 2, 3, 4, 5 and 10 mg/mL) and 20 µL of pH 5 sodium phosphate buffer were mixed in 96-well plate and incubated at 37 °C for 10 min; 10 µL of enzyme solution (β-glucosidase Sigma Chemical Co., 5 mg/mL) were added and the mixture was incubated for another 30 min at 37 °C. 140 µL of pH 10 buffer 50 mM was added to stopped the reaction. Positive control contained, a mixture of solvents instead of the extract; while in the negative control, pH 10 buffer was added at the beginning of the test in order to block enzyme activity. Absorbance was read at 410 nm and the activity was calculated using the following formula:

% enzymatic inhibition = 100 - [\frac{Abs test - Abs negative control}{Abs positive control} \times 100]

Chokki M., Cudălbeanu M., Zongo C., Dah-Nouvlessounon D., Ghinea I.O., Furdui B., Raclea R., Savadogo A., Baba-Moussa L., Avamescu S.M., Dinica R.M, & Baba-Moussa F. (2020). Exploring Antioxidant and Enzymes (A-Amylase and B-Glucosidase) Inhibitory Activity of Morinda lucida and Momordica charantia Leaves from Benin. Foods, 9(4), 434.

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Purification of Phospholipases and Lipases

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Dromedary (DrPlA₂-IB), porcine (PPPlA₂-IB), and stingray group IB phospholipases (SPLA₂-IB) were purified and prepared as described in our previous works [18 (link), 19 (link)] and De Haas et al. [20 (link)], respectively. Dromedary (DrPL), stingray (SPL), and porcine (PPL) lipases were also purified according to protocols optimized in previous works [21 (link)–23 (link)]. The enzymes β-glucosidase, α-amylase, and XO were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Alonazi M., Horchani H., Alwhibi M, & Ben Bacha A. (2020). Cytotoxic, Antioxidant, and Metabolic Enzyme Inhibitory Activities of Euphorbia cyparissias Extracts. Oxidative Medicine and Cellular Longevity, 2020, 9835167.

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Glycosidic Bond Composition of CCK-Oligosaccharides

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CCK-oligosaccharides were treated with various carbohydrate-hydrolyzing enzymes to determine the composition of the glycosidic bonds. CCK-oligosaccharides (1%, w/v) were treated with the following enzymes and concentrations: 10 mU of α-amylase, 100 mU of α-glucosidase, 520 mU of amyloglucosidase (Sigma-Aldrich), 1.4 U of pullulanase M1, 10 mU of lichenase (Megazyme, Chicago, IL, USA), 100 mU of β-glucosidase, and 10 mU of β-1,3-d-glucanase (Sigma-Aldrich). The CCK-oligosaccharides were reacted with enzymes at 37 °C for 1 h and the products were determined by TLC.

Lee S., Park J., Jang J.K., Lee B.H, & Park Y.S. (2019). Structural Analysis of Gluco-Oligosaccharides Produced by Leuconostoc lactis and Their Prebiotic Effect. Molecules, 24(21), 3998.

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Saccharomyces cerevisiae Ethanol Production

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SSF was conducted based on NREL protocol TP-510-42630 (Dowe and McMillan 2008 ). The yeast implemented was Saccharomyces cerevisiae D₅A (S. cerevisiae Meyen ex E.C. Hansen ATCC^® 200062TM). The enzyme loading was 15 units/g cellulose, at 7.0 g cellulose/g biomass, 105 units of cellulase enzyme from Trichoderma reesei (Sigma Aldrich, C2730-50ML) was loaded into each 100 mL flask, and 50 units of β-glucosidase from almonds was also added. The flasks were incubated at 35 °C, 130 rpm. The ethanol production data (% ethanol, g/L, % cellulose conversion) for 0, 24, 96, and 160 h were also determined as a function of time (in Additional file 1). Post-SSF, the solid residue was separated from the fermentation broth via vacuum filtration using a Büchner funnel fitted with Whatman Grade 1 filter paper (Sigma Aldrich, WHA1001150) and a side-arm flask. The solid portion of the pretreatment residue was washed with 500 mL of water three times to remove residual yeast fermentation broth and was freeze-dried with a VirTis Freezemobile 25 L freeze dryer for 48 h.

Le R.K., Das P., Mahan K.M., Anderson S.A., Wells T J.r., Yuan J.S, & Ragauskas A.J. (2017). Utilization of simultaneous saccharification and fermentation residues as feedstock for lipid accumulation in Rhodococcus opacus. AMB Express, 7, 185.

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Enzymatic Characterization of β-Glucosidase

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β-glucosidase
(1 mg/L, Sigma-Aldrich, Vienna, Austria) was incubated with tyramine, p-coumaric acid, caffeic acid, catechin, lignosulfonic acids
(18 and 52 kDa), and humic acids (1 and 0.1 g/L, respectively; all
compounds were purchased from Sigma-Aldrich) in 50 mM Tris-HCl (pH
7.0). SinATYR (1 mg/L) was added to the mixture for
samples with TYR activity. Samples solutions were incubated at 4 °C
and β-glucosidase activity has been determined after 24, 48,
72, and 96 h.
4-Methylumbelliferyl-β-d-glucopyranoside
(Sigma-Aldrich) was used to measure β-glucosidase activity.
Fluorescence measurements were performed on a TECAN infinite M200
reader (Tecan, Salzburg, Austria, excitation wavelength: 365 nm, emission
wavelength: 455 nm)^{77 (link)} in triplicate. For
each measurement, 10 μL of sample solution was mixed with 5
μL of 10 mM 4-methylumbelliferyl-β-d-glucopyranoside
(dissolved in 100% 2-methoxyethanol). The solution was filled up to
200 μL with H₂O and adjusted to 50 mM Tris-HCl (pH
7.0). As a control sample, β-glucosidase (1 mg/L) was incubated
at 4 °C in 50 mM Tris-HCl (pH 7.0) without additional phenolics.

Panis F, & Rompel A. (2023). Biochemical Investigations of Five Recombinantly Expressed Tyrosinases Reveal Two Novel Mechanisms Impacting Carbon Storage in Wetland Ecosystems. Environmental Science & Technology, 57(37), 13863-13873.

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