The enzymatic hydrolysis was performed according to the method described by Tironi and Añón [58 (link)]. Briefly, 5 g of the protein extract was diluted in 100 mL of deionized water (milli Q 18.2 MΩ*cm, Manufacturer, Darmstadt, Germany). For alcalase hydrolysis (H1), prior to the reaction, the pH of the solution was adjusted to 10, and alcalase (≥2.4 U/g, Anson Units; Sigma-Aldrich (St. Loui, MO, USA) was added at concentration of 8 µL/100 mg of the sample. For flavourzyme hydrolysis (H2), the pH of the reaction media was adjusted to 7 and flavourzyme (≥500 U/g; Sigma Aldrich, St. Louis, MO, USA) was added at concentration of 5 µL/100 mg of the sample. Finally, when both enzymes were used in a two-step continuous hydrolytic process (H3), after 2 h of hydrolysis with alcalase, the enzyme was inactivated by heating to 85 °C for 10 min. The solution was adjusted to pH 7 and hydrolysis with flavourzyme was carried out. For all three methods, hydrolysis was followed up during 4 h and progress of the reaction was monitored every 20 min. The aliquots were heated to 85 °C during 10 min and frozen until further use.
Alcalase
Manufactured by Merck Group
Sourced in United States, China, Denmark
Alcalase is a commercial enzyme product manufactured by Merck Group. It is a serine endopeptidase that hydrolyzes peptide bonds in proteins. Alcalase has a broad specificity and can be used in various industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Flavourzyme, by Merck Group (12 mentions)
Papain, by Merck Group (10 mentions)
Pepsin, by Merck Group (10 mentions)
Trypsin, by Merck Group (9 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (5 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (5 mentions)
Neutrase, by Merck Group (4 mentions)
α amylase, by Merck Group (3 mentions)
Potassium bromide, by Merck Group (3 mentions)
Savinase, by Merck Group (3 mentions)
β mercaptoethanol, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Trolox, by Merck Group (2 mentions)
Reduced glutathione, by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Sodium tetraborate, by Merck Group (2 mentions)
Bacillus licheniformis, by Merck Group (2 mentions)
Thermolysin, by Merck Group (2 mentions)
α glucosidase, by Merck Group (2 mentions)
55 protocols using alcalase
1
Enzymatic Hydrolysis of Protein Extract
2
Fluorescent Peptide Synthesis and Characterization
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Example 1
Peptide 1 QC-1-AAAFALAC-IRDye800CW was obtained by standard solid phase peptide synthesis methods. Fluorescent agent IRDye800CW and non-fluorescent agent (quencher) QC-1 were obtained from LI-COR (Nebraska, USA).
Alcalase, Sigma Aldrich P4860—stock contains 150 mg/mL alcalase.
Fluorescence was monitored over time using a Cary Eclipse Instrument, settings: Ex. Wavelength: 720 nm; Em. Wavelength: 785 nm; Ex. Slit 10 nm; Em. Slit 10 nm; Ave. Time 0.1 s; Ex. Filter and Em. Filter: Auto
3
Optimizing Chickpea Protein Hydrolysis
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A response surface analysis (RSA) with a central composite design was performed. A worksheet with randomly selected hydrolysis reactions was generated. The hydrolysis was carried out using alcalase (≥2.4 U/g, Sigma-Aldrich, St. Louis, MO, USA). The experimental design is shown in Table 1 . The following processing variables were optimized: (1) enzyme/substrate concentration (U/g), (2) time (h), and (3) temperature (°C). Three levels were considered for each variable. The central conditions were taken from a previous study with minor modifications to expand the experimental region [14 (link)]. ACE-I inhibition was the response variable.
Fifteen mg of the chickpea protein isolate was solubilized in 1 mL of BIS-TRIS Propane buffer (20 mM; pH 11) (Sigma-Aldrich, St. Louis, MO, USA). Afterwards, the samples were sonicated for 10 min (Ultrasonic Homogenizer Model 150 V/T; power = 40; pulser = 30%) and shaken at 1000 rpm (50 °C) for another 10 min. Before the addition of alcalase (≥2.4 U/g, Sigma-Aldrich, St. Louis, MO, USA), the temperature of the solution was adjusted according to the experimental design (Supplementary Materials Table S1 ). The hydrolysis reactions were carried out using a Thermomixer (Eppendorf™, Hamburg, Germany), and the reactions were stopped by heating the samples at 85 °C for 15 min. Finally, the samples were stored at −20 °C until their use.
Fifteen mg of the chickpea protein isolate was solubilized in 1 mL of BIS-TRIS Propane buffer (20 mM; pH 11) (Sigma-Aldrich, St. Louis, MO, USA). Afterwards, the samples were sonicated for 10 min (Ultrasonic Homogenizer Model 150 V/T; power = 40; pulser = 30%) and shaken at 1000 rpm (50 °C) for another 10 min. Before the addition of alcalase (≥2.4 U/g, Sigma-Aldrich, St. Louis, MO, USA), the temperature of the solution was adjusted according to the experimental design (
4
Oat Flour Characterization and Enzyme Assays
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Medium bran oat flour (i.d. 112-001) with a particle size percentage distribution of 2.00 mm (0.8%), 0.841 mm (61.5%), 0.595 mm (32.1%), 0.420 mm (5.0%), and Pan (0.6%) was donated by Richardson Milling (Portage La Prairie, MB, Canada). The enzymes (α-amylase, Flavourzyme®, Alcalase®, and Papain, sodium tartrate, sodium dodecyl sulfate, cupric sulfate pentahydrate, Trolox, 1,10-phenanthroline, iron(II) sulfate heptahydrate, hydrogen peroxide (H2O2), Tris-HCl, Tris-Base, potassium bromide, pyrogallol, reduced glutathione, L-serine, 3,5-dinitrosalicylic acid, sodium potassium phosphate tartrate, starch, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Ltd. (Oakville, Ontario, Canada). The solvents, including concentrated hydrochloric acid, methanol and Folin-Ciocalteau Phenol reagent, fluorescein, and 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH), were purchased from Fisher Scientific Co. (Nepean, Ontario, Canada).
5
Enzymatic Extraction of Legume Proteins
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Enzymatic-assisted extraction (EAE), according to the procedure described by Prandi et al.10 (link), was performed in duplicate on legume by-products (chickpeas, green peas, and white beans) using commercial proteases, alcalase (AL) and papain (PA) (Sigma–Aldrich, St. Louis, MO, USA) and starting from 10 g of each legume sample. The legume matrices were finely minced with a kitchen grinder. The reaction media phosphate buffer 10 mM was brought to pH 7.5 for the hydrolysis with alcalase and to pH 6.5 for the reaction with papain. Then, 10 g of finely minced legume matrix were added to 50 ml of phosphate buffer at appropriate pH for each enzyme, together with 1% enzyme per w of legume residue. (w/w for papain which is solid, v/w for alcalase, which is a water solution). Protease activity is 300 U/ml for papain and 0.52 U/ml for alcalase respectively. The enzymatic extraction was carried out under constant stirring (with magnetic stirrer) in a water bath for 2 h, at 60 °C for the reaction carried out with alcalase, and at 65 °C for the reaction carried out with papain. The enzymes were thermally inactivated by warming up at 90 °C for 10 min. The hydrolysates were then centrifuged at 3220 g, at 4 °C for 30 min and the protein supernatant was separated from the pellet and lyophilized.
6
Sturgeon Cartilage Collagen Peptide Extraction
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Cartilages of Siberian sturgeon (A. baerii) were kindly provided by Thousand Island Lake Sturgeon Technology Co., Ltd. (Hangzhou, China). HUVECs were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), trypsin, Alcalase, NAC, DPPH, papain, and pepsin were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). Flavorzyme and Sephadex G-25 was purchased from Shanghai Source Poly Biological Technology Co., Ltd. (Shanghai, China). Collagen peptides of SCP1 to SC13 with a purity higher than 98% were synthesized in Shanghai Apeptide Co., Ltd. (Shanghai, China).
7
Enzymatic Extraction of Seaweed Compounds
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Specimens of the red seaweed (Rhodophyta, Florideophyceae), Osmundea pinnatifida (Ceramiales) Rhodomelaceae family, and of the brown seaweed (Heterokontophyta, Phaeophyceae), Sargassum muticum (Fucales) Sargassaceae family, were harvested in Buarcos bay (Figueira da Foz, Portugal), and cleaned and dried according to Rodrigues et al., (2015b) [5 (link)]. Enzymatic extraction of O. pinnatifida by Viscozyme L (Sigma-Aldrich, St. Louis, MO, USA) and of S. muticum by Alcalase (Sigma-Aldrich) was performed according to procedures described by Rodrigues et al., (2015a) [3 (link)]. In this study, the authors used lyophilized aliquots of the same extracts obtained by Rodrigues et al., (2015a) [3 (link)] in order to enable accurate comparison and correlation analyses.
8
Enzymatic Sericin Hydrolysis and Isolation
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Enzymatic modification of sericin was performed using Alcalase® (EC 3.4.21.62; Sigma-Aldrich, St. Louis, MO, USA) as previously reported [20 (link)]. Briefly, lyophilized sericin powder, a generous gift from Ruenmai-baimon, Ltd., Surin Province, Thailand, was dispersed in de-ionized water and further heated at 95 °C for complete solubilization for 10 min. The solution was put on ice for cooling down to room temperature. The hydrolysis by Alcalase® was performed at enzyme/substrate ratio: 2, pH 8 at 60 °C for 3 h as recommended by the manufacturer’s instructions. Then, the reaction was heated until 100 °C for 5 min to stop enzymatic function of Alcalase® followed with being immediately chilled on ice. Then, the supernatant was collected through 3500× g (4 °C) for 30 min and further dialyzed overnight with the phosphate buffer at 4 °C. The obtained solution was then freeze-dried to collect the powder of sericin hydrolysates that were kept at −20 °C until use.
9
Antioxidant Properties of Royal Jelly
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Fresh royal jelly (RJ) from Apis mellifera L. was obtained from the Honey Bee Farm (Changhua, Taiwan). Fresh RJ was dissolved in 0.01 M phosphate buffered saline (pH 7.2) and heated in a water bath (90 °C) for 30 min to eliminate its enzymatic activity. It was then lyophilized and stored at −20 °C until use.
The enzymes used for protein hydrolysis were alcalase (2.4 Anson units (AU)/g) and flavourzyme (0.5 unit/g) (Sigma Chemicals Co., St. Louis, MO, USA), respectively. TBA (2-thiobarbituric acid, minimum 98%), 8-hydroxy-2′-deoxyguanosine, 2′-deoxyguanosine monohydrate (2′-dG; 99–100%), bleomycin sulphate and calf thymus DNA were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals and solvents used were of analytical grade.
The enzymes used for protein hydrolysis were alcalase (2.4 Anson units (AU)/g) and flavourzyme (0.5 unit/g) (Sigma Chemicals Co., St. Louis, MO, USA), respectively. TBA (2-thiobarbituric acid, minimum 98%), 8-hydroxy-2′-deoxyguanosine, 2′-deoxyguanosine monohydrate (2′-dG; 99–100%), bleomycin sulphate and calf thymus DNA were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All other chemicals and solvents used were of analytical grade.
10
Enzymatic Lipid Modification Protocol
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Four genes (Photobacterium lipolyticum M37 lipase [25 (link)28 (link, link, link)], Proteus vulgaris K80 lipase [29 (link)], Bacillus stearothermophilus L1 lipase [30 (link)], Staphylococcus haemolyticus L62 lipase [31 (link)]) isolated previously by our laboratory were used in this study. Candida antarctica lipase B (CalB) enzyme was purchased from Novozyme. Alcalase (Bacillus licheniformis protease), diethyl malate, p-nitrophenyl caprylate, silica gel (Davisil Grade 635, pore size 60Å, 60-100 mesh), and 1-butanol were purchased from Sigma-Aldrich. Acetic acid was purchased from Kanto. Potassium permanganate and formic acid were purchased from Daejung. Sodium hydroxide and potassium carbonate were purchased from Jusei. Acetonitrile was purchased from Duksan.
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