Chicken feet from Gallus gallus domesticus were provided by a local farm (Granja Gaià, La Riera de Gaià, Spain). Protamex® (Novozymes, Bagsværd, Denmark) (EC 3.4.21.62 and 3.4.24.28, 1.5 AU/g from Bacillus licheniformis and Bacillus amyloliquefaciens), was kindly provided by Novozymes (Bagsværd, Denmark). ACE (angiotensin-converting enzyme, EC 3.4.15.1), N-Hippuryl-His-Leu (Hip-His-Leu), Captopril (PubChem CID: 44093) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and o-aminobenzoylglicil-p-nitrofenilalanilprolina (o-Abz-Gly-p-Phe(NO2)-Pro-OH, PubChem CID: 128860) was provided by Bachem Feinchemikalien (Bubendorf, Switzerland). Acetylcholine (PubChem CID: 187), methoxamine hydrochloride (PubChem CID: 6081) and heparin heparin (PubChem CID: 772) were purchased from Sigma-Aldrich (Madrid, Spain). All other chemical solvents used were of analytical grade.
Protamex
Manufactured by Novozymes
Sourced in Denmark, China, United States
Protamex is a microbial enzyme produced by Novozymes for use in lab applications. It functions as a protease, which is an enzyme that catalyzes the breakdown of protein molecules.
Automatically generated - may contain errors
Lab products found in correlation
Alcalase, by Novozymes (23 mentions)
Neutrase, by Novozymes (14 mentions)
Flavourzyme, by Novozymes (14 mentions)
Alcalase 2.4 l, by Novozymes (6 mentions)
Alcalase 2.4 l fg, by Novozymes (5 mentions)
Pepsin, by Merck Group (5 mentions)
Papain, by Novozymes (3 mentions)
Flavourzyme 500mg, by Novozymes (3 mentions)
Ficin, by Merck Group (3 mentions)
Flavorzyme, by Novozymes (2 mentions)
Trypsin, by Novozymes (2 mentions)
Trypsin, by Merck Group (2 mentions)
Flavourzyme 1000 l, by Novozymes (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Neutrase 0.8 l, by Novozymes (2 mentions)
Leucine, by Merck Group (2 mentions)
2 2 azino bis, by Merck Group (2 mentions)
Cholera toxin, by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
51 protocols using protamex
1
Chicken Feet Protease Extraction and ACE Inhibition
2
Enzymatic Hydrolysis of Proteins
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Alcalase, Flavorzyme, Neutrase, and Protamex were purchased from Novozymes (Bagsvaerd, Denmark). Bromeline and Papain were purchased from Daesong Sangsa (Seoul, Korea). All chemicals for antioxidant and anti-aging tests were purchased from the Sigma-Aldrich Chemical Company (St. Louis, MS, USA). All other reagents and solvents used in this study were of analytical grade.
3
Polyclonal Antibody Production for Dairy Proteins
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Casein (purity > 85%), ɑ‐LA (purity > 85%), β‐LG (purity > 85%), alkaline phosphatase‐conjugated goat anti‐rabbit IgG antibodies, Freund's complete and incomplete adjuvant, o‐phthalaldehyde (OPA), dithiothreitol (DTT), o‐phenylenediamine dihydrochloride (OPD), Papain and Pepsin were purchased from Sigma‐Aldrich. Alcalase, Neutrase, Protamex, and Flavourzyme were purchased from Novozymes. Three types of rabbit serum comprising polyclonal antibodies targeting CN, ɑ‐LA, β‐LG were prepared at the Shenyang Agricultural University.
4
Enzymatic Hydrolysis of Blood Proteins
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The blood proteins obtained in the previous steps were subjected to single
protease hydrolysis using the following proteases: alcalase, flavourzyme,
neutrase, protamex, papain, and trypsin (Novozymes, Bagsvaerd, Denmark). The
characteristics of commercial enzymes used in this study were decribed inTable 1 . In brief, 1 g of blood protein was
dissolved in 50 mL of 25 mM sodium phosphate buffer (0.2 M monobasic sodium
phosphate+0.2 M dibasic sodium phosphate), which was adjusted to pH 7.0
with 1 N NaOH and 1 N HCl. The mixture was homogenized with a homogenizer (T-25
basic, Ika Works, Wilmington, NC, USA). To determine the optimum hydrolysis time
for each protease, blood protein solutions were hydrolyzed with each protease in
a 50℃ water bath for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. The proteases
were added at a ratio of 1% for a particular blood protein. After
completion of hydrolysis, the solutions were heated in boiling water for 3 min
to inactivate the proteases. The mixture was centrifuged at 8,000×g for
25 min using Supra 22K (Hanil Science Industrial, Incheon, Korea). The
supernatant was stored at –20℃ until use.
protease hydrolysis using the following proteases: alcalase, flavourzyme,
neutrase, protamex, papain, and trypsin (Novozymes, Bagsvaerd, Denmark). The
characteristics of commercial enzymes used in this study were decribed in
dissolved in 50 mL of 25 mM sodium phosphate buffer (0.2 M monobasic sodium
phosphate+0.2 M dibasic sodium phosphate), which was adjusted to pH 7.0
with 1 N NaOH and 1 N HCl. The mixture was homogenized with a homogenizer (T-25
basic, Ika Works, Wilmington, NC, USA). To determine the optimum hydrolysis time
for each protease, blood protein solutions were hydrolyzed with each protease in
a 50℃ water bath for 30 min, 1 h, 2 h, 3 h, 4 h, or 5 h. The proteases
were added at a ratio of 1% for a particular blood protein. After
completion of hydrolysis, the solutions were heated in boiling water for 3 min
to inactivate the proteases. The mixture was centrifuged at 8,000×g for
25 min using Supra 22K (Hanil Science Industrial, Incheon, Korea). The
supernatant was stored at –20℃ until use.
5
Enzymatic Digestion of Cow Milk Proteins
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Fresh CM samples were obtained from 120 healthy Holstein cows from a farm near Shenyang (Liaoning, China). Fresh CM samples had fat removed by highspeed centrifugation at 15,000 × g for 30 min at 4°C and were stored at -80°C. Alcalase, Protamex, Flavourzyme, and Neutrase were acquired from Novozymes. Papain and pepsin were acquired from Sigma-Aldrich. Cholera toxin (CT) was obtained from Sigma-Aldrich. We purchased ELISA kits (mouse IgG 1 and IgG 2a ) from eBioscience.
6
Mackerel Protein Hydrolysate Production
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Mackerel hydrolysate fractions were produced at Merinov (Gaspé, QC, Canada) according to a procedure adapted from [4 (link)]. Briefly, 100 kg of ground mackerel fished in 2016 was added to an equal amount of demineralized water (w/w), and the total volume was heated to 40–43 °C. Then, 100 g Protamex (Novozymes, Bagsvaerd, Denmark) was added to start the hydrolysis. After 150 min of hydrolysis at 40–43 °C, the tank temperature increased to 90 °C, to inactivate proteases. The liquid fraction was decanted using a clarifying decanter and then centrifuged at 11,000 g to separate suspended insoluble matter and lipids from the hydrolysate. The hydrolysate was then ultrafiltered (spiral membrane with molecular weight cut off of 10 kDa) to separate proteins and peptides according to molecular mass. Permeate from the 10 kDa membrane was nano-filtered at 200 Da (Model R, GEA filtration, Hudson, WI, USA) to obtain a 10 kDa–200 Da retentate. The nano-filtration retentate was spray-dried and stored at 4 °C until analyzed.
7
Isolation and Characterization of Carp Skin Collagen
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H. Molitrix skin was supplied by Hubei Zhongke Agriculture Co., Ltd. (Jingzhou, China). Adenosine diphosphate (ADP), trypsin -EDTA solution was purchased from Solarbio (Beijing, China). Alcalase and Protamex® were purchased from Novozymes (Beijing, China). Collagen type I from rat tail tendon, pancreatin from porcine, and pepsin from porcine gastric mucosa were purchased from Sigma-Aldrich (Shanghai, China). Phosphate buffer solution (PBS) was purchased from HyClone (Beijing, China). Dulbecco modified Eagle’s minimal essential medium (DMEM), Fetal bovine serum (FBS), Hank’s buffered saline solution (HBSS), nonessential amino acids (NEAA), penicillin, and streptomycin were purchased from Gibco (Life Technologies, Grand Island, NY, USA). Sodium hydroxide (NaOH), trisodium citrate (Na3C6H5O7·2H2O), hydrochloric acid (HCl), sodium bicarbonate (NaHCO3), and other chemicals were all analytically pure grade and purchased from Sinopharm chemical reagent Co., LTD (Beijing, China).
8
Antiplatelet agents in hemostasis
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Alcalase and Protamex were purchased
from Novozymes Biotechnology Co., Ltd. (Beijing, China). The 5-HT
ELISA kit was purchased from IBL international (German). The β-TG
ELISA kit was obtained from Cloud-Clone Corp (Wuhan, China). Thrombin
and ADP were purchased from Beijing Solarbio Science and Technology
Co., Ltd. (Beijing, China). Aspirin and clopidogrel hydrogen sulfate
were purchased from MCE (Shanghai, China). Other chemicals used were
analytical grade or better.
from Novozymes Biotechnology Co., Ltd. (Beijing, China). The 5-HT
ELISA kit was purchased from IBL international (German). The β-TG
ELISA kit was obtained from Cloud-Clone Corp (Wuhan, China). Thrombin
and ADP were purchased from Beijing Solarbio Science and Technology
Co., Ltd. (Beijing, China). Aspirin and clopidogrel hydrogen sulfate
were purchased from MCE (Shanghai, China). Other chemicals used were
analytical grade or better.
9
Enzymatic Hydrolysis of Proteins
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Based on a literature survey with focus on obtained bioactivities (antihypertensivity, antioxidativity and other) [6] , [14] and our earlier experience, eight different proteolytic enzymes were selected for enzymatic hydrolysis (Table 1 ). Corolase® PP and Corolase® 7089 (both from AB Enzymes GmbH), Protamex® (Novozymes A/S), Papain FG and Bromelain 400 GDU/g (both from Enzybel), Trypsin (Sigma Aldrich), Protex 6L (Genecor) and Seabzyme L 200 (Specialty Enzymes & Biotechnologies) were received from the producer.
10
Snow Crab Hydrolysate Fractionation
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Snow crab hydrolysate fractions were produced at Merinov, the Quebec Fisheries and Aquaculture Innovation Centre (Gaspé, QC, Canada) according to a procedure by Beaulieu et al. [20] (link). Briefly, 100 kg of grinded snow crab by-products were added to equal amount of demineralized water (w/w), the total volume was heated to 45°C. Then, 100 g Protamex (Novozymes, Bagsvaerd, Denmark) were added to start the hydrolysis. After 120 minutes hydrolysis at 45°C, the tank temperature was increased to 90°C, to inactivate proteases.
The liquid fraction was decanted using a clarifying decanter and then centrifuged at 11,000 g to separate suspended insoluble matter and lipids from the hydrolysate. The hydrolysate was then ultrafiltered (spiral membrane with cut off of 10 kDa) to separate proteins and peptides according to the molecular mass. Permeate from the 10 kDa membrane at 200 Da was nano-filtered (Model R, GEA filtration, Hudson, WI, USA) to obtain a 10 kDa -200 Da retentate (SCAMPs). Nano-filtration retentate was spray-dried and kept at 4°C until analyses.
The liquid fraction was decanted using a clarifying decanter and then centrifuged at 11,000 g to separate suspended insoluble matter and lipids from the hydrolysate. The hydrolysate was then ultrafiltered (spiral membrane with cut off of 10 kDa) to separate proteins and peptides according to the molecular mass. Permeate from the 10 kDa membrane at 200 Da was nano-filtered (Model R, GEA filtration, Hudson, WI, USA) to obtain a 10 kDa -200 Da retentate (SCAMPs). Nano-filtration retentate was spray-dried and kept at 4°C until analyses.
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