Coriander (Coriandrum sativum L.) seeds were purchased from a local food store in Warsaw, Poland, whereas almonds (Prunus dulcis) and hazelnuts (Corylis avellana L.) were obtained from the Hebar Company, Poland. The yeast strain of Y. lipolytica W29 (ATCC20460) was obtained from the culture collection of the Food and Microbiology Processing and Engineering Laboratory (GPMA) at the University of Burgundy/AgroSup, France. The strain was stored in 10% glycerol in freezing conditions (−80 °C). All medium ingredients (agar–agar, yeast extract, peptone, and glucose) were obtained from BTL Łódź, Poland. Sodium chloride, methyl and ethyl alcohol (99.8%), chloroform, ethyl acetate, n-hexane, toluene, and anhydrous magnesium sulfate were purchased from POCH, Poland, a division of Avantor Performance Products (Center Valley, PA, USA). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), Folin-Ciocalteu’s phenol reagent, and gallic acid were obtained from Sigma-Aldrich Chemicals, Poznan, Poland. p-Nitrophenyl laurate was synthesized at our laboratory from lauroyl chloride (Sigma-Aldrich) and p-nitrophenol (POCH), according to a method described by Vogel et al. [12 ]. All reagents used were of analytical grade.
P nitrophenyl laurate
Manufactured by Merck Group
Sourced in United States
P-nitrophenyl laurate is a chemical compound used as a substrate in enzymatic assays. It is commonly employed in the measurement of lipase and esterase enzyme activity.
Automatically generated - may contain errors
Lab products found in correlation
P nitrophenyl palmitate, by Merck Group (6 mentions)
P nitrophenyl caprate, by Merck Group (5 mentions)
P nitrophenyl caproate, by Merck Group (4 mentions)
P nitrophenyl caprylate, by Merck Group (4 mentions)
P nitrophenyl myristate, by Merck Group (3 mentions)
P nitrophenyl butyrate, by Merck Group (3 mentions)
Gallic acid, by Merck Group (2 mentions)
Orlistat, by Merck Group (2 mentions)
α glucosidase, by Merck Group (2 mentions)
Acarbose, by Merck Group (2 mentions)
P nitrophenyl acetate, by Merck Group (2 mentions)
P nitrophenyl decanoate, by Merck Group (2 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Folin ciocalteu reagent, by Merck Group (2 mentions)
Methanol, by Merck Group (2 mentions)
Lauroyl chloride, by Merck Group (1 mentions)
Folin ciocalteu s phenol reagent, by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
Kod plus neo kit, by Toyobo (1 mentions)
P nitrophenyl butyrate c4, by Merck Group (1 mentions)
15 protocols using p nitrophenyl laurate
1
Coriander, Almonds, and Hazelnuts Characterization
2
Substrate Specificity Assay Protocol
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The artificial substrates with different carbon-chain length (C2-C16), including p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl caprylate (C8), p-nitrophenyl caprate (C10), p-nitrophenyl laurate (C12), p-nitrophenyl myristate (C14), and p-nitrophenyl palmitate (C16), were obtained from Sigma-Aldrich (St. Louis, MO, United States). Restriction enzymes were purchased from TaKaRa (Dalian, China). The KOD-Plus-Mutagenesis Kit and KOD-Plus-Neo Kit were purchased from Toyobo (Japan). Other reagents and chemicals used in this study were sourced from local vendors and were of analytical grade.
3
Cloning and Characterization of a Novel Cold-Adapted Esterase from Lysinibacillus sp.
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An open reading frame (ORF) (654 bp and 217 amino acids) from Lysinibacillus sp. encoding a novel cold-adapted esterase (EstRag) was artificially synthesized by GenScript Biotech ® CO., USA. pET-28b ( +) was used as the expression vector. Whilst E. coli BL21 (DE3) Rosetta (Promega Co., USA) was utilized as the cloning and expression host in this study. Lauria-Bertani (LB) broth was used for the activation and growing purposes of E. coli (BL21) DE3 Rosetta strain with an agitation speed of 180 rpm, at 37 °C for overnight. Substrates (Sigma-Aldrich Co., St Louis, USA) used for the enzyme assay were p-nitrophenyl acetate (p-NP-C2), p-nitrophenyl butyrate (p-NP-C4), p-nitrophenyl caproate (p-NP-C6), p-nitrophenyl caprylate (p-NP-C8), and p-nitrophenyl laurate (p-NP-C12). Imidazole was purchased from Loba Chemie PVT, Mumbai, India. Isopropyl-β- D-1-thiogalactopyranoside (IPTG), protein ladder, and kanamycin were purchased from Bioline, USA.
4
Phytochemical Analysis of S. orientalis Linne
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The S. orientalis linne plant materials were bought from Yuanshan Company (Kaohsiung City, Taiwan), and its nucleotide sequence was determined and deposited in the GenBank database with accession number JN987228 [69 (link)]. Acarbose, α-amylase (porcine pancreatic Type IV-B), angiotensin converting enzyme (ACE, rabbit lung), l -ascorbic acid, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT), captopril, (+)-catechin, 3,5-dinitrosalicylic acid (DNS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), DL-dithiothreitol (DTT), Folin-Ciocalteu reagent, gallic acid, Girard’s reagent T, glucose, α-glucosidase (Saccharomyces cerevisiae), glyoxal, Hippuryl-His-Leu acetate salt (HHL), lipase (porcine pancrease Type II), nitroblue tetrazolium (NBT), 4-nitrophenyl-α-d -glucopyranoside (PNPG), p-nitrophenyl laurate, orlistat, and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO, USA). Bovine serum albumin (BSA) was purchased from Fluka Biochemika (Buchs, Switzerland). All other chemicals were of reagent or analytical grade.
5
Immobilized Yarrowia Lipolytica Yeast Lipase
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Lyophilized powder of marine Yarrowia lipolytica yeast lipase from our laboratory was used. WDA918 (macroporous acrylic acid series weakly acidic cation exchange resin) was purchased from Anhui Wandong Chemical Co., Ltd., China. LX-1000EP (epoxy resin) and LX-1000EA (amino resin) were acquired from Xi'an Lanxiao Technology Co., Ltd., China. Glutaraldehyde AR (50% in H2O) was purchased from Aladdin Industrial Inc. Tris(hydroxymethyl)aminomethane (Tris) was from Amresco Co. P-Nitrophenyl laurate was purchased from Sigma-Aldrich. Ultrapure water generated through a UNIQUE-S15 facility was used throughout the experiments.
6
Lipase Activity of RmL with Propeptide
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Example 2
The hydrolytic activity of RmL with and without propeptide on various fatty acids was investigated in a kinetic assay using p-nitrophenyl acyl esters as substrate. The 100 mM stock solutions in DMSO of the substrates: p-Nitrophenyl butyrate (C3), p-Nitrophenyl caproate (C6), p-Nitrophenyl caprate (C10), p-Nitrophenyl laurate (C12) and p-Nitrophenyl palmitate (C16) (all from Sigma-Aldrich Danmark NS, Kirkebjerg Allé 84, 2605 Brondby; Cat. no.: C3:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) were diluted to a final concentration of 1 mM into assay buffer (50 mM Tris; pH 7.7; 0.4% TritonX-100).
RmL with and without propeptide and appropriate controls: Buffer (negative), Lipex™ (positive) RmL with and without propeptide in 50 mM Hepes pH8.0; 10 ppm TritonX-100; 20 mM CaCl2 were added to the substrate solution in the following final concentrations: 0.01 mg/mL; 5×10−3 mg/mL; 2.5×10−4 mg/mL; and 1.25×10−4 mg/mL in 96-well NUNC plates (Cat. No: 260836, Kamstrupvej 90, DK-4000, Roskilde). Release of p-nitrophenol by hydrolysis of p-nitrophenyl acyl was monitored at 405 nm for 5 minutes in 10 second intervals on a Spectra max 190 (Molecular Devices GmbH, Bismarckring 39, 88400 Biberach an der Riss, GERMANY).
RmL showed activity in all conditions tested with a maximal activity at C10. RmL with the propeptide bound showed no activity at any of the conditions tested.
7
Microbial Enzyme Characterization Protocol
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Microbulbifer thermotolerans DAU221 was deposited in the Korean Culture Center of Microorganisms (KCCM 43021, 16S rDNA sequence GenBank ID KC571186 ) and cultured in Marine Broth 2216 (Difco, Detroit, MI, USA). Plasmids pUC118 and pCC1FOS (Epicentre, Madison, USA) were used to construct the genomic library, and pCold I (TaKaRa, Kyoto, Japan) was used as the protein expression vector. Escherichia coli (E. coli) JM109 and EPI300-T1 were used for cloning, and BL21 (DE3) was used for protein expression. E. coli strains were grown at 37°C in Luria-Bertani (LB) broth supplemented with ampicillin (50 μg mL−1) or chloramphenicol (12.5 μg mL−1) when required. Tributyrin, p-nitrophenyl acetate (C2), p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl caprylate (C8), p-nitrophenyl caprate (C10), p-nitrophenyl laurate (C12), p-nitrophenyl myristate (C14), p-nitrophenyl palmitate (C16), and p-nitrophenyl stearate (C18) were purchased from Sigma-Aldrich (USA).
8
Lipase Hydrolytic Specificity Assay
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The lipase hydrolytic specificities were assayed with the chromogenic p-Nitrophenyl ester substrates: p-Nitrophenyl caprylate (C8), p-Nitrophenyl decanoate (C10), p-Nitrophenyl laurate (C12), and p-Nitrophenyl palmitate (C16) (Sigma-Aldrich Co, St Louis, MO, USA). The reaction mixtures contained 1 mL of the cold buffer (20 mM Tris–HCl, pH 7.4, 0.25 M sucrose) and 100 μL substrate (3 mM p-Nitrophenyl ester in 2-propanol). The reactions were started by the addition of the substrate at 37 °C, followed by an incubation at 37 °C for 30 min. The p-Nitrophenyl that was released was monitored at 410 nm. A standard curve of p-Nitrophenyl-esters was used to calculate the activity.
9
Inhibitory Effects of Natural Compounds
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Methanol, acetonitrile, formic acid, and Folin–Ciocalteu reagent were purchased from Merck (Darmstadt, Germany). Pancreatic lipase (from porcine pancreas, 163 U/mg, EC: 3.1.1.3), α-glucosidase from Saccharomyces cerevisiae (Type I, ≥ 10 units/mg protein), p-nitrophenyl–α-D-glucopyranoside (pNPG, purity ≥ 99.0%), acarbose (95%), orlistat (purity ≥ 97.0%), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), p-nitrophenyl laurate (purity ≥ 98.0%), and 2′,7′-Dichlorofluorescin diacetate (DCFH-DA, purity ≥ 97.0% were purchased from Sigma (Sigma-Aldrich, Shanghai, China). Rutin (purity ≥ 98.0%), isorhamnetin-3-O-Rutinoside (also known as Narcissoside, purity ≥ 98.0%), and quercetin-3-O-glucoside (purity ≥ 98.0%) were obtained from Chengdu Must Bio-technology Co., Ltd. (Chengdu, China). All of the other reagents that were used were of analytical grade.
10
Enzymatic Assay for Para-nitrophenyl Esters
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Para-nitrophenyl esters (C2-C16), p-nitrophenyl acetate (pNPC2), p-nitrophenyl butyrate (pNPC4), p-nitrophenyl caprylate (pNPC8), p-nitrophenyl caprate (pNPC10), p-nitrophenyl laurate (pNPC12), p-nitrophenyl myristate (pNPC14), p-nitrophenyl palmitate (pNPC16), isopropyl-β-D-thiogalactopyranoside (IPTG), chloramphenicol, kanamycin, and all other reagents were obtained from Sigma (Hampshire) and New England Biolab, Hertfordshire, (UK) and were of analytical grade.
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