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.
P nitrophenyl caprate
Manufactured by Merck Group
Sourced in Sao Tome and Principe, United States, United Kingdom
P-nitrophenyl caprate is a chemical compound used as a substrate in various biochemical and analytical applications. It is a colorless to pale yellow crystalline solid. The compound is often used in enzymatic assays, specifically for the detection and quantification of esterase or lipase activity.
Automatically generated - may contain errors
Lab products found in correlation
P nitrophenyl palmitate, by Merck Group (5 mentions)
P nitrophenyl laurate, by Merck Group (5 mentions)
P nitrophenyl caproate, by Merck Group (4 mentions)
P nitrophenyl caprylate, by Merck Group (3 mentions)
P nitrophenyl myristate, by Merck Group (3 mentions)
P nitrophenyl butyrate, by Merck Group (3 mentions)
P nitrophenyl acetate, by Merck Group (2 mentions)
Kod plus neo kit, by Toyobo (1 mentions)
P nitrophenyl butyrate c4, by Merck Group (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
Tributyrin, by Merck Group (1 mentions)
Pcold 1, by Takara Bio (1 mentions)
Pcc1fos, by Illumina (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Isopropyl β d thiogalactopyranoside, by Merck Group (1 mentions)
5 protocols using p nitrophenyl caprate
1
Substrate Specificity Assay Protocol
2
Lipase Activity of RmL with Propeptide
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
3
Microbial Enzyme Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
4
Enzymatic Assay for Para-nitrophenyl Esters
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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.
5
Kinetic Assay for Lipase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 1
The hydrolytic activity of a lipase may be determined by a kinetic assay using p-nitrophenyl acyl esters as substrate.
A 100 mM stock solution in DMSO for each of the substrates p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl caprate (C10), p-nitrophenyl laurate (C12) and p-nitrophenyl palmitate (C16) (all from Sigma-Aldrich Danmark A/S, Kirkebjerg Allé 84, 2605 Brøndby; Cat. no.: C3:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) is diluted to a final concentration of 1 mM 25 mM in the assay buffer (50 mM Tris; pH 7.7; 0.4% Triton X-100).
The lipase variants, the parent lipase and appropriate controls, e.g., Lipolase™ (SEQ ID NO: 2) in 50 mM Hepes; pH 8.0; 10 ppm Triton X-100; +/−20 mM CaCl2) are added to the substrate solution in the following final protein concentrations: 0.01 mg/ml; 5×10−3 mg/ml; 2.5×104 mg/ml; and 1.25×104 mg/ml in 96-well NUNC plates (Cat. No. 260836, Kamstrupvej 90, DK-4000, Roskilde). The buffer is also run as a negative control. Release of p-nitrophenol by hydrolysis of a p-nitrophenyl acyl may be 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). The hydrolytic activity towards one or more substrates of a variant may be compared to that of the parent lipase.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!