Enzymatic activity assay of d-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

2006 ◽  
Vol 67 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Montserrat Andújar-Sánchez ◽  
Francisco Javier Las Heras-Vázquez ◽  
Josefa María Clemente-Jiménez ◽  
Sergio Martínez-Rodríguez ◽  
Ana Camara-Artigas ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Isothermal Titration Calorimetry ◽  
Activation Parameters ◽  
Titration Calorimetry ◽  
Activity Assay ◽  
Thermodynamic Activation Parameters ◽  
Hydrolysis Of ◽  
Enzymatic Activity Assay

Simple and Rapid Determination of Phytase Activity

1994 ◽  
Vol 77 (3) ◽  
pp. 760-764 ◽  
Author(s):  
Adrianus J Engelen ◽  
Fred C Van Der Heeft ◽  
Peter H G Randsdorp ◽  
Ed L C Smtt
Keyword(s):  
Enzymatic Activity ◽  
Rapid Method ◽  
Rapid Determination ◽  
Phytase Activity ◽  
Sodium Phytate ◽  
Microbial Phytase ◽  
Hydrolysis Of

Abstract A simple and rapid method is described for determining the enzymatic activity of microbial phytase. The method is based on the determination of inorganic orthophosphate released on hydrolysis of sodium phytate at pH 5.5.

scholarly journals Identification of Small Molecule Inhibitors of the Deubiquitinating Activity of the SARS-CoV-2 Papain-Like Protease: in silico Molecular Docking Studies and in vitro Enzymatic Activity Assay

2020 ◽  
Vol 8 ◽  
Author(s):  
Eleni Pitsillou ◽  
Julia Liang ◽  
Katherine Ververis ◽  
Kah Wai Lim ◽  
Andrew Hung ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Enzymatic Activity ◽  
Small Molecules ◽  
Binding Site ◽  
Protein Docking ◽  
Activity Assay ◽  
Epicatechin Gallate ◽  
Inhibitor Binding ◽  
Enzymatic Activity Assay

COVID-19 is an ongoing pandemic caused by the SARS-CoV-2 virus with important political, socio-economic, and public health consequences. Inhibiting replication represents an important antiviral approach, and in this context two viral proteases, the SARS-CoV-2 main and papain-like proteases (PLpro), which cleave pp1a and pp1ab polypeptides, are critical. Along with protease activity, the PLpro possesses deubiquitinating activity, which is important in immune regulation. Naphthalene-based inhibitors, such as the well-investigated GRL-0617 compound, have been shown to possess dual effects, inhibiting both protease and deubiquitinating activity of the PLpro. Rather than binding to the canonical catalytic triad, these type of non-covalent inhibitors target an adjacent pocket, the naphthalene-inhibitor binding site. Using a high-throughput screen, we have previously identified the dietary hypericin, rutin, and cyanidin-3-O-glucoside compounds as potential protease inhibitors targeting the naphthalene-inhibitor binding site. Here, our aim was to investigate the binding characteristics of these compounds to the PLpro, and to evaluate deubiquitinating activity, by analyzing seven different PLpro crystal structures. Molecular docking highlighted the relatively high affinity of GRL-0617 and dietary compounds. In contrast binding of the small molecules was abolished in the presence of ubiquitin in the palm subdomain of the PLpro. Further, docking the small molecules in the naphthalene-inhibitor binding site, followed by protein-protein docking revealed displacement of ubiquitin in a conformation inconsistent with functional activity. Finally, the deubiquitinating activity was validated in vitro using an enzymatic activity assay. The findings indicated that the dietary compounds inhibited deubiquitinase activity in the micromolar range with an order of activity of GRL-0167, hypericin >> rutin, cyanidin-3-O-glucoside > epigallocatechin gallate, epicatechin gallate, and cefotaxime. Our findings are in accordance with mechanisms and potential antiviral effects of the naphthalene-based, GRL-0617 inhibitor, which is currently progressing in preclinical trials. Further, our findings indicate that in particular hypericin, rutin, and cyanidin-3-O-glucoside, represent suitable candidates for subsequent evaluation as PLpro inhibitors.

Viscometric Determination of p-Glucanase and Endoxylanase Activity in Feed

1996 ◽  
Vol 79 (5) ◽  
pp. 1019-1025 ◽  
Author(s):  
Adrianus J Engelen ◽  
Fred C Van Der Heeft ◽  
Peter H G Randsdorp
Keyword(s):  
Enzymatic Activity ◽  
Standard Addition ◽  
Glycosidic Bonds ◽  
Hydrolysis Of ◽  
Feed Samples

Abstract A method is described for viscometric determination of enzymatic activity of β-glucanase and endoxylanase in feed samples. The method is based on determination of the decrease in viscosity as a result of hydrolysis of glycosidic bonds in β-glucan and xylan at pH 3.5. This method does not require a blank sample (feed without enzyme addition), and it does not need standard addition for reliable quantitation.

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