A colorimetric protease activity assay method using engineered procaspase-3 enzymes

2016 ◽  
Vol 8 (33) ◽  
pp. 6270-6276 ◽  
Author(s):  
Dokyung Yang ◽  
Hyeon Ji Park ◽  
Tae Hyeon Yoo
Keyword(s):  
Protease Activity ◽  
Assay Method ◽  
Activity Assay ◽  
Platform Technology ◽  
Protease Assay ◽  
Diagnosis Of Diseases

A protease assay platform technology based on engineered proenzymes has been developed for diagnosis of diseases.

Engineering β-lactamase zymogens for use in protease activity assays

2014 ◽  
Vol 50 (70) ◽  
pp. 10155-10157 ◽  
Author(s):  
Hajin Kim ◽  
Hyun Kyung Yoon ◽  
Tae Hyeon Yoo
Keyword(s):  
Protease Activity ◽  
Assay Method ◽  
Protease Assay

In this report, we engineered β-lactamase zymogens and developed a sensitive protease assay method based on the precursor enzymes.

Azocoll protease activity assay

2007 ◽  
Author(s):  
Naxin Jiang ◽  
Nguan Soon Tan ◽  
Bow Ho ◽  
Jeak Ling Ding
Keyword(s):  
Protease Activity ◽  
Activity Assay

scholarly journals A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

2021 ◽  
Author(s):  
Gang Liu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Direct Method ◽  
Total Concentration ◽  
Colorimetric Method ◽  
Industrial Applications ◽  
Assay Method ◽  
Activity Assay ◽  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity

Abstract Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.

Chronoamperometric Biosensor for Protease Activity Assay and Inhibitor Screening

2017 ◽  
Vol 29 (10) ◽  
pp. 2395-2400 ◽  
Author(s):  
Loubna El Harrad ◽  
Aziz Amine
Keyword(s):  
Protease Activity ◽  
Activity Assay ◽  
Inhibitor Screening

scholarly journals A new two-mode fluorescence signal amplification strategy for protease activity assay based on graphene oxide

RSC Advances ◽  
2017 ◽  
Vol 7 (76) ◽  
pp. 47983-47989 ◽  
Author(s):  
Fangfang Wang ◽  
Jie Gao ◽  
Jianwei Zhao ◽  
Wenyue Zhang ◽  
Jie Bai ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Signal Amplification ◽  
Protease Activity ◽  
Fluorescence Signal ◽  
Activity Assay ◽  
Fluorescence Signal Amplification ◽  
Amplification Strategy

A new graphene oxide-based two-mode fluorescence signal amplification strategy for the detection of protease activity has been established.

scholarly journals Improved Surface Display of Human Hyal1 and Identification of Testosterone Propionate and Chicoric Acid as New Inhibitors

2020 ◽  
Vol 13 (4) ◽  
pp. 54 ◽  
Author(s):  
Isabelle Lengers ◽  
Fabian Herrmann ◽  
Marc Le Borgne ◽  
Joachim Jose
Keyword(s):  
Enzyme Activity ◽  
Single Cell ◽  
Testosterone Propionate ◽  
Surface Display ◽  
Assay Method ◽  
Activity Assay ◽  
Enzyme Activity Assay ◽  
Chicoric Acid ◽  
Ic50 Value ◽  
Order Of Magnitude

Degradation of high molecular weight hyaluronic acid (HA) in humans is mainly catalyzed by hyaluronidase Hyal1. This enzyme is involved in many pathophysiological processes and therefore appears an interesting target for drug discovery. Until now, only a few inhibitors of human Hyal1 are known due to obstacles in obtaining active enzymes for inhibitor screening. The aim of the present work was to provide a convenient enzyme activity assay and show its feasibility by the identification of new inhibitors. By autodisplay, Escherichia coli F470 can present active Hyal1 on its surface. In this study, the inducible expression of Hyal1 on the cell surface of E. coli under the control of a rhamnose-dependent promoter (Prha) was performed and optimized. Enzyme activity per single cell was increased by a factor of 100 compared to the constitutive Hyal1 surface display, as described before. An activity of 6.8 × 10−4 mU per single cell was obtained under optimal reaction conditions. By this modified activity assay, two new inhibitors of human Hyal1 were identified. Chicoric acid, a natural compound belonging to the phenylpropanoids, showed an IC50 value of 171 µM. The steroid derivative testosterone propionate showed and IC50 value of 124 ± 1.1 µM. Both values were in the same order of magnitude as the IC50 value of glycyrrhizic acid (177 µM), one of the best known inhibitors of human Hyal1 known so far. In conclusion, we established a new enzyme activity assay for human Hyal1 and identified new inhibitors with this new assay method.

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