Kinetic study of site directed and randomly immobilized his‐tag alkaline phosphatase by flow injection chemiluminescence

Zhang, Juankun; Cass, Anthony E. G.

doi:10.1002/jmr.764

Cited by 4 publications

(2 citation statements)

References 16 publications

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“…Special flow-through-cells are made for continuous flow of solution into and out of the detector and many are commercially available for various detectors [10–15]. This enables flow injection analysis-based systems to couple with various types of detectors (e.g., electrochemical, mass spectrometry, chemiluminescence) [16–19] other than visible absorption spectrophotometers that are usually employed in enzyme kinetic studies.…”

Section: Flow-based Analysis Systemsmentioning

confidence: 99%

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

Hartwell

Grudpan

2012

Journal of Analytical Methods in Chemistry

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Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

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Section: Flow-based Analysis Systemsmentioning

confidence: 99%

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

Hartwell

Grudpan

2012

Journal of Analytical Methods in Chemistry

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“…20 Fundamental enzymology studies have a strong impact on medicine and healthcare. Many techniques, such as stopped-°ow system 21,22 and continuous-°ow system, 23,24 have been used to study enzymology at the bulk level.…”

Section: Introductionmentioning

confidence: 99%

A versatile platform for single-molecule enzymology of restriction endonuclease

Nie

Pan

et al. 2019

J. Innov. Opt. Health Sci.

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Enzymes are the major players for many biological processes. Fundamental studies of the enzymatic activity at the single-molecule level provides important information that is otherwise inaccessible at the ensemble level. Yet, these single-molecule experiments are technically difficult and generally require complicated experimental design. Here, we develop a Holliday junction (HJ)-based platform to study the activity of restriction endonucleases at the single-molecule level using single-molecule FRET (sm-FRET). We show that the intrinsic dynamics of HJ can be used as the reporter for both the enzyme-binding and the substrate-release events. Thanks to the multiple-arms structure of HJ, the fluorophore-labeled arms can be different from the surface anchoring arm and the substrate arm. Therefore, it is possible to independently change the substrate arm to study different enzymes with similar functions. Such a design is extremely useful for the systematic study of enzymes from the same family or enzymes bearing different pathologic mutations. Moreover, this method can be easily extended to study other types of DNA-binding enzymes without too much modification of the design. We anticipate it can find broad applications in single-molecule enzymology.

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Specifically Immobilised Aldo/Keto Reductase AKR1A1 Shows a Dramatic Increase in Activity Relative to the Randomly Immobilised Enzyme

Holland‐Nell¹,

Beck‐Sickinger²

2007

ChemBioChem

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The difference between site-specific and random immobilisation of the aldo/keto reductase AKR1A1 was explored. AKR1A1 was recombinantly expressed as a thioester by the intein strategy. The thioester was selectively modified with a biotin label by the expressed protein ligation method, and subsequent immobilisation on streptavidin templates was performed. Adsorption of wild-type AKR1A1 to streptavidin templates and of biotinylated AKR1A1 to uncoated templates was used to study randomly immobilised enzymes. Investigation of the kinetic parameters revealed remarkably improved activity for the site-specifically immobilised enzyme, which was comparable to that of the wild-type enzyme in solution and 60-300-fold greater than that of the randomly immobilized enzymes. Furthermore, the enzyme was surprisingly stable. No loss of activity was observed for over a week, and even after 50 days more than 35% of activity was maintained.

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Kinetic study of site directed and randomly immobilized his‐tag alkaline phosphatase by flow injection chemiluminescence

Cited by 4 publications

References 16 publications

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

A versatile platform for single-molecule enzymology of restriction endonuclease

Specifically Immobilised Aldo/Keto Reductase AKR1A1 Shows a Dramatic Increase in Activity Relative to the Randomly Immobilised Enzyme

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