scholarly journals Simultaneous screening of multiple substrates with an unspecific peroxygenase enabled modified alkane and alkene oxyfunctionalisations

2021 ◽  
Author(s):  
Anja Knorrscheidt ◽  
Jordi Soler ◽  
Nicole Hünecke ◽  
Pascal Püllmann ◽  
Marc Garcia-Borràs ◽  
...  
Keyword(s):  
Protein Engineering ◽  
High Throughput ◽  
Multiple Substrates ◽  
P Protein

Protein engineering of an unspecific peroxygenase (UPO) was performed with three substrates and six products in parallel by a high throughput GC-MS setup. Modified chemo- and regioselective variants were identified for aliphatic substrates.

scholarly journals Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Science ◽  
2021 ◽  
Vol 373 (6553) ◽  
pp. eabf8761
Author(s):  
C. J. Markin ◽  
D. A. Mokhtari ◽  
F. Sunden ◽  
M. J. Appel ◽  
E. Akiva ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Kinetics ◽  
High Throughput ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Extensive Investigation ◽  
Purification And Characterization ◽  
Multiple Substrates ◽  
Catalytic Effects

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

scholarly journals Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

ACS Omega ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. 1498-1508 ◽  
Author(s):  
Xue W. Diefenbach ◽  
Iman Farasat ◽  
Erik D. Guetschow ◽  
Christopher J. Welch ◽  
Robert T. Kennedy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Engineering ◽  
High Throughput ◽  
Droplet Microfluidics

scholarly journals Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

2021 ◽  
Author(s):  
Allwin McDonald ◽  
Peyton Higgins ◽  
Andrew Buller
Keyword(s):  
Protein Engineering ◽  
Enzyme Engineering ◽  
Enzyme Specificity ◽  
Potential Utility ◽  
Laboratory Equipment ◽  
Multiple Substrates ◽  
Rich Information ◽  
Substrate Promiscuity ◽  
Pharmacologically Active ◽  
Common Laboratory

Abstract Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded scope is a contemporary challenge. Measuring reaction outcomes on mixtures of substrates, called here SUbstrate Multiplexed Screening (SUMS), has long been used to rigorously quantitate enzyme specificity. Despite the potential utility of SUMS to guide engineering of promiscuous enzymes, this approach has not found widespread adoption in biocatalysis. Here, we develop principles of how to design robust SUMS methods that, rather than assess absolute specificity, use heuristic readouts of substrate promiscuity to identify hits for further investigation. This rich information enables engineering of activity for multiple substrates simultaneously and identifies enzyme variants with altered promiscuity, even when overall activity is lower. We demonstrate the effectiveness of SUMS by engineering two enzymes to produce pharmacologically active tryptamines from simple indole precursors in a biocatalytic cascade. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

scholarly journals High-Throughput Thermal Scanning: A General, Rapid Dye-Binding Thermal Shift Screen for Protein Engineering

2009 ◽  
Vol 131 (11) ◽  
pp. 3794-3795 ◽  
Author(s):  
Jason J. Lavinder ◽  
Sanjay B. Hari ◽  
Brandon J. Sullivan ◽  
Thomas J. Magliery
Keyword(s):  
Protein Engineering ◽  
High Throughput ◽  
Dye Binding ◽  
Thermal Shift
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