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 Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

2020 ◽  
Author(s):  
C.J. Markin ◽  
D.A. Mokhtari ◽  
F. Sunden ◽  
M.J. Appel ◽  
E. Akiva ◽  
...  
Keyword(s):  
High Throughput ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Extensive Investigation ◽  
Purification And Characterization ◽  
Functional Architecture ◽  
Microfluidic Platform ◽  
Multiple Substrates ◽  
Catalytic Effects

AbstractSystematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK, a microfluidic platform for high-throughput expression, purification, and characterization of >1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA, we performed >670,000 reactions to determine >5000 kinetic and physical constants for multiple substrates and inhibitors. These constants allowed us to uncover extensive kinetic partitioning to a misfolded state and isolate catalytic effects, revealing spatially contiguous “regions” of residues linked to particular aspects of function. These regions included active-site proximal residues but also extended to the enzyme surface, providing a map of underlying architecture that could not be derived from existing approaches. HT-MEK, using direct and coupled fluorescent assays, has future applications to a wide variety of problems ranging from understanding molecular mechanisms to medicine to engineering and design.One Sentence SummaryHT-MEK, a microfluidic platform for high-throughput, quantitative biochemistry, reveals enzyme architectures shaping function.

High-throughput expression, purification, and characterization of recombinant Caenorhabditis elegans proteins

2003 ◽  
Vol 307 (4) ◽  
pp. 928-934 ◽  
Author(s):  
Raymond Y Huang ◽  
Simon J Boulton ◽  
Marc Vidal ◽  
Steve C Almo ◽  
Anne R Bresnick ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Purification And Characterization

scholarly journals Characterization of Holstein and Normande whole milk miRNomes highlights breed specificities

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
S. Le Guillou ◽  
A. Leduc ◽  
J. Laubier ◽  
S. Barbey ◽  
M.-N. Rossignol ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Milk Production ◽  
High Throughput ◽  
Genetic Background ◽  
Molecular Mechanisms ◽  
High Throughput Sequencing ◽  
Whole Milk ◽  
Mammary Morphogenesis ◽  
Regulatory Properties

AbstractThe concept of milk as a healthy food has opened the way for studies on milk components, from nutrients to microRNAs, molecules with broad regulatory properties present in large quantities in milk. Characterization of these components has been performed in several species, such as humans and bovine, depending on the stages of lactation. Here, we have studied the variation in milk microRNA composition according to genetic background. Using high throughput sequencing, we have characterized and compared the milk miRNomes of Holstein and Normande cattle, dairy breeds with distinct milk production features, in order to highlight microRNAs that are essential for regulation of the lactation process. In Holstein and Normande milk, 2,038 and 2,030 microRNAs were identified, respectively, with 1,771 common microRNAs, of which 1,049 were annotated and 722 were predicted. The comparison of the milk miRNomes of two breeds allowed to highlight 182 microRNAs displaying significant differences in the abundance. They are involved in the regulation of lipid metabolism and mammary morphogenesis and development, which affects lactation. Our results provide new insights into the regulation of molecular mechanisms involved in milk production.

Purification and characterization of alkaline phosphatase from the gut of sea cucumber Stichopus japonicus

2013 ◽  
Vol 79 (3) ◽  
pp. 477-485 ◽  
Author(s):  
Hai-Tao Wu ◽  
Dong-Mei Li ◽  
Bei-Wei Zhu ◽  
Jing-Heng Cheng ◽  
Jin-Jian Sun ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Sea Cucumber ◽  
Purification And Characterization ◽  
Stichopus Japonicus
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