Toward a Generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits

2013 ◽  
Vol 3 (3) ◽  
pp. 163-171 ◽  
Author(s):  
Su-Lim Choi ◽  
Eugene Rha ◽  
Sang Jun Lee ◽  
Haseong Kim ◽  
Kilkoang Kwon ◽  
...  
Keyword(s):  
High Throughput ◽  
Screening System ◽  
Genetic Circuits ◽  
Enzyme Screening

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

scholarly journals Creation of Novel Protein Transduction Domain (PTD) Mutants by a Phage Display-Based High-Throughput Screening System

2006 ◽  
Vol 29 (8) ◽  
pp. 1570-1574 ◽  
Author(s):  
Yohei Mukai ◽  
Toshiki Sugita ◽  
Tomoko Yamato ◽  
Natsue Yamanada ◽  
Hiroko Shibata ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Screening System ◽  
Novel Protein

scholarly journals A high-throughput cellulase screening system based on droplet microfluidics

2014 ◽  
Vol 8 (4) ◽  
pp. 041102 ◽  
Author(s):  
Raluca Ostafe ◽  
Radivoje Prodanovic ◽  
W. Lloyd Ung ◽  
David A. Weitz ◽  
Rainer Fischer
Keyword(s):  
High Throughput ◽  
Droplet Microfluidics ◽  
Screening System
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