scholarly journals Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution

2020 ◽  
Vol 21 (22) ◽  
pp. 8668
Author(s):  
Seung-Gyun Woo ◽  
Seong Keun Kim ◽  
Baek-Rock Oh ◽  
Seung-Goo Lee ◽  
Dae-Hee Lee
Keyword(s):  
High Throughput Screening ◽  
Bacteriophage T4 ◽  
T4 Lysozyme ◽  
Screening System ◽  
Random Mutation ◽  
Secondary Sludge ◽  
Enzyme Screening ◽  
Model Protein ◽  
Genetically Encoded Biosensor ◽  
Lysozyme Evolution

Lysozyme is widely used as a model protein in studies of structure–function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.

scholarly journals Evaluation of Feasibility of Using the Bacteriophage T4 Lysozyme to Improve the Hydrolysis and Biochemical Methane Potential of Secondary Sludge

Energies ◽  
2019 ◽  
Vol 12 (19) ◽  
pp. 3644
Author(s):  
Sangmin Kim ◽  
Seung-Gyun Woo ◽  
Joonyeob Lee ◽  
Dae-Hee Lee ◽  
Seokhwan Hwang
Keyword(s):  
Cell Wall ◽  
Bacterial Cell ◽  
Wastewater Treatment Plants ◽  
Biogas Production ◽  
Bacteriophage T4 ◽  
Suspended Solid ◽  
T4 Lysozyme ◽  
Biochemical Methane Potential ◽  
Secondary Sludge ◽  
Methane Potential

Anaerobic digestion (AD) of secondary sludge is a rate-limiting step due to the bacterial cell wall. In this study, experiments were performed to characterize secondary sludges from three wastewater treatment plants (WWTPs), and to investigate the feasibility of using bacteriophage lysozymes to speed up AD by accelerating the degradation of bacterial cell walls. Protein was the main organic material (67.7% of volatile solids in the sludge). The bacteriophage T4 lysozyme (T4L) was tested for hydrolysis and biochemical methane potential. Variations in the volatile suspended solid (VSS) concentration and biogas production were monitored. The VSS reduction efficiencies by hydrolysis using T4L for 72 h increased and ranged from 17.8% to 26.4%. Biogas production using T4L treated sludges increased and biogas production was increased by as much as 82.4%. Biogas production rate also increased, and the average reaction rate coefficient of first-order kinetics was 0.56 ± 0.02/d, which was up to 47.5% higher compared to the untreated samples at the maximum. Alphaproteobacteria, Betaproteobacteria, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia were major microbial classes in all sludges. The interpretation of the microbial community structure indicated that T4L treatment is likely to increase the rate of cell wall digestion.

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 Purification of Bacteriophage T4 Lysozyme

1968 ◽  
Vol 243 (2) ◽  
pp. 391-397 ◽  
Author(s):  
Akira Tsugita ◽  
Masayori Inouye ◽  
Eric Terzaghi ◽  
George Streisinger
Keyword(s):  
Bacteriophage T4 ◽  
T4 Lysozyme

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 Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery

2018 ◽  
Vol 23 (7) ◽  
pp. 697-707 ◽  
Author(s):  
John Joslin ◽  
James Gilligan ◽  
Paul Anderson ◽  
Catherine Garcia ◽  
Orzala Sharif ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening System ◽  
Preclinical Development ◽  
Drug Identification ◽  
Therapeutic Setting ◽  
Compound Libraries ◽  
Automated Platform

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

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