Nanoscale Enzyme Screening Technologies

2016 ◽  
pp. 769-800
Keyword(s):  
Enzyme Screening

Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

2019 ◽  
Author(s):  
Anja Knorrscheidt ◽  
Pascal Püllmann ◽  
Eugen Schell ◽  
Dominik Homann ◽  
Erik Freier ◽  
...  
Keyword(s):  
Microtiter Plate ◽  
Axial Ligand ◽  
Cell System ◽  
Enzymatic Reactions ◽  
The Novel ◽  
Internal Standards ◽  
E Coli ◽  
Enzyme Screening ◽  
Plate Analysis ◽  
Microtiter Plate Analysis

Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

scholarly journals Metagenomics, gene discovery and the ideal biocatalyst

2004 ◽  
Vol 32 (2) ◽  
pp. 298-302 ◽  
Author(s):  
D.A. Cowan ◽  
A. Arslanoglu ◽  
S.G. Burton ◽  
G.C. Baker ◽  
R.A. Cameron ◽  
...  
Keyword(s):  
New Technologies ◽  
Rapid Development ◽  
Gene Discovery ◽  
Metagenomic Library ◽  
Metagenomic Sequencing ◽  
Optimum Conditions ◽  
Enzyme Screening ◽  
Specific Amplification ◽  
The Ideal

With the rapid development of powerful protein evolution and enzyme-screening technologies, there is a growing belief that optimum conditions for biotransformation processes can be established without the constraints of the properties of the biocatalyst. These technologies can then be applied to find the ‘ideal biocatalyst’ for the process. In identifying the ideal biocatalyst, the processes of gene discovery and enzyme evolution play major roles. However, in order to expand the pool genes for in vitro evolution, new technologies, which circumvent the limitations of microbial culturability, must be applied. These technologies, which currently include metagenomic library screening, gene-specific amplification methods and even full metagenomic sequencing, provide access to a volume of ‘sequence space’ that is not addressed by traditional screening.

Improved enzyme screening by automated fast protein liquid chromatography

1985 ◽  
Vol 151 (2) ◽  
pp. 547-553 ◽  
Author(s):  
Horst Schütte ◽  
Werner Hummel ◽  
Maria-Regina Kula
Keyword(s):  
Liquid Chromatography ◽  
Fast Protein Liquid Chromatography ◽  
Enzyme Screening

scholarly journals 1724. Plasmid-free CRISPR-Cas9 System for Genetic Engineering of Rhizopus delemar

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S632-S632
Author(s):  
Yiyou Gu ◽  
Clara Baldin ◽  
Teklegiorgis Gebremariam ◽  
Abdullah Alqarihi ◽  
Zeinab Mamouei ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Etiologic Agent ◽  
Pcr Analysis ◽  
Free System ◽  
Rhizopus Delemar ◽  
Additional Tool ◽  
Qrt Pcr ◽  
Enzyme Screening ◽  
Serious Infection ◽  
Short Palindromic Repeat

Abstract Background Mucormycosis is a serious infection caused by fungi of the order Mucorales. Rhizopus delemar is the most common etiologic agent of mucormycosis. Pathogenesis studies of mucormycosis have been hampered by poor genetic trackability of the organism, owing to rare chromosomal integration events and multinucleated nature of the cells. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system has been widely used in genetic manipulation through efficient homologous and non-homologous break points in a variety of organisms including R. delemar. However, plasmid-free CRISPR/Cas9 system has not been previously described in the fungus. Here, we introduce a rapid plasmid-free system for inducing orotidine 5’-phosphate decarboxylase (pyrF) gene mutation in R. delemar. Methods Protoplasts of R. delemar 99–880 strain were transformed with 20 nucleotide gRNA targeting the N-terminus of pyrF gene and the Cas9 enzyme. Screening for pyrF auxotrophy was carried out by plating transformed protoplasts on potato dextrose agar (PDA) plates containing 1 mg/mL 5-fluoroorotic acid (5-FOA) and 100 µg/mL uracil. Putative mutant strains were selected for uracil auxotrophy by plating simultaneously on media with or without uracil. pyrF disruption was verified by using PCR and qRT–PCR. Results Approximately100 transformants were generated through plating on 5-FOA plates. Only three transformants did not grow on minimal medium lacking uracil, indicating that they were true pyrF null mutants. PCR analysis showed that these three transformants have undergone nucleotide deletion events within the pyrF gene. The lack of pyrF gene expression was further verified by using qRT–PCR relative to wild-type R. delemar 99–880. Conclusion Similar to the plasmid-based genome manipulation strategy, the plasmid-free CRISPR/Cas9 system can induce gene editing in R. delemar. This rapid and simple approach adds an additional tool in our conquest to understand pathogenesis of mucormycosis. Disclosures All authors: No reported disclosures.

Self-immolative versatile fluorogenic probes for screening of hydrolytic enzyme activity

2016 ◽  
Vol 14 (38) ◽  
pp. 9146-9150 ◽  
Author(s):  
Anna Żądło-Dobrowolska ◽  
Martyna Szczygieł ◽  
Dominik Koszelewski ◽  
Daniel Paprocki ◽  
Ryszard Ostaszewski
Keyword(s):  
Enzyme Activity ◽  
Hydrolytic Enzyme ◽  
Cascade Reaction ◽  
Enzymatic Cleavage ◽  
Enzyme Screening ◽  
Fluorogenic Probes

Self-immolative probes for rapid and sensitive hydrolase detection are reported. This system allows hydrolytic enzyme screening through a cascade reaction triggered by enzymatic cleavage.

scholarly journals Innovative microscale workflow from fungi cultures to Cell Wall‐Degrading Enzyme screening

2019 ◽  
Vol 12 (6) ◽  
pp. 1286-1292 ◽  
Author(s):  
Roxane Raulo ◽  
Egon Heuson ◽  
Ali Siah ◽  
Vincent Phalip ◽  
Renato Froidevaux
Keyword(s):  
Cell Wall ◽  
Degrading Enzyme ◽  
Cell Wall Degrading Enzyme ◽  
Enzyme Screening

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.

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