Screening Systems

Abstract

Enzyme screening technology has undergone massive developments in recent years, particularly in the area of high-throughput screening and microarray methods. Screening consists of testing each sample of a sample library individually for the targeted reaction. This requires enzyme assays that accurately test relevant parameters of the reaction, such as catalytic turnover with a given substrate and selectivity parameters such as enantio- and regioselectivity. Enzyme assays also play an important role outside of enzyme screening, in particular for drug screening, medical diagnostics, and in the area of cellular and tissue imaging. In the 1990s, methods for high-throughput screening of enzyme activities were perceived as a critical bottleneck. As illustrated partly in this chapter, a large repertoire of efficient screening strategies are available today that allow testing of almost any reaction with high-throughput.

Isolation of monoclonal microcarriers colonized by fluorescent E. coli

“…However, as microcarriers containing more than one monoclonal cell are prone to lead to ambiguous results during analysis, for HT experimenting usually compartments containing exactly one cell are desired. Especially for approaches where positive cells or clones are later subjected to rather laborious secondary screening or analysis methods, (e.g., biopanning, protein/DNA extraction or sequencing) (2,6,(21)(22)(23)(24)(25)(26) larger number of false positives will result in costly and nonproductive secondary analyses, which in severe cases may offset the advantages of microcarrier-based approaches.…”

A practical high-throughput screening system for feruloyl esterases: Substrate design and evaluation

“…A microplate screening based on the pH indicator, 4-nitrophenol, has also been reported [16]. However, the establishing and evaluation of a reliable and practical high-throughput screening system is still required, which would otherwise facilitates the directed evolution of FAEs, as well as the novel enzyme mining from environmental microorganisms [17,18].…”

Rapid Methods for High-Throughput Detection of Sulfoxides

“…As a consequence, there is a continuous search for novel or improved biocatalysts. In order to find an appropriate candidate for a process, various sources of enzymes must be screened for activity (23). Therefore, a sensitive, reproducible, accurate, and simple highthroughput screening method is a key prerequisite for the development of biocatalytic processes on an industrial scale (32,39).…”

“…The first class contains assays applicable to testing growing or resting microbial colonies for enzymatic activity directly on agar plates (23), for example, detection of epoxide hydrolase activity on butane oxide by use of safranin O. Oxidation of the 1,2-diol product by Escherichia coli modified the membrane potential and led to accumulation of the red dye in the colonies producing active enzyme (34). In another study, the spontaneous oxidation of substituted catechols to brown-red quinones was used to screen random libraries of whole cells expressing toluene monooxygenases (TMOs) for regioselective oxidation of substituted phenols (12,30).…”

“…The positive clones produced a red halo around the cells. These assays are high-throughput, simple procedures but often require a tailored substrate with a chromophore, such as bromonaphthol or azo-dye (23).…”