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β-Alanine (3-aminopropionic acid) holds great potential in industrial application. It can be obtained through a chemical synthesis route, which is hazardous to the environment. It is well known that l-aspartate-α-decarboxylase (ADC) can convert l-aspartate to β-alanine in bacteria. However, due to the low activity of ADC, industrial production of β-alanine through the green biological route remains unclear. Thus, improving the activity of ADC is critical to reduce the cost of β-alanine production. In this study, we established a dual-fluorescence high-throughput system for efficient ADC screening. By measuring the amount of β-alanine and the expression level of ADC using two different fluorescence markers, we can rapidly quantify the relative activity of ADC variants. From a mutagenesis library containing 2000 ADC variants, we obtained a mutant with 33% increased activity. Further analysis revealed that mutations of K43R and P103Q in ADC significantly improved the yield of β-alanine produced by the whole-cell biocatalysis. Compared with the previous single-fluorescence method, our system can not only quantify the amount of β-alanine but also measure the expression level of ADC with different fluorescence, making it able to effectively screen out ADC variants with improved relative activity. The dual-fluorescence high-throughput system for rapid screening of ADC provides a good strategy for industrial production of β-alanine via the biological conversion route in the future.

Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of nitrilases. Here, we review the various methodologies developed for high-throughput screening of nitrilases and focus on their advantages and limitations.

Low aqueous solubility of active pharmaceutical ingredients (APIs) is one of the most important challenges facing drug development researchers today [1, 2]. With the development of computational chemistry and high throughput screening methods it is possible to obtain a large number of compounds with attractive therapeutic activity. However, at the same time the selection of novel active molecules with suitable biopharmaceutical properties (like solubility, intestinal permeability) becomes a great challenge and a bottleneck in drug development. Statistically, more than 40% of approved drugs and even 70–90% of those under investigations are poorly water-soluble and additional efforts are required to improve their water solubility [3–5].

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.

Natural products or their related derivatives were the most important source of officially approved drugs owning to the huge chemical structure diversities and their biodiversities Compared with traditional combinatorial chemistries and high throughput screening methods genomics driven natural product discovery is a rational and an efficient alternative for the discovery of novel structures In this mini review process of genomics driven natural product discovery is highlighted including the main steps the relevant technologies and bioinformatics tools

Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.

Background: The search for new natural or synthetic products with antioxidant activity is commonly based on methods that involve reduction of either 2,2-diphenyl-1-picrylhydrazyl (DPPH) or 2-2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). However, the reported values of the effective concentrations are highly variable, even in controls. Herein, we optimize and validate both meth-ods of determining antiradical activity. Methods: Optimization was carried out using both a fractionated factorial design and a basic sequential simplex method, by monitoring the reduction percentage. Quercetin or Trolox were used as positive con-trol. Furthermore, for each method, linearity, precision, accuracy, robustness, plate uniformity, signal variability, and Z factor, were established. Results: The optimized conditions for the DPPH method were: DPPH 280 μM in ethanol and 15 min of reaction time in the dark. The linear range was between 7 and 140 μM with an R2 value of 0.9987. The optimized conditions for the ABTS method were: ABTS adjusted to 0.7 absorbance units, 70% concen-tration in ethanol, and a reaction time of 6 min in the dark. The linear range was found to be between 1 and 70% with an R2 = 0.9991. For both methods, the accuracy and precision were within limits and the Z factor value was higher than 0.89. The applicability of each method was assessed by analyzing eight plant extracts. Conclusion: The DPPH and ABTS reduction methods were optimized and validated on a microscale and could be expected to be implemented in any laboratory.

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography, and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation, and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs.

Proteases constitute one of the most important groups of industrial enzymes with a worldwide value expected to reach 2.7 billion US dollars by 2019. Proteases represent a group of enzymes that hydrolyze the peptide bonds of proteins, releasing polypeptides or free amino acids. These enzymes are used in cleaning products, production of leathers, textiles, food and dairy products, in the pharmaceutical and diagnostic industries and for water treatment. Another area of interest regarding proteases is the development of drugs that act as protease inhibitors. This review will briefly describe the general methods used in the detection of proteases and the few studies in the development of high throughput screening methods of proteases and protease inhibitors.

In situ and operando techniques can play important roles in the development of better performing photoelectrodes, photocatalysts, and electrocatalysts by helping to elucidate crucial intermediates and mechanistic steps. The development of high throughput screening methods has also accelerated the evaluation of relevant photoelectrochemical and electrochemical properties for new solar fuel materials. In this chapter, several in situ and high throughput characterization tools are discussed in detail along with their impact on our understanding of solar fuel materials.

One approach to accelerate the discovery process of experimentally determined products is through the use of high-throughput screening (HTS) methods. This paper presents a method for the r...

The ability to image specific molecular targets in vivo would have significant impact in allowing earlier disease detection and in tailoring molecular therapies. One of the rate-limiting steps in the development of novel compounds as reporter probes has been the lack of cell-based, biologically relevant, high throughput screening methods. Here we describe the development and validation of magnetic resonance imaging (MRI) as a technique to rapidly screen compounds that are potential MR reporter agents for their interaction with specific cellular targets. We show that MR imaging can (1) evaluate thousands of samples simultaneously and rapidly, (2) provide exceedingly accurate measurements, and (3) provide receptor binding/internalization data as validated by radioactive assays. The technique allows the screening of libraries of peptide-nanoparticle conjugates against target cells and the identification of conjugates that may be subsequently used as reporter agents in vivo. The technology should greatly accelerate the development of target-specific or cell-specific MR contrast agents.

Reliable and effective virtual high-throughput screening (vHTS) methods are desperately needed to minimize the expenses involved in drug discovery projects. Here, we present an improvement to the negative image-based (NIB) screening: the shape, the electrostatics, and the solvation state of the target protein’s ligand-binding site are included into the vHTS. Additionally, the initial vHTS results are postprocessed with molecular mechanics/generalized Born surface area (MMGBSA) calculations to estimate the favorability of ligand-protein interactions. The results show that docking produces very good early enrichment for phosphodiesterase-5 (PDE-5); however, in general, the NIB and the ligand-based screening performed better with or without the added electrostatics. Furthermore, the postprocessing of the NIB screening results using MMGBSA calculations improved the early enrichment for the PDE-5 considerably, thus, making hit discovery affordable.

The nonmevalonate isoprenoid pathway is an established target for antiinfective drug development. This paper describes high-throughput methods for the screening of 2C-methyl-D-erythritol synthase (IspC protein), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD protein), 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE protein), and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF protein) against large compound libraries. The assays use up to three auxiliary enzymes. They are all monitored photometrically at 340 nm and are robust as documented by Z-factors of >or=0.86. 13C NMR assays designed for hit verification via direct detection of the primary reaction product are also described. Enzyme-assisted methods for the preparation, on a multigram scale, of isoprenoid biosynthesis intermediates required as substrates for these assays are reported. Notably, these methods enable the introduction of single or multiple 13C labels as required for NMR-monitored assays. The preparation of 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate in multigram quantities is described for the first time.

Publisher Summary This chapter discusses the physical separation of enantiopure compound. The importance and value of chiral molecules to the drug discovery industry has provided the impetus for much research in asymmetric organic synthesis. While many drug molecules are chiral and most of these have the desired function in only one enantiomer, the synthesis of large and diverse libraries of enantiopure compounds is not practical. Accordingly, initial screens for drug-like properties are almost always done on racemates. Enantiopure structures of interest are then identified after rounds of winnowing, and the challenge of asymmetric synthesis is directed at particular targets of high value. The physical separation of enantiomers, either by classical resolution techniques or by chromatography over chiral stationary phases, is often a preferred method of preparation of enantiopure molecules. The chapter surveys techniques that are being developed for the rapid measurement of enantiomeric excess of chiral nonracemic organic compounds, and thus serves as an introduction to the later chapters in this volume.