RESUMEN
Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.
Asunto(s)
Evolución Molecular Dirigida , Hidrolasas/genética , Oxidorreductasas/genética , Alcohol Deshidrogenasa/metabolismo , Técnicas Químicas Combinatorias/métodos , Hidrolasas/análisis , Hidrolasas/metabolismo , Lipasa/metabolismo , Oxidorreductasas/análisis , Oxidorreductasas/metabolismoRESUMEN
Enantiomerically pure amino acids, amino alcohols, amines, alcohols, and epoxides play an increasingly important role as intermediates in the pharmaceutical industry and agrochemistry, where both a high degree of purity and large quantities of the compounds are required. The chemical industry has primarily relied upon established chemical methods for the synthesis of these intermediates, but is now turning more and more to enzymatic and biotechnological fermentation processes. For the industrial implementation of many transformations alternative methods are available. The advantages of the individual methods will be discussed herein and exemplified by syntheses of relevant compounds.
RESUMEN
A rapid and reliable test for the determination of hydrolase activity and enantioselectivity comprises the conversion of acetic acid released from acetates to NADH by using a commercially available enzymatic test-kit. The NADH is spectrophotometrically quantified in a microtiter plate format.