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This cationic form reacts with the free fatty acids forming the colored compounds. In the following protocol, agar plates are pink. Clones expressing lipases are detected due to the observation of an orange fluorescent halo surrounding the colonies after UV light irradiation (350 nm). 1. 4). library into SCM2 agar plates 2. Incubate the plates for 16 h at 37 C. 3. Place the plates under UV light (350 nm) and positive clones will appear as colonies surrounded by an orange fluorescent halo. A recent successful example of the application of such protocol is described in Zheng et al.

Place the plates under UV light (350 nm) and positive clones will appear as colonies surrounded by an orange fluorescent halo. A recent successful example of the application of such protocol is described in Zheng et al. [52], where six positive clones were identified by screening 20,000 clones. 15 Functional Screening for General Lipase Activity (Toward Brilliant Green and Olive Oil) The following protocol is based on the method described by Svendsen and collaborators [53], modified in order to be applied over colonies growth in agar plates.

Table 1 Screening assays [33] [44] Non-competitive selectivity, specific substrate required [42, 43]a [41] [37] [36] Ref. Evaluation of the screening results with real substrates necessary Non-competitive selectivity, artificial substrate Non-competitive selectivity, moderate sensitivity, artificial substrate No enantioselectivity, preference for extracellular hydrolases No enantioselectivity, preference for extracellular hydrolases Weaknesses 42 Thomas Classen et al. Autosampler, 10-700/day 10b Liquid HPLC and GC assay, liquid and violable esters HPLC, GC Mass spectrometry NMR spectroscopy Fluorometry Precise quantification, high sensitivity, analysis of multiple products, competitive assays Precise quantification, high sensitivity, analysis of multiple products, competitive assays Precise quantification, analysis of multiple products, competitive assays Ultra-high throughput, high sensitivity, competitive assay b The basis has been set by achiral substrates; the enantioselectivity assay is described in this book chapter These methods are not described in detail in this chapter a Autosampler, 1-10 k/day Liquid assay with deuterium-labelled pseudo-enantiomers, any ester 9b Autosampler, 100-10 k/day Liquid assay with isotope-labelled pseudo-enantiomers, any ester 8b Cell sorter, >1,000 k/day In vivo covalent attachment of products via enzyme display, biotin-tyramide ester, streptavidin and R-phycoerythrin 7b [35] Low throughput, reference compounds needed Degree of abstraction of the pseudoenantiomer (see Fig.