Except for exploring sequenced genomes, yet other interesting approaches for discovering novel BVMOs exist. A recent development in the area of biocatalyst discovery is the exploitation of metagenomic DNA libraries. By this methodology, DNA is directly isolated from a certain sample that is expected to contain a variety of microbes (e.g., soil). Subsequently, the obtained DNA is randomly fragmented and cloned into a suitable vector and expression host. The resulting gene library, containing a huge number of randomly cloned genome fragments, can subsequently be screened for any desired biocatalytic activity. In this way, bacterial enzymes exhibiting specific activities can be found without the need of isolating or cultivating a specific bacterium. This culture-independent methodology has been shown to be very successful to uncover novel biocatalysts and has quickly become a standard approach at academic and industrial institutions. However, no BVMO discovered by metagenome screening has yet been reported. As shown above, BVMOs occur frequently in bacterial genomes and therefore metagenomic gene libraries should typically contain a multitude of BVMOs. However, it is not trivial to screen libraries for specific enzyme activities. Often screening methods rely on visualization of enzyme activity by color formation upon substrate conversion or growth that is triggered by the specifically desired enzyme activity. Several assays that can be used for screening cells for BVMO activity have been reported but none of them appears to be suited for generic screening of a massive number of clones.48-50 The most commonly used assay to detect BVMO activity in vitro is measuring the depletion of NADPH which absorbs light at 340 nm. This approach cannot be used when handling whole cells. Recently, several alternative methods have been reported to detect cells exhibiting BVMO activity. In one report, it was shown that the BVMO-associated ester/lactone product formation can be coupled to an esterase/lactonase which will result in a drop of pH. By using pH indicators, this change of pH could be visualized.51 A disadvantage of this approach is that it is limited by the substrate range of the hydrolytic partner enzyme and very sensitive to other activities that influence the pH. Another reported method for detecting BVMO activity is also dependent on hydrolysis of the formed product. It was shown that HAPMO readily catalyzes conversion of 3-acetylindole to indoxyl acetate. Chemical or enzymatic hydrolysis of this product yields indoxyl which spontaneously reacts with molecular oxygen forming the blue-colored compound, indigo (Fig. 5).52
Recently, two fluorogenic assays were also described that can be used for detecting BVMO activity.4849 However, these methods are biased toward specific chromogenic and fluorogenic substrates and will only yield enzymes that are active toward these and similar compounds.
An effective and generic method of screening of BVMO activity is indispensable for screening metagenome libraries as these libraries are often in the range of 100 000-1 000 000 clones. Therefore, it is a challenge to develop a novel way to efficiently detect BVMO activity in gene libraries. This would facilitate
Figure 5: Chromogenic BVMO assay using 3-acetyl indole resulting in the formation of indigo blue.52
discovery of a new set of BVMOs displaying novel biocatalytic properties and may also disclose new types of BVMOs.
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