Asymmetric synthesis of cis-3,5-diacetoxycyclopent-1-ene using metagenome-derived hydrolases
Graphical abstract
Introduction
The asymmetric hydrolysis of cis-3,5-diacetoxycyclopent-1-ene 1 to the enantiomerically pure monoacetates 1a or 1b is an important step in the synthesis of optically active cyclopentanoids (e.g., prostaglandins, prostacyclins, thromboxanes), and enzymatic reactions have been shown to allow access to both enantiomers.1, 2, 3, 4 However, there is a constant need to identify new biocatalysts offering superior properties with respect to enantiopreference, stereoselectivity, activity, and stability.
A very rich source of novel enzymes is the so-called ‘metagenome’, that is, the collective genomes of ideally all microorganisms in a given habitat.5 Modern molecular biology methods allow the isolation and cloning of this environmental DNA without the need of cultivation of the corresponding organisms.6, 7, 8, 9, 10, 11 Sophisticated screening approaches have been established to allow access to the enormous resource of microbial biocatalysts.12 Many new and diverse enzymes have already been isolated from metagenomic libraries13 to evaluate their potential as industrial biocatalysts, as they needed to be characterized with respect to process relevant parameters, such as activity, stability, and enantioselectivity.
Herein, we report the screening and characterization of novel metagenome-derived hydrolases for their activity and selectivity in the asymmetric hydrolysis of cis-3,5-diacetoxycyclopent-1-ene 1 (Scheme 1).
Section snippets
Results and discussion
For the identification of new and selective enzymes, 83 esterases from the metagenome were investigated. In addition, a commercial preparation of pig liver esterase (Fluka-PLE) and the recombinant γ-rPLE14 served as benchmark enzymes.
First, active enzymes were identified by a microtiterplate-based screening using a pH-indicator assay,15, 16 with bromothymol blue. The 35 metagenome-derived enzymes and the PLEs showed activity and were then subjected to analytical scale reactions (10 mM
Conclusions
Herein, we have shown that enzymes derived from environmental DNA exhibit excellent and complementary enantiopreference in the asymmetric hydrolysis of cis-3,5-diacetoxycyclopent-1-ene and even surpass the performance of pig liver esterase, which was reported3 to give only 80.3% ee.
General
All chemicals were purchased from Fluka (Buchs, Switzerland) at the highest purity available. All metagenomic esterases were produced by B.R.A.I.N. AG and used as glycerol-stabilized crude cell extracts. Plasmids containing the genes for the esterases A3,11 CE11 CL1, and CL2 were obtained from K.-E. Jaeger (Juelich, Germany) and Professor W. Streit (Hamburg, Germany). The commercial PLE preparation was purchased from Fluka (Buchs, Switzerland) and the recombinant γ-PLE was produced as described.
Acknowledgments
We thank the Deutsche Bundesstiftung Umwelt (Osnabrück, Germany) for financial support (AZ13141). We are especially grateful to Professor K.-E. Jaeger and Professor W. Streit for supplying four esterases.
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