Enhancing the Enantioselectivity of an Epoxide Hydrolase by Directed Evolution

Reetz, Manfred T.; Torre, Claudia; Eipper, Andreas; Lohmer, Renate; Hermes, Marcus; Brunner, Birgit; Maichele, Andrea J.; Bocola, Marco; Arand, Michael; Cronin, Annette; Genzel, Yvonne; Archelas, A.; Furstoss, R.

doi:10.1021/ol035898m

Cited by 156 publications

(73 citation statements)

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“…The potential for biocatalytic application of epoxide hydrolases was significantly increased with the discovery of microbial epoxide hydrolases (41), which are easier to produce in large quantities. The cloning and overexpression of several enantioselective epoxide hydrolases, e.g., from Agrobacterium radiobacter (35), Aspergillus niger (3), and potato plants (40), not only facilitated large-scale production of these enzymes but also made it possible to improve their biocatalytic properties by site-directed or random mutagenesis (34,36,43).Since many microbial genome sequences are available in the public domain, it is useful to screen these databases for genes that might encode new enzymes with interesting properties.Novel epoxide hydrolases can be identified by performing a BLAST search of the genomic databases, using amino acid sequences of known epoxide hydrolases as queries. This approach will result in putative epoxide hydrolases but also in amino acid sequences from structurally and mechanistically related enzymes, such as esterases and dehalogenases (33), which can be filtered out using conserved epoxide hydrolase sequence motifs that define the active site (Fig.…”

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Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Loo

Kingma

Arand

et al. 2006

Appl Environ Microbiol

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109

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Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative ␣/␤-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.Enantiopure epoxides and vicinal diols are valuable intermediates in the synthesis of a number of pharmaceutical compounds. Epoxide hydrolases (EC 3.3.2.3) catalyze the conversion of epoxides to the corresponding diols. If they are enantioselective, they can be used to produce enantiopure epoxides by means of kinetic resolution (5). In the past, when only epoxide hydrolases from mammalian sources were known (12), the use of epoxide hydrolases in biocatalysis was hampered by their poor availability and insufficient catalytic performance, such as a low turnover rate or poor enantioselectivity. The potential for biocatalytic application of epoxide hydrolases was significantly increased with the discovery of microbial epoxide hydrolases (41), which are easier to produce in large quantities. The cloning and overexpression of several enantioselective epoxide hydrolases, e.g., from Agrobacterium radiobacter (35), Aspergillus niger (3), and potato plants (40), not only facilitated large-scale production of these enzymes but also made it possible to improve their biocatalytic properties by site-directed or random mutagenesis (34,36,43).Since many microbial genome sequences are available in the public domain, it is useful to screen these databases for genes that might encode new enzymes with interesting properties.Novel epoxide hydrolases can be identified by performing a BLAST search of the genomic databases, using amino acid sequences of known epoxide hyd...

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confidence: 99%

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confidence: 99%

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Loo

Kingma

Arand

et al. 2006

Appl Environ Microbiol

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109

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“…Mass spectrometry (MS) can be used when a target product has a different mass or fragmentation pattern from the product(s) of the wild-type enzyme(s). Using MS, it is possible to screen up to 10,000 samples per day (182), and MS screening has been used to for the directed evolution of enzymes with altered product profiles or enantioselectivity (166,168). However, due to the large capital investment required (Ͼ$1 million), automated, high-throughout MS-based screening equipment is found primarily in well-funded industrial research laboratories.…”

Section: Prospects and Challenges For Diversifying Other Pathways By mentioning

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Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Umeno

Tobias

Arnold

2005

Microbiol Mol Biol Rev

195

148

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Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed

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“…In addition, it is acknowledged that the toxic effects of DON are associated with its 12, 13-epoxy structure and removal of this epoxide group entails a significant loss of toxicity (Awad et al, 2010), and destroying its epoxy structure can largely reduce the DON toxicity. To date, several conventional methods including chemical, physical and microbiological treatments (Bretz et al, 2006;Abolmaali et al, 2008;Avantaggiato et al, 2004) have been attempted to reduce the toxicity of DON, and microbiological and enzymatic treatments may be the best choice for reducing the toxicity of DON (Reetz et al, 2004). Shim et al (1997) isolated an Agrobacterium-Rhizobium strain E3-39 from soil samples, which could transform DON into3-ketoDON under aerobic conditions.…”

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confidence: 99%

Isolation and identification of an extracellular enzyme from Aspergillus Niger with Deoxynivalenol biotransformation capability

Yang

et al. 2017

Emir J Food Agric

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In this study, the purification and characterization of an extracellular enzyme form Aspergillus niger was performed. With an optimized protocol, it was conducted a 42.6-fold purification with a yield of 26.2%. The purified lipase had a monomeric molecular weight of 40.5kDa and an isoelectric point of 6.01, and its maximum enzyme activity could be achieved at 40°C and pH 7.5-9.0. The enzyme could be activated by Ca2+, Mg2+ and Fe2+, while its activity could be inhibited by Zn2+ and Cu2+. Additionally, organic compounds exerted an inhibitory effect on the enzyme activity in a descending order of methanol, ethanol, DMSO, EDTA, acetone. Meanwhile, the specificity analysis of the enzyme indicated a preference to tributyrin and vegetable oils as well as long-chain fatty acid methyl esters (C12-C18). Most importantly, this enzyme could successfully transform deoxynivalenol (DON). Using HPLC analysis，it was detected a biotransformation rate of more than 70%.The liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular weight of the transformation product was 18.0 larger than that of DON, indicating that DON could be hydrolyzed by the enzyme. Overall, the proposed method here provides a new avenue for reducing the toxicity of DON, which appears to have a wide application outlook for DON biotransformation.

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Enhancing the Enantioselectivity of an Epoxide Hydrolase by Directed Evolution

Cited by 156 publications

References 37 publications

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

Isolation and identification of an extracellular enzyme from Aspergillus Niger with Deoxynivalenol biotransformation capability

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