The Adrenaline Test for Enzymes

Wahler, Denis; Reymond, Jean‐Louis

doi:10.1002/1521-3773(20020402)41:7<1229::aid-anie1229>3.0.co;2-5

Cited by 74 publications

(44 citation statements)

References 52 publications

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“…Substrate profiling of the various epoxide hydrolases was done using the adrenaline test (46). Initial testing of various vicinal diols in a reaction with IO 4 Ϫ and adrenaline revealed that adenochrome was rapidly produced with all diols that are formed from the epoxides shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…2) or an indirect assay that measures periodate depletion by its reaction with a diol (substrates 1, 2, 4, 7, 8, 11 to 14, 16, 17, and 19 to 25). In the latter assay, the diol that is formed by epoxide hydrolase activity reacts with periodate, and the amount of remaining periodate is measured by incubating it with adrenaline to yield a colored product (46). Using these assays, it appeared that of the 12 epoxide hydrolases that were tested, 8 were active with one or more epoxides (Table 4).…”

Section: Vol 72 2006 Novel Epoxide Hydrolases From Genomic Databasementioning

confidence: 99%

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Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Loo

Kingma

Arand

et al. 2006

Appl Environ Microbiol

108

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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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Section: Methodsmentioning

confidence: 99%

Section: Vol 72 2006 Novel Epoxide Hydrolases From Genomic Databasementioning

confidence: 99%

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Loo

Kingma

Arand

et al. 2006

Appl Environ Microbiol

108

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“…To Scheme 1 Hydrolytic desymmetrization catalyzed by mutants of limonene epoxide hydrolase (LEH) generated by different saturation mutagenesis strategies ensure 95 % library coverage, the screening of 2912 × 3 variants of library A and 1294 × 3 variants of library B would be required. Experimentally, we assayed only 2912 and 1294, respectively, using the adrenaline on-plate pretest for activity (Wahler and Reymond 2002) followed by chiral GC analysis of the active variants.…”

Section: Residue Selection and Library Designmentioning

confidence: 99%

Exploring productive sequence space in directed evolution using binary patterning versus conventional mutagenesis strategies

Sun

Salas

Siirola

et al. 2016

Bioresour. Bioprocess.

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Background: Recent methodology development in directed evolution of stereoselective enzymes has shown that various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation of small and smart mutant libraries requiring minimal screening. Methods:In this endeavor, limonene epoxide hydrolase (LEH) has served as an experimental platform, the hydrolytic desymmetrization of cyclohexene oxide being the model reaction with formation of (R,R)-and (S,S)-cyclohexane-1,2-diol. This system has now been employed for testing reduced amino acid alphabets based on the Hecht concept of binary patterning, with and without additional hydrophobic amino acids. Results and Conclusions:It turns out that in binary pattern based saturation mutagenesis as applied to LEH, polar amino acids are seldom introduced. When applying binary patterning in combination with additional hydrophobic amino acids as building blocks in iterative saturation mutagenesis, excellent LEH variants were evolved for the production of both (R,R)-and (S,S)-diols (80-97 % ee), but again the introduction of polar amino acids occurs rarely. Docking computations explain the source of enhanced and inverted stereoselectivity. Some of the best variants are also excellent catalysts in the hydrolytic desymmetrization of other meso-epoxides, although both enantiomeric diols are not always accessible.

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“…A variety of other high -throughput screening assays have been reported based on the chemoselective detection of the reaction products from untagged esters. These include fl uorogenic hydrazone formation with acetaldehyde produced by lipase/esterase -catalyzed transesterifi cation of vinyl esters in organic solvents [24] , the detection of acetic acid produced from acetate esters using an enzyme kit [25] , or the back titration of periodate with adrenaline after oxidation of the glycerol release from triglyceride hydrolysis [26,27] .…”

Section: Lipases and Esterasesmentioning

confidence: 99%

“…On the other hand various indirect assays have been reported to detect either the unreacted epoxide [46] or the carbonyl product resulting from periodate cleavage of the 1,2 -diol product [26,47,48] . These assays are suitable for fl uorescence or colorimetric assays for the hydrolysis of any epoxide of interest.…”

Section: Epoxide Hydrolasesmentioning

confidence: 99%