Denis Wahler

Novel methods for biocatalyst screening.

There have been a number of recent advances in catalysis assays applicable for screening biocatalyst libraries in high-throughput format. These include instrumental assays such as high-performance liquid chromatography, mass spectrometry, capillary electrophoresis and IR-thermography, reagent-based assays producing spectroscopic signals (UV/VIS or fluorescence) in response to reaction progress, and assays based on fluorogenic or chromogenic substrates. These fluorogenic substrates enable the assaying of a variety of enzymes in enantioselective and stereoselective manner, including alcohol dehydrogenases, aldolases, lipases, amidases, epoxide hydrolases and phosphatases.

High-throughput screening for biocatalysts.

Recent progress in high-throughput enzyme assays includes new examples of fluorogenic and chromogenic substrates, fluorescence resonance energy transfer substrates, and applications of the pH and pM indicator methods. Recent developments of Horeaus pseudo-enantiomer derivatisation method to screen enantioselectivities in high-throughput have also been reported.

The Adrenaline Test for Enzymes

Enzyme assays[1] play a key role in the search for novel enzymes,[2] which are in great demand as components of consumer products, industrial processes, diagnostics, and analytical reagents.[3] While many enzyme assays are based on chromogenic or fluorogenic substrates, it is often desirable to have assays that produce a recordable signal indirectly and thus avoid the incorporation of a chromophore into the substrate itself. Such indirect assays are possible with sensors that either record changes in physicochemical parameters such as temperature,[4] pH,[5] or pM[6] upon reaction progress, or that respond by selective recognition of product over substrate through noncovalent interactions.[7, 8] The classical enzyme-coupled enzyme assays achieve the same goal through selective degradation of the reaction product by one or more secondary enzymes, most often by means of a redox chain that leads to the formation of NADH, which is analyzed spectrophotometrically at 340 nm.[9] Enzyme-coupled assays are, however, expensive and incompatible with varying technically important parameters such as cosolvents, pH, and temperature. Herein we report a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products by back-titration of the oxidant with adrenaline. The assay uses inexpensive, commercially available reagents, and offers a simple solution for assaying a variety of industrially important enzymes with their natural substrates. We recently reported a series of enzyme substrates that released a colored or fluorescent product by oxidation of the primary enzyme reaction product by alcohol dehydrogenase[10] or sodium periodate.[11] Considering that sodium periodate reacts rapidly with any 1,2-diol or related periodate-sensitive functional groups independent of the presence of a chromophore in the molecule, we reasoned that this oxidant should also be applicable to nonchromogenic and nonfluorogenic substrates if used in a back-titration mode. Thus, any periodate-sensitive reaction product formed from a periodate-resistant substrate should be detectable by adding first a measured amount of sodium periodate, and second a chromogenic or fluorogenic reagent to quantitate the unreacted sodium periodate by optical density (OD) measurements (Scheme 1).

Enzyme Fingerprints of Activity, and Stereo- and Enantioselectivity from Fluorogenic and Chromogenic Substrate Arrays

A series of stereochemically and structurally diverse fluorogenic and chromogenic substrates for hydrolytic enzymes has been synthesized and used to characterize enzyme activity profiles of esterases, lipases, proteases, peptidases, phosphatases, and epoxide hydrolases. The substrates used are particularly resilient to nonspecific reactions due to their mechanism of activation. The activities recorded with the individual substrates are therefore remarkably reproducible, and enable us to use the overall pattern of activity as a specific fingerprint for the enzyme sample. Fingerprints of activity, and enantio- and stereoselectivity are displayed as arrays of color-scale squares that are easily analyzed visually. Such fingerprints might be useful for quality control, enzyme discovery, and possibly for addressing the issue of functional convergence in enzymes.

Substrate Arrays as Enzyme Fingerprinting Tools

The most fundamental property of any catalyst is selectivity. Selectivity is defined in terms of reaction types, substrate and product range, stereochemical preferences, and operating conditions. The activity pattern of a catalyst, which defines its function, is more important in practical terms than its actual chemical structure. Activity cannot be deduced accurately from structure because of the limited predictive value of chemical theories. Therefore, characterization of a catalyst is an experimental exercise that amounts to recording its activity for a series of substrates and reaction conditions. In contrast to the description of chemical structures, there is no generally accepted formalism for the description of catalytic activity patterns, which can represent extremely diverse and potentially infinitely large data sets. Herein, we discuss the use of substrate arrays as tools for the rapid characterization of enzyme activities and the potential use of such arrays in enzyme discovery and quality control applications.

Enzyme assay and activity fingerprinting of hydrolases with the red-chromogenic adrenaline test

The adrenaline test for enzymes is a colorimetric enzyme assay based on the quantification of periodate-sensitive reaction products such as 1,2-diols and 1,2-aminoalcohols by back-titration of the oxidant with adrenaline to produce adrenochrome as an easily detectable red product. The test uses commercial reagents and is suitable for screening the activity of various hydrolases. It is demonstrated here for testing epoxide hydrolases, lipases and esterases, and for activity fingerprinting of these enzymes across substrate series. The complete assay requires 2–3 h.

Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid.

Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87, I263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid.

Directed evolution of a 13-hydroperoxide lyase (CYP74B) for improved process performance

The performance of a 13-hydroperoxide lyase from guava, an enzyme of the CYP74 family, which is of interest for the industrial production of saturated and unsaturated C6-aldehydes and their derivatives, was improved by directed evolution. Four rounds of gene shuffling and random mutagenesis improved the functional expression in E. coli by offering a 15-fold higher product yield factor. The increased product yield factor relates to an improved total turnover number of the variant enzyme, which also showed higher solubility and increased heme content. Thermal stability was also dramatically improved even though there was no direct selection pressure applied for evolving this trait. A structure based sequence alignment with the recently solved allene oxide synthase of Arabidopsis thaliana showed that most amino acid alterations occurred on the surface of the protein, distant of the active site and often outside of secondary structures. These results demonstrate the power of directed evolution for improving a complex trait such as the total turnover number of a cytochrome P450, a critical parameter for process performance that is difficult to predict even with good structural information at hand.

Cover Picture: Chem. Eur. J. 14/2002

The cover picture shows a two-dimensional color code that can be used to represent catalytic activity (intensity) and enantio- or stereo-selectivity (green/red balance) for the action of enzymes on individual fluoro- and chromogenic substrates in a 6×5 array. The patterns obtained serve as activity fingerprints for each enzyme. The illustration shows the selectivity fingerprints for (from left to right) Pseudomonas cepacia lipase, Candida antarctica lipase, Electrophorus electricus acetylcholine esterase, and Rhizomucor miehei lipase, together with the structure of the enzymes. Color coding is illustrated by connecting two squares in each fingerprint to the corresponding color in the reference grid (bottom). For more details see the article by J.-L. Reymond et al. on p. 3211 ff.