Multienzyme Profiling of Thermophilic Microorganisms with a Substrate Cocktail Assay

Abstract

Labeled substrates for 16 different catalytic activities were combined into a cocktail reagent for multienzyme functional profiling, called PHENO-ZYM TM . The assay involves a single reaction followed by determination of substrate consumption by HPLCanalysis. The method allows a rapid identification of multiple enzyme activities, and is compatible with a diversity of growth media and reaction conditions (pH, temperature). The PHENOZYMTM cocktail was used to analyze the activity of 16 enzyme activities in a series of microbial strains, including thermophilic microorganisms. The functional profiles were used for a functional classification of the different microbial strains tested by hierarchical cluster analysis. The resulting "phylo-enzymatic" tree revealed associations consistent with the known phylogenetic classification of the strains. The influence of the culture medium on the enzyme activity profiles was also apparent.

Combinatory Library of Microorganisms in the Selection of Reductive Activity Applied to a Ketone Mixture: Unexpected Highlighting of an Enantioselective Oxidative Activity

“…The incubation of a cocktail of substrates targeting different enzymatic activities has been already used to study the biocatalytic abilities of several microorganisms. This method allowed functional profiling to be obtained in one test for each strain [ 19 ]. To our knowledge, no biological or enzymatic activity has been sought by setting up a selection test on both a mixture of substrates and a mixture of microorganisms.…”

Enzyme assays

“…Similarly, the classical APIZYM substrate palette for microbial characterization can be formulated as a cocktail reagent, allowing 16 different enzyme reactivities to be determined in a single analysis. 217 A cocktail of fluorescent umbelliferyl glycosides was recently used to characterize various glycosidases using an HPLC-based assay. 218 Substrate cocktails can also be analyzed by mass spectrometry in a variety of setting including enantioselectivity determination using a mixture of two isotopically labeled pseudo-enantiomers, [219][220][221][222][223][224][225] for glycosyl transferase reaction using substrate mixtures [226][227][228][229][230] or for protease activity determination using a mixture of peptide substrates.…”

Substrate Arrays for Fluorescence‐Based Enzyme Fingerprinting and High‐Throughput Screening

“…We have reported cocktails to analyze lipases and esterases and proteases 33 . We also demonstrated cocktail profiling of microorganisms in a setup resembling the API ZYM series, but allowing the analysis of thermophilic microorganisms 34 . Substrate cocktails currently under development in our laboratory have proven useful in the characterization of novel enzymes from the metagenome 35 …”