C O M M U N I C A T I O N S
Acknowledgment. This work was supported by the University
of Berne and the Swiss National Science Foundation. We thank
Jacob Kofoed for assistance in data processing.
Supporting Information Available: Synthetic procedures and
structures of all substrates, abbreviations of enzyme names, and activity
fingerprints of all enzymes measured (PDF). This material is available
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(18) The differentiating ability of our lipase cocktail is similar in its optically
pure form (40 enantiomers in two HPLC analyses). An earlier report of
lipase reactivity analysis by GC using a mixture of five different
triglyceride substrates did not allow significant enzyme differentiation;
see: Berger, M.; Schneider, M. P. Biotechnol. Lett. 1991, 13, 641-645.
Figure 2. Distance matrix generated by statistical treatment of enzyme
fingerprints. Similarity between samples is measured by Euclidian distance
in multidimensional space defined by each substrate activity and represented
by color coding (black for the highest similarity and white for the lowest).
b
aSubstrate total concentration 2 mM. Identical reactions.
The similarities between the different fingerprints obtained were
investigated by multivariate analysis softwares Winidams or Vista.16
The enzymes were grouped by hierarchical clustering using the
group average method on the basis of standardized Euclidean
distances (Figure 2).17 Most lipases and esterases, which are often
very similar, could be readily distinguished from one another. Even
very similar enzyme pairs such as different preparations of pig liver
esterase differed by a reproducible reactivity difference on at least
one of the substrates in the cocktail.
Cocktail fingerprinting of enzyme activities is a robust and
operationally simple method. Functional fingerprinting across as
few as 20 substrates as shown here should be sufficient to
differentiate between similar enzymes in most cases.18 It should
be noted that the choice of a reactive substrate type for the enzyme
class under study is essential so that the cocktail produces enzyme-
specific fingerprints in all cases. If too many substrates would react
only rarely with an enzyme, the cocktail would return indistinguish-
able “zero” fingerprints for most enzymes. The cocktail used here
is particularly well-suited for lipases and esterases and generates
activity fingerprints even for very dilute enzymes or for enzymes
with low activities. The method can be readily extended to other
enzyme types using the appropriate substrates and should function
with other separative instruments for analysis. Furthermore, cocktail
fingerprinting can be adapted to any operational parameters for the
enzyme. Data from such functional fingerprinting can be acquired
on a large scale by automated analysis and might provide new
insight into the divergent or convergent evolution of enzyme
function in different organisms. Substrate cocktails might also find
applications as enzyme identification reagents for quality control
of enzymes or enzyme-containing products or for medical diag-
nostics.
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J. AM. CHEM. SOC. VOL. 126, NO. 36, 2004 11117