Aldol® Novel Chromogenic Enzyme Substrates for Microbiological Assays

Introduction. Chromogenic enzyme substrates yielding colored precipitates after enzymatic cleavage of the enzyme labile group are widely used for the detection and localization of specific enzyme activity in various fields such as in clinical diagnostics, food and environmental testing, bacterial taxonomy, histology, and immunological assays. However, currently employed substrates have their limitations. For example the broadly used indoxyl substrates depend on the presence of oxygen, other oxidizers or toxic diazonium salts. Moreover, they produce toxic intermediates. Alternative substrates not dependent on oxygen need metal ions to form colored precipitates.

The Aldol® Family of Substrates

To overcome common drawbacks of conventional substrates we developed a novel type of chromogenic enzyme substrates in the Biosynth synthesis and microbiology laboratory. In contrast to the classical indoxyl substrates (like X-Gal) these new Aldol® substrates do not depend on oxidative dimerization for color development.
We synthesized a set of substrates by attaching various enzyme labile groups (comprising beta-D-galactoside, acetate, alpha-D-glucoside, beta-D-glucuronide, phosphocholine, and N-acetyl-beta-D-galactosaminide) to this new dye precursors. The specific cleavage of these substrates was demonstrated in assays with purified enzymes as well as with various microorganisms. The following examples demonstrate the unique features of the Aldol® family of substrates.

A. Aldol® Substrates Work under Aerobic and Anaerobic Culture Conditions



Strong colors under aerobic as well as anaerobic Culture Conditions. Streptococcus gallolyticus on blood agar containg beta-D-glucosidase substrates grown under aerobic and anaerobic conditions. Whereas staining with X-beta-D-glucoside is very weak under anaerobic conditions, the Aldol® substrate stains equally well under aerobic and anaerobic conditions.

B. Aldol® Adds New Colors to the Paintbox of Microbiologists - Providing an Extended Colour Range


Color range from yellow to red. Mixture of Enterobacter cloacae (galactosidase positive, colored) and Shigella boydii  (galactosidase negative, colorless) on Nutrient Agar containing various Aldol-beta-D-galactosidase substrates.

C. Aldol®  Produces Colorimetric and Fluorogenic Signals

Fuorescence of Aldol® products.  ALDOL-455-beta-D-galactoside on blood agar with a mixture of K. pneumoniae (galactosidase positive) and S. enteritidis (galactosidase negative) under aerobic and  anaerobic conditions.

Soluble fluorophore. ALDOL-459-acetate yielded a soluble green fluorescent product (at pH of 6.5 and above) after cleavage by esterase. Left: without enzyme; right: with esterase.

The Aldol® Indicator Platform

Our Aldol® substrates can be applied in many different setups in analytics or diagnostics.

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Application note:

Use of Aldol® substrates with Gram positive bacteria

We are constantly developing our patented ALDOL® substrates; we explore additional applications for this platform and continue to optimize existing protocols. One of our recent findings shows that ALDOL® substrates inhibit the growth of certain Gram positive bacteria.
To eliminate the inhibitory effect, we recommend adding a low concentration of Tween® 80 to the growth media for any applications aiming at the differentiation of Gram positive bacteria.

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Overview of ALDOL® Types

The substrates yield different dyes after cleavage (solubility of the chromophor after enzymatic cleavage is given for water based media):

Aldol® 495 substrates: yielding insoluble dye with brown-orange color, green fluorescence in polypropylene, emulsions, Petrifilm surface etc.. (for special applications, best fluorescence in hydrophobic matrices of these dyes)

Aldol® 515 substrates: yielding insoluble dye with dark red color, fluorescence on certain membranes, oil, Petrifilm surface etc. (best visible color of these dyes)

Aldol® 518 substrates: yielding insoluble dye with dark red color, fluorescence on certain membranes, oil, Petrifilm surface etc. (best visible color of these dyes), very similar to Aldol® 515

Aldol® 484 substrates: yielding insoluble dye with dark red color

Aldol® 470 substrates: yielding insoluble dye with yellow color, fluorescence on certain membranes, oil, Petrifilm surface etc. (weaker fluorescence than 495 and 515)

Aldol® 458 substrates: yielding soluble dye (in water based media when pH >6, will precipitate at pH < 6) with yellow color and green fluorescence (for applications where soluble dye is needed after enzymatic cleavage); Advantage of Aldol 458 towards MeU is its green fluorescence, which is less prone to background compared to the blue fluorescence of MeU.

Summary. In conclusion, this new class of chromogenic enzyme substrates proved useful in applications where auxiliary agents such as oxygen, diazonium salts or metal ions interfere with the assay, or as substrates offering alternative colors (yellow, orange, red) to the presently available substrates. Some of the substrates even combined chromogenic and fluorogenic properties, further expanding potential applications for these substrates.

At a Glance - Benefits of Aldol® Chromogenic / Fluorogenic Enzyme Substrates:

  • No oxygen required: Anaerobic incubation
  • No auxiliary agents (e.g. metal ions, diazonium salts) required: Simpler, faster assays, less toxic
  • No toxic intermediates: Better bacteria growth, imporved sensitivity of the assays
  • No or only little diffusion: Precise staining
  • Yellow, orange and red colors: Expanding available color range
  • Chromogenic and fluorogenic: Enables versatile detection methods, including digital imaging

Please visit Products to see all of Biosynth's Aldol® Substrates.