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Enzyme Conjugation with Small Molecules

Enzymes are often conjugated with small molecules for different purposes. Using industrial enzyme development as an example, enzyme conjugation with small molecules are applied to neutralize or invert charges on the enzyme surface. The effect of modification is based on the nature of the small molecule, the modification degree, and the enzyme used. Thermo-stability is usually the objective. For example, modification of horseradish peroxidase (HRP) with anhydrides showed improved thermostability. In addition, enzymes have been used to introduce linkers with different lengths, followed by preparation of enzyme conjugates for immunoassay. HRP is one of the most commonly used enzymes for enzyme-linked immunosorbent assay (ELISA). In pharmaceutical industry, conjugation of small molecules to enzyme could also contribute in development of medical diagnostics and drug delivery systems. 

Conjugation with small molecules usually occurs as enzyme-catalyzed covalent addition of some chemical groups to an amino acid residue of the target enzyme. This modification of enzyme can be categorized as i) cofactor attachment and ii) attachment of small functional groups, such as methyl, acetyl, sulfate, and phosphate groups. Different types of reactions may be employed. For example, cofactor attachment includes biotinylation, phosphopantetheinylation, lipoic-acid attachment, and tetrapyrrole/heme ligation; attachment of small functional groups includes methylation, N-acetylation, phosphorylation, sulfurylation and hydroxylation.   

Creative Enzymes is a leading figure in the field. We offer services for enzyme conjugation, especially with small molecules. We satisfy various customer needs by using both chemical and enzymatical strategies. Conjugation with specified enzyme-to-molecule ratios could also be realized. Finally, with a streamline of purification and characterization procedures, we deliver enzyme conjugates with high purity and reliable structural data. A typical conjugation project includes the following key steps:


  1. Milczek, E.M. (2018) Commercial Applications for Enzyme-Mediated Protein Conjugation: New Developments in Enzymatic Processes to Deliver Functionalized Proteins on the Commercial Scale. Chemical Reviews. 118(1): 119-141.
  2. Minten, I.J., Abello, N., Schooneveld-Bergmans, M.E.F., Van Den Berg, M.A. (2014) Post-production modification of industrial enzymes. Applied Microbiology and Biotechnology. 98(14): 6215-6231.

Related sections

Enzyme Engineering and Modification

Enzyme conjugates with small molecule such as anhydrides.Figure 1 Reaction of anhydrides with a primary amine of an enzyme.
(Applied Microbiology and Biotechnology 2014)

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