Peptide Conjugation for Enzyme Engineering

The application of peptide conjugation technology in enzyme engineering provides a new way to improve the stability, activity, and specificity of enzymes. By conjugating peptides to enzyme molecules, enzyme functions can be enhanced and optimized, and their applications in industry, biomedicine, and environmental protection can be expanded. Here are a few examples of how peptide conjugation can be used in enzyme engineering.

Improves enzyme activity

The conjugation of peptides to nucleic acid biomolecules such as DNA can improve the activity of enzymes, and this technology has a wide application prospect in the field of enzyme engineering and bioengineering. For example, researchers used in vitro screening techniques to identify a tetrapeptide that can be specifically conjugated to DNA to increase human neutrophil elastase (HNE) inhibition. Hydronuclear magnetic resonance data and comparative analysis of selected sequences showed that high-affinity binding DNA alone had no inhibitory effect on the enzymatic activity of HNE. Surprisingly, however, this DNA-peptide conjugate obtained by ligating DNA with the tetrapeptide N-methoxysuccinyl-Ala-Ala-Pro-Val is nearly 5 orders of magnitude more inhibitive of HNE than the peptide alone.

Table 1. Peptide conjugation service at Creative Peptides

Detection of enzyme activity

Peptide conjugation technology provides an efficient and accurate method for detecting enzyme activity by reacting specific markers with enzymes and is widely used in biomedical research. For example, Lowe Stuart B's team reported enzyme activity by synthesizing enzyme-specific peptide sequences with orthogonal end functionalization to attach to quantum dots with different emission spectra, reporting enzyme activity by binding gold nanoparticle-peptide conjugates or FRET receptor dye-labeled antibodies, thus avoiding cross-reactivity and signal overlap. This specific peptide sequence-conjugated assay allows for the simultaneous detection of different classes of enzymes, allowing for the development of a universal platform for high-throughput enzyme screening.

Improve enzyme selectivity

Esterases are used in the pharmaceutical industry to synthesize chiral drugs, and their stereoselectivity is critical to the quality of the product. The researchers used peptide conjugation technology to conjugate a chiral recognition peptide to an esterase, which significantly improved the selectivity of esterases for specific chiral substrates. In this way, the modified esterase can be used to produce high-purity chiral drugs more efficiently.

Expand the application of enzymes

Peptide conjugation can also confer new functions on enzymes. For example, researchers conjugated peptides with cell-penetrating capabilities to enzymes that allow these enzymes to efficiently penetrate cell membranes and enter the interior of the cell to function. This has important applications in gene editing and cell therapy. It is well known that stealth coatings effectively extend the circulating life of nanomaterials in the blood, which facilitates systemic delivery, but also limits their cellular internalization, which in turn prevents effective tumor targeting and accumulation. In this study, the researchers addressed this dilemma by developing an enzyme-responsive zwitterionic stealth peptide coating that responds to matrix metalloproteinase-9 (MMP-9) overexpressed in the tumor microenvironment. The multifunctional peptide is composed of cell penetrating peptide, MMP-9 enzyme cleavable sequence and zwitterion sequence. With this coating to protect photothermal gold nanorods (AuNRs), the results showed satisfactory systemic circulatory life and significantly enhanced tumor cell uptake, resulting in a significantly improved photothermal treatment effect in a mouse model.

Fig.1 Schematic diagram of the preparation of multifunctional peptide coating encapsulated AuNRs and their accumulation in tumors. (Wu Liming, et al., 2019)

Development of peptides with enzyme-like activity

Peptides and their conjugates (bound to lipids, macromolecular N-terminus, or other groups) can self-assemble into nanostructures that mimic the binding sites of native enzymes to catalyze a variety of reactions. For example, a research team in Brazil developed a series of proline-based amyloid peptides and lipopeptides in 2020 that form self-assembled micelles, nanoribbons, and fibril structures and exhibit excellent catalytic activity and high selectivity for aldol reactions in aqueous solutions.

Summary

Peptide conjugation is widely used in enzyme engineering to improve the stability, activity, and specificity of enzymes, and to enhance the performance and longevity of enzymes under extreme conditions by binding peptides to enzymes. In the future, with the continuous progress of biotechnology, peptide conjugating is expected to further promote the development of enzyme engineering in the fields of medicine, agriculture, industry, and environmental governance, bringing more innovations and breakthroughs.

References

1. Lin, Yun, et al., Peptide conjugation to an in vitro-selected DNA ligand improves enzyme inhibition. Proceedings of the National Academy of Sciences 92.24 (1995): 11044-11048.
2. Lowe, Stuart B., et al., Multiplex sensing of protease and kinase enzyme activity via orthogonal conjugating of quantum dot-peptide conjugates. ACS nano 6.1 (2012): 851-857.
3. Pelin, Juliane NBD, et al.,Self-Assembly, Nematic phase formation, and organocatalytic behavior of a proline-functionalized lipopeptide. ACS applied materials & interfaces 12.12 (2020): 13671-13679.
4. Hamley, Ian W., Biocatalysts based on peptide and peptide conjugate nanostructures. Biomacromolecules 22.5 (2021): 1835-1855.
5. Wu, Liming, et al., Enzyme-responsive multifunctional peptide coating of gold nanorods improves tumor targeting and photothermal therapy efficacy. Acta Biomaterialia 86 (2019): 363-372.