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Novel biosensors based on optimized glycine oxidase
Elena Rosini1, Luciano Piubelli, Gianluca Molla
1Dipartimento di Biotecnologie e Scienze della Vita, Università degli studi dell'Insubria, Varese, Italy; The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano, ICRM CNR Milano, Università degli studi dell'Insubria, Milano, Italy.
Engineered glycine oxidase (GO) enzymes can now detect glycine and sarcosine, crucial metabolites in prostate cancer. This innovation offers a rapid, low-cost biosensor for analyzing these amino acids in biological samples.
Area of Science:
- Biochemistry
- Enzyme Engineering
- Analytical Chemistry
Background:
- Glycine functions as a neurotransmitter, while elevated sarcosine indicates prostate cancer progression.
- Developing precise analytical methods for these small amino acids is crucial for diagnostics.
Purpose of the Study:
- To engineer Bacillus subtilis glycine oxidase (GO) for enhanced detection of glycine and sarcosine.
- To create specific biosensors for quantifying these metabolites in biological samples.
Main Methods:
- In silico analysis and site-saturation mutagenesis of 11 positions in glycine oxidase.
- Kinetic analysis of single-point and multiple-mutation variants.
- Development of a fluorescence-based biosensor using engineered GO variants.
Main Results:
- Engineered variants A54R, H244K-N-Q-R, Y246W, and M261R showed improved glycine kinetics.
- The H244K/M261R variant exhibited a 5.4-fold increase in maximal activity on glycine.
- The M49L variant demonstrated a 6-fold increase in kinetic efficiency for sarcosine.
- Specific variants (H244K GO for glycine, M49L GO for sarcosine) were developed for a fluorescence-based biosensor.
Conclusions:
- Engineered glycine oxidase variants offer enhanced activity and specificity for glycine and sarcosine.
- A novel, inexpensive, and rapid fluorescence-based biosensor was developed for detecting glycine and sarcosine at low micromolar concentrations (≤ 0.5 μm).
- This biosensor provides a valuable tool for analyzing these key metabolites in biological samples, with potential applications in cancer diagnostics.
