An enzyme-centric approach for constructing an amperometric l-malate biosensor with a long and programmable linear...

Area of Science:

• Biochemistry and biosensor technology
• Enzyme engineering and directed evolution
• Food and beverage analysis

Background:

• L-malate is crucial for flavor and acidity in food and beverages, especially wine.
• Current enzyme biosensors for l-malate have limited linear ranges, requiring sample dilution.
• A need exists for more versatile and sensitive l-malate detection methods.

Purpose of the Study:

• To discover and engineer novel malate-oxidizing enzymes for improved biosensor performance.
• To develop an l-malate biosensor with a tunable linear range and high sensitivity.
• To validate the biosensor's functionality in real-world food and beverage samples.

Main Methods:

• Database mining, gene synthesis, and recombinant expression to identify and produce enzymes.
• Spectrophotometric assays to characterize enzyme activity and suitability for biosensors.
• Development and optimization of an amperometric biosensor prototype using a novel enzyme.

Main Results:

• A bespoke biocatalyst, Ascaris suum malic enzyme with mutation R181Q [AsME(R181Q)], was engineered.
• The AsME(R181Q) biosensor demonstrated an ultra-wide linear range (50-200 mM) in its initial configuration.
• Sensitivity was enhanced five-fold with Mn2+ and six-fold with citrate, yielding a 1-10 mM linear range.
• Interference from ascorbate was mitigated using ascorbate oxidase, enabling accurate measurements in undiluted wine.

Conclusions:

• An enzyme-centric approach successfully yielded a novel l-malate biosensor with adaptable sensitivity and linear range.
• The developed biosensor accurately quantifies l-malate in undiluted wine samples, outperforming previous methods.
• This work paves the way for advanced electrochemical biosensors with enhanced functionality for food and beverage applications.