A novel potentiometric biosensor for selective L-cysteine determination using L-cysteine-desulfhydrase producing Trichosporon jirovecii yeast cells coupled with sulfide electrode

摘要

Trichosporon jirouecii yeast cells are used for the first time as a source of L-cysteine desulfhydrase enzyme (EC 4.4.1.1) and incorporated in a biosensor for determining L-cysteine. The cells are grown under cadmium stress conditions to increase the expression level of the enzyme. The intact cells are immobilized on the membrane of a solid-state Ag2S electrode to provide a simple L-cysteine responsive biosensor. Upon immersion of the sensor in L-cysteine containing solutions, L-cysteine undergoes enzymatic hydrolysis into pyruvate, ammonia and sulfide ion. The rate of sulfide ion formation is potentiometrically measured as a function of L-cysteine concentration. Under optimized conditions (phosphate buffer pH 7, temperature 37±1°C and actual weight of immobilized yeast cells 100 mg), a linear relationship between L-cysteine concentration and the initial rate of sulfide liberation (dE/dt) is obtained. The sensor response covers the concentration range of 0.2-150 mgL~(-1) (1.7-1250 mu mol L~(-1)) L-cysteine. Validation of the assay method according to the quality control/quality assurance standards (precision, accuracy, between-day variability, within-day reproducibility, range of measurements and lower limit of detection) reveals remarkable performance characteristics of the proposed biosensor. The sensor is satisfactorily utilized for determination of L-cysteine in some pharmaceutical formulations. The lower limit of detection is ~1 mu molL~(-1) and the accuracy and precision of the method are 97.5% and ±1.1%, respectively. Structurally similar sulfur containing compounds such as glutathione, cystine, methionine, and D-cysteine do no interfere.