Salidroside synthesis via glycosylation by -D-glucosidase immobilized on chitosan microspheres in deep eutectic solvents

This study aimed to establish a novel method for catalysing salidroside synthesis by immobilized 18-D-glucosidase with deep eutectic solvents (DESs) as a new green non-aqueous medium. To this end, the catalytic properties of 18-D-glucosidase (i.e. activity, stability, and enzymatic properties) were analysed within sixteen different DESs systems. Although most DESs significantly improved enzyme stability, they also reduced enzymatic activity. This was verified at the molecular level by employing fluorescence spectroscopy to evaluate conformational changes in 18-D-glucosidase molecules. Subsequently, chitosan microspheres were used to immobilize 18-D-glucosidase; the structures of the microspheres and immobilized enzyme were observed by scanning electron microscopy (SEM). Through single factor experiments and application of a response surface design, the optimal conditions for production of salidroside by the reaction of tyrosol with immobilized 18-D-glucosidase were established. These conditions comprised 80 vol% ChCl/G (1:2), 50 degrees C reaction temperature, 5.8 pH, 100 h reaction time, 45 U/mL enzyme dosage, and a tyrosol/D-glucose molar ratio of 10. Under these conditions, the average conversion rate of the tyrosol substrate was 31.6%. Moreover, a salidroside crude product was obtained with a purity > 70% (g/g) and a substrate conversion rate of immobilized 18-D-glucosidase > 50% of its initial value after five repeated uses. Collectively, this study establishes a novel green reaction system for the efficient preparation of salidroside and other glycoside compounds, while also providing a theoretical foundation for the analysis of 18-D-glucosidase catalytic reactions in DESs systems.