Hydrolyzation of mogrosides: Immobilized β-glucosidase for mogrosides deglycosylation from Lo Han Kuo

Abstract

An immobilized enzyme system for bioconversion of Lo Han Kuo (LHK) mogrosides was established. β-Glucosidase which was covalently immobilized onto the glass spheres exhibited a significant bioconversion efficiency from pNPG to pnitrophenol over other carriers. Optimum operational pH and temperature were determined to be pH 4 and 30°C. Results of storage stability test demonstrated that the glass sphere enzyme immobilization system was capable of sustaining more than 80% residual activity until 50 days, and operation reusability was confirmed for at least 10 cycles. The Michaelis constant (K _m) of the system was determined to be 0.33 mM. The kinetic parameters, rate constant (K) at which Mogrosides conversion was determined, the τ ₅₀ in which 50% of mogroside V deglycosylation/mogroside IIIE production was reached, and the τ complete of complete mogroside V deglycosylation/mogroside IIIE production, were 0.044/0.017 min^-1, 15.6/41.1 min, and 60/120 min, respectively. Formation of the intermediates contributed to the kinetic differences between mogroside V deglycosylation and mogroside IIIE formation.

Keywords: Lo Han Kuo; glucosidase; immobilized enzyme; mogroside IIIE; mogroside V.

Figure 1

Catalytic behaviors of the suspended β‐glucosidase and the immobilized β‐glucosidase system using three different carriers (glass spheres, nylon pellets, cellulose beads) at 30 and 50°C. The relative activity was defined as the ratio of Conc.(t)/Conc.(s), where Conc.(t) represents the concentration of the p‐nitro‐phenol solution after t min of glucosidase system treatment and Conc.(s) represents the concentration of p‐nitro‐phenol converted completely from p NPG in the solution

Figure 2

Glass spheres without (a) and with (b) β‐glucosidase immobilization

Figure 3

The observed full survey scan (1) and N 1s (2) ESCA spectra to examine the glucosidase immobilization: (a) without and (b) with β‐glucosidase immobilized on glass spheres. The characteristic elements and functional groups responsible for corresponding ESCA signals are indicated by arrows

Figure 4

Effect of pH (a) and temperature (b) on the activity of suspension and immobilized enzyme systems. One Unit activity corresponds to the amount of enzyme which hydrolyzes 1 μmol p‐nitro‐phenyl‐β‐D‐glucopyranoside per minute

Figure 5

Effect of the concentration of p NPG on the activity of the suspension enzyme (a) and the immobilized enzyme (b). The reaction mixture was incubated at 30°C for 10 min. The velocity corresponds to the amount of p‐nitro‐phenyl hydrolyzed by enzyme per minute

Figure 6

Bioconversion of mogrosides in Luo Han Kuo fruit extract solution by the suspension β‐glucosidase (a) and the immobilized glucosidase system (b). MG‐V, S‐I, MG‐IV and MG‐III represent Mogroside V, Siamenoside I, Mogroside IV, and Mogroside IIIE, respectively

Figure 7

Determination of kinetic parameters for the free (a) and the immobilized (b) β‐glucosidase in different concentration LHK solution. The reaction mixture was incubated at 30°C for 30 min. The velocity corresponds to the amount of glucose released by glucosidase per minute

Figure 8

Storage stability of immobilized β‐glucosidase from 1 to 50 days