Boosting Fructosyl Transferase's Thermostability and Catalytic Performance for Highly Efficient Fructooligosaccharides (FOS) Production

Abstract

Achieving enzymatic food processing at high substrate concentrations can significantly enhance production efficiency; however, related studies are notably insufficient. This study focused on the enzymatic synthesis of fructooligosaccharides (FOS) at high temperature and high substrate concentration. Results revealed that increased viscosity and limited substrate solubility in high-concentration systems could be alleviated by raising the reaction temperature, provided it aligned with the enzyme's thermostability. Further analysis of enzyme thermostability in real sucrose solutions demonstrates that the enzyme's thermostability was remarkedly improved at higher sucrose concentrations, evidenced by a 10.3 °C increase in melting temperature (T_m) in an 800 g/L sucrose solution. Building upon these findings, we developed a novel method for enzymatic FOS synthesis at elevated temperatures and high sucrose concentrations. Compared to existing commercial methods, the initial transglycosylation rate and volumetric productivity for FOS synthesis increased by 155.9% and 113.5%, respectively, at 65 °C in an 800 g/L sucrose solution. This study underscores the pivotal role of substrate concentration, incubation temperature, and the enzyme's actual status in advancing enzyme-catalyzed processes and demonstrates the potential of enzymatic applications in enhancing food processing technologies, providing innovative strategies for the food industry.

Keywords: engineering process optimization; enzymatic catalysis efficiency; enzyme-catalyzed FOS synthesis; high substrate concentration; thermostability of enzymes.

Figure 1

Physiochemical properties of sucrose solutions. (A) Solubility of sucrose in water across varying temperatures. Data sourced from Engineering Toolbox https://www.engineeringtoolbox.com/sugar-solubility-water-d_2193.html (accessed on 18 June 2024). (B) Viscosity profiles of sucrose solutions at various concentrations and temperatures. Information adapted from Engineering Toolbox “https://www.engineeringtoolbox.com/sugar-solutions-dynamic-viscosity-d_1895.html” (accessed on 18 June 2024). (C) The potential relationship between the solubility and viscosity of sucrose solutions at various concentrations and temperatures using data referenced from [45,46].

Figure 2

Effects of temperature on FTase activity. (A) Examination of the optimal temperature for FTase activity across a range of temperatures. (B) Thermostability analysis of the enzyme following incubation at various temperatures for 1 h, with residual activities assessed using the specified enzyme activity assay conducted in triplicate. (C) Extended thermostability assessment involving incubation of the enzyme at different temperatures for up to 32 h, followed by determination of residual activities, also performed in triplicate. The letters a–h indicate statistically significant differences in decreasing orders of magnitude (p < 0.01).

Figure 3

The physicochemical characteristics of denaturation of FTase in sucrose solutions. (A) Temperature-fluorescence profiles of FTase in various concentrations of sucrose solutions, monitored using a real-time PCR instrument. Fluorescent dyes were employed to bind to protein structures, enhancing fluorescence signals to compare melting temperatures (T_m) and assess potential structural deformations in the protein. (B) Representative thermodenaturation curves of FTase in the presence of different sucrose concentrations, analyzed using circular dichroism. (C) Analysis of the relationship between α_w and T_m in solutions with varying sugar concentrations. (D) Determination of the equilibrium constant (K_unf) and α_w in various sugar solutions at different concentrations. (E) Correlation between free energy difference (ΔΔG) and sugar concentration. The letters a–f indicate statistically significant differences in decreasing orders of magnitude (p < 0.01).

Figure 4

FOS synthesis characteristics with different concentrations of sucrose at different temperatures. (A) Initial rate of FOS synthesis. (B) The relationship between [S]/V and [S] with substrate inhibition effects. The letters a–g and A–E indicate statistically significant differences in decreasing orders of magnitude (p < 0.01).

Figure 5

Newly developed process for high-efficiency FOS preparation. (A) Comparison of FOS yields at 65 °C with a sucrose concentration of 800 g/L (solid rectangles) versus 45 °C with a sucrose concentration of 500 g/L (solid circles). (B) Time-course changes in contents (rectangles) and productivities (circles) during FOS preparation at 65 °C with 800 g/L sucrose (open symbols) versus 45 °C with 500 g/L sucrose (solid symbols). The letters a–g indicate statistically significant differences in decreasing order of magnitude (p < 0.01).