Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification

Abstract

Background: Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size.

Results: The performance of a commercially available feruloyl esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that K_m was not affected by the immobilization but k_cat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction.

Conclusions: These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.

Keywords: E-FAERU; Enzyme reusability; Enzyme stability; Feruloyl esterase selectivity; Kinetic parameters; Mesoporous silica.

Fig. 1

Reaction scheme of feruloyl esterase catalysis in a buffer-butanol mixture where both transesterification reaction converting methyl ferulate (MFA) to butyl ferulate (BFA) and hydrolysis reaction converting MFA to ferulic acid (FA) take place

Fig. 2

Effect of immobilization pH and of enzyme concentration on loading and enzymatic activity. a Effect of immobilization pH on the loading using different buffers. b Effect of enzyme concentration on the loading. c Influence of the immobilization pH and buffer on the BFA/FA molar ratio. The results are compared to those obtained for the free enzyme at pH 7.0 in the respective buffers. d Influence of the immobilization pH on the BFA specific activity. The results are compared to those obtained for the free enzyme at pH 7.0 in the respective buffers. All reactions were in initial velocity. All values are averages of triplicates and the error bars represent one standard deviation

Fig. 3

Effect of the reaction pH for free and immobilized enzyme. a Effect on the BFA/FA molar ratio. b Effect on the BFA specific activity. (Reactions were performed in a 1-butanol (92.5%) phosphate-citrate buffer (7.5%) mixture. The pH of the buffer fraction was varied from 6.0 to 8.0. Immobilization was performed at pH 6.5 and only the reaction pH was varied.) All reactions were in initial velocity. The results are the average of triplicate samples, and the error bars represent one standard deviation

Fig. 4

Influence of the water content on the selectivity of free and immobilized enzyme. a Effect on the BFA/FA ratio. b Effect on the BFA activity. All reactions were in initial velocity. The results are averages of triplicate samples, and the error bars represent one standard deviation

Fig. 5

Determination of pH and temperature optima for the four reaction systems studied a Optimum pH. Reaction rates measured using phosphate-citrate buffer at pH 5.0–8.0 and 40 °C. b Optimum temperature. The temperature was varied from 15 to 80 °C and the reaction was performed using phosphate-citrate buffer at pH 7.0. All reactions were in initial velocity. The results are the average of triplicate samples, and the error bars represent one standard deviation

Fig. 6

Evaluation of the reusability of the immobilized enzyme over 10 cycles of 48 h each. a Relative residual hydrolytic and transesterification activities. b Evolution of the BFA/FA ratio during the experiment. All reactions were in initial velocity. Data are averages of triplicates. Error bars represent one standard deviation