Enzymatic Preparation of l-Malate in a Reaction System with Product Separation and Enzyme Recycling

Abstract

Reaction coupling separation systems using calcium fumarate as a substrate can break the reaction equilibrium and promote the production of l-malate. However, the low reusability and stability of fumarase limit its further application. In this study, partially purified fumarase of Thermus thermophilus (87.0 U mg−1) was immobilized within konjac-κ-carrageenan beads. An amalgamation of konjac and carrageenan gum (2%) was used to form the beads, and polyethylene polyamine (0.2%) and glutaraldehyde (0.1%) were used as the cross-linking agents. The pH and temperature profiles of free and immobilized fumarases were remarkably similar. The diffusion limit of immobilized fumarase caused a decline in the maximal velocity (Vmax), whereas the kinetic constant (Km) value increased. Optimization of the parameters for biotransformation by immobilized fumarase showed that 88.3% conversion of 200 mM calcium fumarate could be achieved at 55 °C within 8 h. The beads were stored for 30 days at 4 °C with minimal loss in activity and were reusable for up to 20 cycles with 78.1% relative activity. By recycling the reaction supernatant, a total amount of 3.98 M calcium fumarate was obtained with a conversion of 99.5%, which is the highest value ever reported.

Immobilization of fumarase from thermophilic eukaryotic red alga <i>Cyanidioschyzon merolae</i> on ceramic carrier

“…Immobilized CmFUM preserved 94.5% of its initial activity (day 1) after 30 days of storage at 4°C, which showed that the immobilized enzyme was more stable than free CmFUM stored at 2 mg/mL or and stored at 4℃ preserved 91.3% of its initial activity after 30 days, which aligns with our results (Li et al, 2022). We observed that more than 86% of initial enzyme activity remained after ten repeated reactions, indicating the high reusability of CmFUM immobilized on Toyonite 200 (Fig.…”

“…4). In a similar study, Li et al (2022) reported that an immobilized fumarase exhibited more than 90% residual activity after ten assays, and although our result was slightly lower, it remains comparable (Li et al, 2022). The stability and reusability of CmFUM immobilized on Toyonite 200 are consistent with those of other reported immobilized fumarases, suggesting its potential for various industrial applications.…”

“…The binding of hydroxy groups on the carrier to areas close to the substrate binding site might interfere with access or binding of substrate to the substrate binding site, thereby leading to an increased Km value. However, although an increase in Km value was observed after immobilization, the lower Km value of immobilized CmFUM for fumarate compared to other immobilized fumarases suggests a superior affinity of immobilized CmFUM for fumarate (Atay et al, 2022;Giorno et al, 2001;Li et al, 2022;Marconi et al, 2001, Table 3). On the other hand, decrease in kcat was significant and was thought to be a major factor associated with the decrease in CmFUM activity following immobilization.…”

“…As shown in Tables 1 and 2, the specific activity and kcat/Km of immobilized CmFUM significantly decreased to 0.016-0.033-fold compared to the free state. Li et al (2022) reported that the Km value of Thermus thermophilus-derived fumarase immobilized in a konjac-κ-carrageenan matrix using an entrapment technique and cross-linked with polyethylene polyamine and glutaraldehyde increased approximately threefold after immobilization, and the reaction velocity decreased to about 0.5-fold. Beeckmans and Kanarek (1982) also reported that in an experiment where fumarase from pig heart was immobilized on Sepharose 4B, the specific activity decreased to 0.3-fold upon immobilization.…”

“…Subsequent studies have focused on the immobilization of purified fumarase (Beeckmans and Kanarek, 1982;Giorno et al, 2001;Marconi et al, 2001). Recently, Li et al (2022) reported that fumarase from Thermus thermophilus immobilized on konjac-κ-carrageenan beads showed improved stability during storage and high reusability, although its enzymatic activity decreased (Li et al, 2022). Atay et al (2022) have reported that immobilized fumarase from pig heart on silanized nanostructures shows improved storage stability and increased activity (Atay et al, 2022).…”