Efficient immobilization of thermophilic rhamnosidase dual-network hybrid-gel with high enzymatic activity and stability.

Thermophilic enzymes possess numerous favorable properties for biotransformation, including high catalytic activity, exceptional thermal stability, and ease of purification and preparation. However, the challenge arises from the lack of suitable enzyme immobilization materials that exhibit both thermal tolerance and reusability, thereby hindering the effective and recurrent utilization of thermophilic enzymes. In this study, a specially designed dual-network hybrid-gel consisting of acrylamide (AM) and sodium alginate (SA) was developed to serve as a thermotolerant and reusable carrier for the efficient immobilization of a thermotolerant α-rhamnosidase IagRha. The optimal amounts of AM and SA were determined to be 5 % and 2 %, respectively. Following the step of acrylamide crosslinking to establish the skeleton of hybrid-gel, a subsequent immersion in a Ca2+ solution facilitated the formation of ionic bonds between SA and Ca2+, thereby adjusting the internal voids of the hydrogel, which effectively enhanced the thermal stability and enzyme immobilization efficiency of the hybrid-gel. This innovative approach resulted in the achievement of 93.65 % recovery of enzyme activity, along with remarkable enhancements in enzymatic properties. The immobilized α-rhamnosidase, AM-SA-IagRha, demonstrated a significant 1.34-fold increase in the Vmax value, rising from 5.28 U/mg to 7.06 U/mg, compared to free IagRha. Furthermore, AM-SA-IagRha exhibited excellent thermostability, with a half-life of 99.0 min at 90 °C, which was 1.57-fold higher than that of free IagRha. After four cycles of reuse, the productivities of rutin and epmdin C by the immobilized enzyme were 1.91-fold and 2.53-fold that of the free enzyme, respectively. Additionally, the enzyme activity of AM-SA-IagRha remained at 90.16 % after storage at 4 °C for 30 days, and retained at 85.63 % after 20 cycles of reuse, demonstrating exceptional storage stability and reusability. The optimal expression conditions for IagRha were identified in TB medium incubated at 37 °C without IPTG, resulting in an enzyme activity of 6.32 U/mL. These significant findings underscore the potential of AM-SA-IagRha for industrial applications, particularly in high-temperature biotransformation.