Preparation And Application Of β-glucuronidase-CaHPO₄ Hybrid Nanoflowers

Glycyrrhetinic acid 3-O-mono-β-D-glucuronide（GAMG） as a natural sweetener and Chinese medicine ingredients has broad application prospects in food industry and medical field. Due to the high impurities concentration and low yield in the chemical synthesis process, our lab previously heterologously expressed the gene of β-glucuronidase from Penicillium purpurogenum Li-3 in E.coli（PGUS-E1） which can directly hydrolyse glycyrrhizin（GL） into GAMG. Due to the poor stability of free enzyme and the reduction of enzyme activity in the traditional enzyme immobilized methods, in this paper, PGUS-E1 was immobilized based on the method of protein-inorganic hybrid assembly. The main research results are as follows:First, the effect of different divalent metal ion(Co²⁺、Mg²⁺、Ni²⁺、Zn²⁺ and Ca²⁺) on PGUS-E1 activity were investigated, which showed that the relative activity of PGUS-E1 was 116% with the presence of Ca²⁺, so Ca²⁺ was chosen as inorganic metal ions to prepare the hybrid nanoflowers. Through the optimization of immobilization conditions, the optimum preparation conditions of CaHPO₄-PGUS-E1 hybrid nanoflowers was obtained: Ca²⁺（1.6 mM）, PB（8 mM）, PGUS-E1（0.4 mg·mL-1）, NaCl（18 mM）, pH（6.5）, time（12 h）, temperature（4 °C）. Under these optimum conditions, 1 mL reaction system can generate 1.2 mg nanoflowers with immobilization efficient of 71.2%, loading capacity of 35.60 mg g-1 and the relative enzyme activity of 118%.The microstructure and catalytic performance of CaHPO₄-PGUS-E1 hybrid nanoflowers were also characterized. SEM images indicated that the nanoflowers were in the shape of rounded flower-like multi-layer hierarchical structure. Zeta potential and particle size analysis showed that the average particle size of nonaflowers was 3637.4 nm, and the distribution of particle size was uniform.The immobilization of FITC-labeled enzyme in the nanoflowers was confirmed through the confocal laser scanning microscopy（CLSM）. FTIR spectrum also indicated the presence of-CONH bonds introduced by the immobilized enzyme. Furthermore, compared with the free enzyme, kcat/Km of immobilized enzyme was declined by 57%. Fifty percent of the original activity of immobilized enzyme was remained after incubation at 55 °C for 10 h, however the enzyme activity of free enzyme had falen below 25% under the same treatment, which indicated that the hybrid flower can significantly increase the thermal stability of enzyme.Finally, the CaHPO₄-PGUS-E1 hybrid nanoflowers was applied to synthesize glycyrrhetic acid 3-O-mono-β-D-glucuronide（GAMG） through glycyrrhizin（GL） hydrolysis. Under optimal reaction conditions, 0.74 mg GL could be hydrolized into GAMG by per gram of nanoflowers per hour and the reaction conversion rate is close to 100%, however the conversion rate of free enzyme is only 83%, which indicated that the nanoflowers could boost the performance of PGUS-E1. In addition, the reaction rate was accelerated with the increase of nanoflowers mass. After eight continuous batches, the nanoflower retained 60% of the initial enzyme activity, which showed that the nanoflowers had a good reusability. The above studies showed that the immobilization of PGUS-E1 through enzyme-inorganic hybrid assembly has significant advantages, and our approach provides a promising alternative for the production of GAMG by immobilized PGUS-E1.