Continuous production of glycyrrtinic acid 3-O-mono-β-D-glucuronide by immobilized cells

Microbial immobilization is widely used in many areas such as bioengineering, biochemical separation and analysis. Glycyrrhizin (GL) can be transformed into glycyrrtinic acid 3-O-mono-β-D-glucuronide (GAMG) by Penicillium purpurogenum Li-3 screened in the previous study. Penicillium purpurogenum Li-3 cells were immobilized into calcium alginate beads for GAMG production. However, the low mechanical strength and thus poor utilizable properties of the calcium lginate beads limited their applications. The mechanical strength of calcium alginate beads was improved by adding natural substances as well as chemical treating. Subsequently, the catalytic activities of the modified calcium alginate immobilized cells were characterized. At last, the continuous production of GAMG by modified immobilized cells in a self-designed packed bed reactor was further investigated. The obtained results were as follows: Firstly, diatomite was confirmed to be useful for improving the mechanical strength of calcium alginate beads with an optimal concentration of 4%. The so obtained modified immobilized cells were reused for 23 times in shake flask experiments. Subsequently, the modified immobilized cells were employed for GAMG production. After 48 h reaction, the output of GAMG was up to 1.62 g/L and the conversion of GL was 54%. The cost of the added diatomite was relatively low so that it can be used for further large-scale production. Secondly, the effects of pH, temperature and synergistic agent Tween-80 on the conversion efficiency and storage stability of the modified immobilized cells were studied. The results showed that the modification of immobilized cells made no difference on their pH and temperature, which were 5.4 and 35℃, respectively. However, the enzyme activity was enhanced compared with the original immobilized cells. In detail, the conversion efficiency of GL by the modified immobilized cells was improved when 0.12% Tween-80 was added into the substrates, leading to the increase of GAMG output by 44% compared with that in substrates without Tween-80 supplemented. On the other hand, only 30% of the enzyme activity was lost in the modified immobilized cells when stored in physiological saline for 6 weeks at 4℃ while the free normal cells began autolysis after 2 weeks with the enzyme activity decreased to 34%. The enhanced and higher storage stability of the modified immobilized cells contributed to the continuous production. Finally, the influences of substrates concentrations and flow rates on the production of GAMG by the modified immobilized cells in packed bed reactor followed by the continuous production of GAMG were studied. The reactor was packed with 440 mL modified immobilized cells and the porosity was 0.55. Subsequently, the so constructed packed reactor was employed for GAMG production. The highest conversion efficiency of GL was up to 33.5% with GAMG output of 0.19 g/L, which was obtained when the balance time of batch reaction was 24 h with 0.72 g/L GL supplemented as substrates at a flow rate of 0.34 mL/min. Furthermore, continuous production of GAMG by the modified immobilized cells in an advanced packed bed reactor was explored. With a retention time of 27 h, the modified immobilized cells worked continuously for 20 d and maintained high enzyme activity. In this way, the daily output of GAMG was approximately 0.193 g/L with the conversion efficiency of 34% from GL and the space-time yield of 13.7 μmol/(L·h). Continuous production of GAMG by the modified immobilized cells in an advanced packed bed reactor laid the foundation for the efficient synthesis of GAMG from GL by whole-cell method.