Search

• Content of poorly soluble naringin substrate was increased by resin adsorption. • Resin adsorption improve naringin biotransformation efficiency. • Liter-scale hydrolysis of naringin at high concentration was performed. • The enzymatic product naringenin could be purified from the resin. Naringenin, a natural plant flavonoid found in citrus fruits, has been reported to exhibit a wide range of pharmacological functions, including anticancer, antioxidant, antiatherogenic, antithrombotic, and vasodilator activities. Naringenin can be produced from the naringinase (NGase)-catalyzed enzymatic hydrolysis of naringin. However, the poor solubility of naringin in aqueous systems considerably limits the efficiency of naringenin biocatalysis. In this work, a novel substrate adsorption system was proposed for naringin adsorption to increase the efficiency of naringin hydrolysis and naringenin production. Three Amberlite macroporous resins, namely, XAD-4, XAD-7HP and XAD-16, were investigated for their naringin adsorption capacities and effects on NGase hydrolysis. Results indicated that the physical properties of the resins played a critical role in naringin adsorption and naringenin enzymatic synthesis. Naringin hydrolysis was carried out using free and adsorbed substrates. The substrate adsorption strategy could increase the catalytic efficiency at a high naringin concentration. In addition, the reaction conditions for enzymatic naringenin synthesis were optimized, and naringenin was prepared at a liter scale with a high substrate concentration. These results suggested that substrate adsorption is a promising strategy to increase the enzymatic hydrolysis efficiency of naringenin in aqueous systems. [ABSTRACT FROM AUTHOR]

Naringinase (NGase) is a multi-compound enzyme with the subunits of α-L-rhamnosidase (Rha) and β-D-glucosidase (BGL). The biotransformation of naringin into the more valuable naringenin was impeded by the uncoordinated activity and instability of natural naringinases. In this study, the efficient artificial fusion naringinases of Rha from Spirochaeta thermophila (StRha) and BGL from Pyrococcus furiosus (PfBGL) were constructed and utilized in biosynthesis of naringenin. Fusion naringinases StRha-PfBGL (DL-NGase), StRha-(GGGGS)-PfBGL (FL-NGase) and StRha-(EAAAK)-PfBGL (RL-NGase) were obtained by fusing StRha and PfBGL through direct fusion or mediated by peptide linkers. DL-NGase showed more coordinated activity and better thermostability than FL-NGase and RL-NGase. The optimal temperature of StRha and PfBGL of DL-NGase was above 65 °C, and both retained above 75% of the initial activities at 55 °C for 2 h. In addition, fusion naringinases exhibited better catalytic performance than the mixed free enzyme systems, and the catalytic efficiency of DL-NGase was increased by 49.8%. Furthermore, DL-NGase whole-cell catalyst was applied for the high-efficient biosynthesis of naringenin, and the final naringenin yield was 13.5 mg/mL with a time-space yield of 2.25 mg/mL/h. These results demonstrate the great potential of artificial fusion naringinases for the efficient bioconversion of naringin to naringenin. [Display omitted] • Artificial fusion naringinases were successfully constructed for the first time. • Artificial fusion naringinases show better operating temperature and thermostability. • Artificial fusion naringinases improve the biosynthesis efficiency of naringenin. [ABSTRACT FROM AUTHOR]

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The K m of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while k cat / K m value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin. • A novel α-L-rhamnosidase from Spirochaeta thermophila was characterized. • St-Rha showed excellent thermostability and methanol tolerance. • The highest production yield of prunin was obtained using methanol cosolvent systems. • Mechanism of methanol tolerance for α-L-rhamnosidase was studied for the first time. [ABSTRACT FROM AUTHOR]

The pods of Caesalpinia spinosa consists of gallotannins, which represent more than 50 % of its weight. The traditional chemical procedure used to produce gallic acid (GA) from tara gallotannins is a high-pollution process. Although biological procedures, including microbial fermentation and enzymatic hydrolysis, are environmentally friendly, insufficient production efficiency significantly hinders industrial application. In this study, we proposed a novel strategy for the simultaneous extraction and enzymatic hydrolysis of tara gallotannins with intermittent feeding. A recombinant thermostable tannase (Tan1) derived from Aspergillus oryzae was first constructed to adapt to the high-temperature simultaneous extraction and enzymatic hydrolysis process. Tan1 showed competent activity yield (83.11 U/mL) and excellent heat resistance, exhibiting the highest activity at 80 °C and half-lives at 60 °C for more than 6 h. The production efficiencies of GA using different enzymatic hydrolysis strategies were compared. The simultaneous extraction and enzymatic hydrolysis strategy combined with intermittent feeding minimized the inhibition of tara gallotannins on Tan1 activity and resulted in an increased GA yield. Using this strategy, 192 g/L tara powder (intermittent feed for 6 times) was treated with Tan1 at 60 °C for 177 min, resulting in 78.5 g/L GA output which was the highest among the currently reported values obtained using biological protocols for GA production. • A thermal stable tannase with high activity was successfully expressed in Pichia pastoris. • A novel strategy was proposed to minimize inhibition effect of tannic acid on tannase. • Highest yield was obtained among biological approaches for gallic acid production. [ABSTRACT FROM AUTHOR]

Naringinase is a kind of glycosidase that catalyzes the biotransformation of naringin into naringenin. It is a multi-compound enzyme that provides the activity of α- L -rhamnosidase and β-glucosidase. In this study, an artificial naringinase system was constructed by co-immobilizing α- L -rhamnosidase (Aspergillus oryzae FJ0123, Ao-rha) and β-glucosidase (Thermotoga maritima MSB8, Tm-glu) on magnetic silica-based chitosan microspheres (MSC). The molar ratio of enzymes and the preparation conditions were optimized for improving synergistic effect and immobilization efficiency. Under optimized conditions (the molar ratio of Ao-rha and Tm-glu, 3:1; temperature, 25 °C; glutaraldehyde concentration, 2.0%; pH, 3.0; time, 9 h), the immobilization yield and activity recovery were 61.4% and 37.3% for Ao-rha and 90.1% and 56.3% for Tm-glu, respectively. Biochemical characterization indicated that MSC-naringinase had better adaptability and stability than free enzymes. The activity of MSC-naringinase was maintained at 58.7% after 10 times of recycling. The catalytic conditions were also investigated. The cascade reactions of naringin to prunin and prunin to naringenin by using MSC-naringinase resulted in a yield of 0.62 mg/mL and a conversion rate of 96.9%, respectively, without prunin accumulation. These results indicated the great potential of MSC-naringinase as an efficient artificial naringinase system for cooperative enzymatic synthesis of naringenin. [Display omitted] • Artificial naringinase system consisting of α- L -rhamnosidase and β-glucosidase was prepared. • Direct conversion of naringin to naringenin was achieved by MSC-naringinase. • After 10 cycles, MSC-naringinase retained 58.7% of its initial activity. • The conversion rate of naringin to naringenin reached 96.9%. [ABSTRACT FROM AUTHOR]

Tannase is widely used in tea beverage processing because of its ability to catalyze the hydrolysis of hydrolysable tannins or gallic acid esters and effectively improve the quality of tea extracts through enzymatic extraction. A new thermophilic tannase was cloned from Aspergillus niger FJ0118 and characterized. The tannase exhibited an optimal reaction temperature of 80 °C and retained 89.6% of the initial activity after incubation at 60 °C for 2 h. The enzymatic extraction of green tea at high temperature (70 °C) for a short time (40 min) was devised on the basis of the superior thermal stability of tannase. The enzymatic reaction significantly increased the total polyphenol content of green tea extract from 137 g·kg−1 to 291 g·kg−1. The enzymatic reaction effectively degraded the ester catechins into non-ester catechins compared with the water extraction method. Results suggested that the thermally stable tannase exhibited potential applications in the enzymatic extraction of green tea beverage. [ABSTRACT FROM AUTHOR]