Efficient production of the anti-aging drug Cycloastragenol: insight from two Glycosidases by enzyme mining.

The telomerase activator cycloastragenol (CA) is regarded as a potential anti-aging drug with promising applications in the food and medical industry. However, one remaining challenge is the low efficiency of CA production. Herein, we developed an enzyme-based approach by applying two enzymes (ß-xylosidase: Xyl-T; ß-glucosidase: Bgcm) for efficient CA production. Both key glycosidases, mined by activity tracking or homology sequence screening, were successfully over-expressed and showed prominent enzymatic activity profiles, including widely pH stability (Xyl-T: pH 3.0-8.0; Bgcm: pH 4.0-10.0), high catalytic efficiency (kcat/Km: 0.096 mM-1s-1 (Xyl-T) and 3.08 mM-1s-1 (Bgcm)), and mesophilic optimum catalytic temperature (50 °C). Besides, the putative catalytic residues (Xyl-T: Asp311/Glu 521; Bgcm: Asp311/Glu 521) and the potential substrate-binding mechanism of Xyl-T and Bgcm were predicted by comprehensive computational analysis, providing valuable insight into the hydrolysis of substrates at the molecular level. Notably, a rationally designed two-step reaction process was introduced to improve the CA yield and increased up to 96.5% in the gram-scale production, providing a potential alternative for the industrial CA bio-production. In essence, the explored enzymes, the developed enzyme-based approach, and the obtained knowledge from catalytic mechanisms empower researchers to further engineer the CA production and might be applied for other chemicals synthesis. KEY POINTS: • A ß-xylosidase and a ß-glucosidase were mined to hydrolyze ASI into CA. • The two recombinant glycosidases showed prominent catalytic profiles. • Two-step enzymatic catalysis for CA production from ASI was developed. Graphical abstract.

Co-Immobilizing Two Glycosidases Based on Cross-Linked Enzyme Aggregates to Enhance Enzymatic Properties for Achieving High Titer Icaritin Biosynthesis.

Icaritin is a rare and high-value isopentane flavonoid compound with remarkable activities. Increasing yields while reducing cost has been a great challenge in icaritin production. Herein, we first reported a high titer icaritin biosynthesis strategy from epimedin C through co-immobilizing α-l-rhamnosidase (Rha1) and ß-glucosidase (Glu4) using cross-linked enzyme aggregates (CLEAs). The created CLEAs exhibited excellent performances in terms of catalytic activity, thermal stability, pH stability, and reusability. Notably, Rha1-CLEAs (Ki: 1 M) and Glu4-CLEAs (Ki: 0.1 M) were more tolerant to sugars (glucose or rhamnose) than free enzymes (0.1 M for Rha1 and 0.007 M for Glu4) by immobilization, achieving the highest icaritin productivity under the highest substrate concentration ever reported. Finally, about 34.24 g/L icaritin could be obtained from 100 g/L epimedin C within 8 h, indicating the great potential for industrialization. This study also provides a promising strategy for the low-cost production of other high-value aglycone compounds by solving poor stability and sugar inhibition of glycosidase.

Enhancing stability and by-product tolerance of ß-glucuronidase based on magnetic cross-linked enzyme aggregates.

ß-glucuronidase is an important catalyst which is highly specific for ß-glucuronides. Here, we constructed magnetic cross-linking ß-glucuronidase aggregates (MCLEAs) to for the production of glycyrrhetinic acid (GA). Before crosslinking via glutaraldehyde, we used carbodiimide to enhance the interaction between enzymes and carboxyl-functionalized Fe3O4, efficiently improving the activity recovery. Compared to free enzymes, both kcat and kcat/Km enhanced, indicating that crosslinking and aggregation brought higher catalytic efficiency to enzymes. MCLEAs enhanced pH and thermal stabilities and retained 63.3% of catalytic activity after 6 cycles. More importantly, it was first found that the glucuronic acid tolerance of ß-glucuronidase after the formation of MCLEAs enhanced 221.5% in 10 mM of glucuronic acid. According to the Raman spectroscopy, the ordered structure of ß-glucuronidase increased from 43.9% to 50.6% after immobilization, which explained the increased stability and tolerance. To sum up, MCLEAs provided an efficient strategy for immobilization of enzymes, which enhanced stability and glucuronic acid tolerance of enzymes. It might be an effective solution to the serious inhibition caused by by-products during the preparation of aglycone from natural glycosides, having a significant applied prospect in industry.

Highly Selective Entrapment of His-Tagged Enzymes on Superparamagnetic Zirconium-Based MOFs with Robust Renewability to Enhance pH and Thermal Stability.

Metal-organic frameworks (MOFs), as a kind of poriferous nanoparticle, are promising candidates for enzyme immobilization to enhance their stability and reusability. However, most MOFs could not specifically immobilize enzymes and regenerate easily, which inevitably leads to serious high consumption and environmental pollution. In this study, renewable and magnetic MOFs were first constructed to specially immobilize His-tagged enzymes from the cell lysates without purification. The immobilized ß-glucuronidase exhibited wider pH adaptability and temperature stability. The relative activity of immobilized ß-glucuronidase was still maintained at ∼80% after eight cycles. Importantly, after simple treatment, the immobilization capacity of regenerated MOFs after simple treatment was restored to more than 90% in the first three times. The specific magnetic MOFs were proven to be an efficient and renewable platform for one-step immobilization and purification of His-tagged enzymes, showing great potential in industrial applications of nanotechnology and biocatalysis.