Engineered Glycosidase for Significantly Improved Production of Naturally Rare Vina-Ginsenoside R7.

Ginsenosides are the main bioactive ingredients in plants of the genus Panax. Vina-ginsenoside R7 (VG-R7) is one of the rare high-value ginsenosides with health benefits. The only reported method for preparing VG-R7 involves inefficient and low-yield isolation from highly valuable natural resources. Notoginsenoside Fc (NG-Fc) isolated in the leaves and stems of Panax notoginseng is a suitable substrate for the preparation of VG-R7 via specific hydrolysis of the outside xylose at the C-20 position. Here, we first screened putative enzymes belonging to the glycoside hydrolase (GH) families 1, 3, and 43 and found that KfGH01 can specifically hydrolyze the ß-d-xylopyranosyl-(1 → 6)-ß-d-glucopyranoside linkage of NG-Fc to form VG-R7. The I248F/Y410R variant of KfGH01 obtained by protein engineering displayed a kcat/KM value (305.3 min-1 mM-1) for the reaction enhanced by approximately 270-fold compared with wild-type KfGH01. A change in the shape of the substrate binding pockets in the mutant allows the substrate to sit closer to the catalytic residues which may explain the enhanced catalytic efficiency of the engineered enzyme. This study identifies the first glycosidase for bioconversion of a ginsenoside with more than four sugar units, and it will inspire efforts to investigate other promising enzymes to obtain valuable natural products.

Engineering Isopropanol Dehydrogenase for Efficient Regeneration of Nicotinamide Cofactors.

Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 × 106-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases.

Research and application status of ecological floating bed in eutrophic landscape water restoration.

Ecological floating bed (EFB) has become the preferred technology due to its reputation of green economy, convenience, and efficiency in treating eutrophic landscape water. Based on the statistical analysis of abundant literatures, this paper systematically summarizes the component elements, design parameters, purification mechanism, purification ability, strengthening methods and the correlations among various parameters of EFB, and points out some issues existing in the current research and applications. The results show that the coverage of 5% ~ 38% and water depth of 60 ~ 110 cm should be recommended for EFB design. The microbial transformation-sedimentation contributes mostly to the removal of pollutant, leading to the contribution rate of 51.9% ± 26.4% to nitrogen (N) removal and 50.8% ± 20.4% to phosphorus (P) removal in water respectively. Meanwhile, the average purification abilities of EFB for carbon (C), N and P in water are 4.59 ± 3.82, 0.43 ± 0.35 and 0.04 ± 0.04 g m-2 d-1 respectively. The purification effect is relatively superior when the initial concentration of C, N and P in water is close to C: N: P = 115: 11: 1. In order to enhance the EFB purification efficiency, the methods of artificial aeration, biological chain extension, functional filler introduction, and composite EFB construction can be used. Furthermore, the purification ability of EFB per unit area is correlated positively with water temperature and initial pollutant concentration (r ≥ 0.577, p < 0.01), and correlated negatively with EFB coverage (r ≤ -0.598, p < 0.01). The future research of EFB should focus on enhancing its purification efficiency and seasonal adaptability, studying the mechanism of algae inhibition by allelochemicals, and exploring the harvesting management and resource utilization of plants. This paper provides more reasonable design parameters, feasible management strategies and prospective research directions for environmental managers and researchers who would like to adopt EFB to purify eutrophic landscape water.