Cloning and Direct Evolution of a Novel 7-O-Glycosyltransferase from Cucurbita moschata and Its Application in the Efficient Biocatalytic Synthesis of Luteolin-7-O-glucoside.

Luteolin-7-O-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-O-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a kcat/Km value of 772 s-1·M-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-O-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-O-glucoside, which holds broad application prospects in the food and pharmaceutical industry.

Screening and characterization of a GH78 α-l-rhamnosidase from Aspergillus terreus and its application in the bioconversion of icariin to icaritin with recombinant ß-glucosidase.

In this study, a GH78 α-L-rhamnosidase AtRha from Aspergillus terreus CCF3059 was screened and expressed in Pichia pastoris KM71H. The maximum enzyme activity of AtRha was 1000 U/mL after 12 days. AtRha was most active at 65 °C and pH 6.5, displaying excellent thermal stability and pH stability. The kinetic parameters Km, Vmax, kcat and kcat/Km values for pNPR were 0.481 mM, 659 µmol/min·mg, 1065 s-1 and 2214 s-1mM-1, respectively. AtRha could be inhibited by Fe2+, Hg2+ and Cu2+. Moreover, it displayed good tolerance to organic reagents with 52.6% activity in 15%(w/v) methanol. AtRha can hydrolyze icariin containing the α-1 rhamnoside linkage. Furthermore, AtRha and ß-glucosidase TthBg3 showed excellent selectivity to cleave the rhamnose at the 3rd position and the glucosyl at the C-7 group of icariin, which established an effective and green method to produce the more pharmacological active icaritin. In addition, the optimal enzyme addition schemes and the reaction conditions were screened and optimized. After a two-stage transformation under optimized conditions, 0.5 g/L of icariin was transformed into 0.25 g/L of icaritin, with a corresponding molar conversion rate of 91.2%. Our findings provide a new, specific and cost-effective method for the production of icaritin in the industry.

High-level expression of a novel multifunctional GH3 family ß-xylosidase/α-arabinosidase/ß-glucosidase from Dictyoglomus turgidum in Escherichia coli.

A novel ß-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of ß-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with ß-xylosidase, α-arabinofuranoside, α-arabinopyranoside and ß-glucosidase activities, and Kcat/Km was 5245.316 mM-1 s-1, 2077.353 mM-1 s-1, 1626.454 mM-1 s-1, and 470.432 mM-1 s-1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.