Jianlin Chu

An efficient novel glycosylation of flavonoid by β-fructosidase resistant to hydrophilic organic solvents.

An effective approach was successfully developed to isolate glycosidase with resistance of hydrophilic organic solvent, simultaneously with acceptor specificity of the target substrate. By this approach, an efficient solvent tolerant glycosidase producing bacterium Arthrobacter nicotianae XM6 was obtained. The β-fructosidase from strain XM6 shows high activity and stability in 10-25% DMSO and 10-20% methanol with 90-99% yields of puerarin glycosides. The addition of hydrophilic solvents not only greatly promoted the solubility of puerarin, but also regulated main products from multifructosyl puerarin to monofructosyl puerarin with increasing solvent concentration. Extraordinary highly efficient synthesis of puerarin glycosides (111.3 g/L of monofructosyl puerarin and 35.6 g/L of difructosyl puerarin) was attained in 25% DMSO solvent system from 110.4 g/L puerarin, which resulted a great facility for purification in large-scale process. The most novelty was that the β-fructosidase did not hydrolyze almost the newly formed glycosides using simply sucrose as donor.

Efficient enzymatic synthesis of mangiferin glycosides in hydrophilic organic solvents

β-Fructofuranosidase from Arthrobacter arilaitensis NJEM01 showed high activity and stability in 15–25% (v/v) methanol, 15–20% (v/v) DMSO, and 15–20% (v/v) ethanol with 91.2–99.7% total molar yields of mangiferin glycosides. The addition of hydrophilic organic solvents not only greatly promoted the solubility of the substrate, but also revealed a regulatory effect on mangiferin glycoside formation with increasing solvent concentration. Extraordinarily high concentrations and high yields of mangiferin glycosides were attained in the 20% DMSO solvent system from 47.6 g L−1 mangiferin with β-fructofuranosidase using sucrose as the donor, which greatly simplified the preparation process. β-Fructofuranosidase showed an exquisite regioselectivity for C-glucosylflavone.

Isolation, identification and pharmacokinetic analysis of fructosyl puerarins from enzymatic glycosylation.

A method of using high-speed counter-current chromatography (HSCCC) was established for preparative isolation and purification of puerarin glycosides from the crude sample after enzymatic glycosylation of puerarin. Four fructosyl puerarins were successfully purified for the first time by HSCCC with a two-phase-solvent system composed of n-butanol-acetic acid-water (4:1.5:6, v/v/v). A total of 5mg of puerarin (1), 20mg of β-d-fructofuranosyl-(2→6)-puerarin (2), 41mg of β-d-difructofuranosyl-(2→6)-puerarin (3), 18mg of β-d-trifructofuranosyl-(2→6)-puerarin (4) and 15mg β-d-tetrafructofuranosyl-(2→6)-puerarin (5) were obtained in one-step separation from 100mg of the crude sample with purities of 98.5%, 98.3%, 98.9%, 97.8%, 97.5% and 97.2%, respectively. Among them, compounds 2-5 are novel compounds, and their chemical structures were identified by HRMS, (1)H NMR, (13)C NMR and 2D NMR. Pharmacokinetic analysis showed that β-d-fructofuranosyl-(2→6)-puerarin (2) was able to maintain higher plasma concentrations and have a longer mean residence time in the blood than puerarin.

High production of succinyl isoflavone glycosides by Bacillus licheniformis ZSP01 resting cells in aqueous miscible organic medium

To achieve efficient production of succinyldaidzin and succinylgenistin, resting cells of a solvent‐stable strain Bacillus licheniformis ZSP01 were used to react with pure isoflavones or soybean flour extract in a reaction medium with 10% dimethyl sulfoxide. Strikingly, 0.8 mM daidzein, 0.8 mM genistein, 2.0 mM daidzin, and 2.0 mM genistin were transformed to succinyl isoflavone glycosides in 27 H (yield >90%). The soybean flour extract (6.1%, w/v) contained 0.32 mM daidzein, 0.84 mM daidzin, 0.38 mM genistein, and 1.04 mM genistin. Over 95% of total isoflavones (daidzein, daidzin, genistein, and genistin) in the soybean flour extract were converted to succinyl isoflavone glycosides after 27 H. Strain ZSP01 shows both high glycosylation and succinylation activities. These results suggest that B. licheniformis ZSP01 could be useful for the efficient production of succinyl soybean isoflavone glycosides.

Characteristics of an Organic Solvent-Tolerant β-Fructofuranosidase from Arthrobacter arilaitensis NJEM01 and Efficient Synthesis of Prebiotic Kestose

An organic solvent-tolerant β-fructofuranosidase (β-FFase) from Arthrobacter arilaitensis NJEM01 was purified, characterized, cloned, and overexpressed in Escherichia coli. The mature β-FFase contained 495 amino acid residues with an estimated molecular mass of 55 kDa. The purified β-FFase from strain NJEM01 was very stable in the buffer systems (pH 5.0-9.5) and showed high stability below 45 °C. Furthermore, the enzyme exhibited relatively high solvent stability in various aqueous organic mixtures and retained nearly 100% of its initial activity after incubation for 10 days in 20% (v/v) DMSO. In addition, the β-FFase exhibited high transfructosylation activity, synthesized prebiotic products of mainly 6-kestose (up to 476 g/L), and showed fructosyl receptor specificity to C-glucosyl flavone. A relatively high yield of FOS was achieved by the β-FFase from bacterium with a high concentration of sucrose. It made the β-FFase an exploitable biocatalyst for the production of glycosides of natural products and prebiotic kestose.

An efficient production of high-pure xylooligosaccharides from corncob with affinity adsorption-enzymatic reaction integrated approach

Xylooligosaccharides (XOS) are high value-added ingredients for functional foods and they have potential use as prebiotics. In order to reduce the production cost of XOS, we constructed a fusion enzyme consisting of expansin and endo-xylanase from B. subtilis Lucky9 to produce high-pure XOS with affinity adsorption-enzymatic reaction integrated approach. By optimization of inserting linker, the specific xylanase activity of fusion enzyme with R2 linker was increased 1.28-fold than that of native xylanase. Electrophoretic pure fusion enzyme was separated from crude enzyme extracts by affinity adsorption on corncob substrate, with 90.3% recovery in optimized conditions. Then, the fusion enzyme-corncob mixture was directly hydrolyzed. The yield of XOS reached 6.91mg/mL. The production of XOS with high purity of 91.7% was obtained by one step of centrifugation. This affinity adsorption-enzymatic reaction integrated XOS production approach provides a green route to alternative XOS traditional enzymatic production.