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Bioglycosylation is an efficient strategy to improve the biological activity and physicochemical properties of natural compounds for therapeutic drug development. In this study, two caffeic acid phenethyl ester (CAPE) glucosides (G-CAPE and 2G-CAPE) were synthesized by transglycosylation with dextransucrase from Leuconostoc mesenteroides 0326 with CAPE as an acceptor and sucrose as a donor. The products were purified and the structures were characterized. The physicochemical properties, anti-inflammatory activity, and cytotoxicity of the two CAPE glucosides were measured. The water solubility of G-CAPE and 2G-CAPE is 35 and 90 times higher, respectively, than that of CAPE. Compared to CAPE, the monoglycoside product showed superior anti-inflammatory effects, and its inhibition rate of NO, IF-6, and TNF-α is 93.4%, 76.81%, and 56.58% in RAW 264.7 macrophages, respectively, at 20 μM. Also, the cytotoxicity of both products was significantly improved. These glycosylation-modified CAPEs circumvent some of the flaws in CAPE application in anti-inflammatory drugs.

Dextran produced by dextransucrase hold strong potential for industrial applications. The exact determinants of the linkage specificity of glucansucrase enzymes have remained largely unknown. Previous studies have investigated the relationships between structure and linkage specificity of the dextransucrase DSR from Leuconostoc mesenteroides by the site-directed mutagenesis of the catalytic pocket. The glycosidic linkage of dextran produced by mutant enzymes changed slightly by 3% to 20%. The mutagenesis dextransucrases were constructed by inserting an amino acid into a catalytic pocket to investigate the product specificities of dextransucrase thoroughly. The sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to two sequences (Motif II and Motif IV) being targeted, which were inserted by saturation mutagenesis and simultaneously recombined between A552 and V553, D662, and S663. Variants with catalytic activity were screened of the library, which synthesizes high molecular weight α-glucans with different proportions of α(1-4) linkages, which ranges from 0% to 52%. Mutant sequence analysis, biochemical characterization, and molecular modeling studies revealed the mechanism of product specificities. The mutant dextransucrase, which synthesizes hyperbranched dextran, were obtained by the novel mutagenesis method. The different properties of dextran provide the foundation for subsequent studies and application., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dextransucrase (DSR, EC2.4.5.1) from Leuconostoc mesenteroides 0326 which catalyzes sucrose to produce dextran, are popularly used in food and medicine industries. However, its poor kinetic stability especially at higher temperatures is a limiting factor for developing industrial applications. The aim of this study is to improve the enzyme activity and thermal stability of dextransucrase by single, double and triple mutations of proline and lysine. In this work, Pro-473, Pro-678, and Pro-856 were selected as engineering targets and individually replaced with serine. Lys-378, Lys-725, and Lys-955 were replaced with threonine, respectively. Mutant P473S/P856S (MW: 170kDa) was selected as the highest enzymatic activity mutant from fourteen mutants. Specifically, the mutant P473S/P856S showed a significant increase in thermal inactivation with a 7.4-fold increase in half-life at 35°C and a 2-fold increase in catalytic efficiency compared with the wild-type. The results of structural simulation are demonstrated that the new intramolecular hydrogen bonds in mutated enzymes increased structural stability, thereby increasing the thermal stability. The thermal stable mutants have an enormous application potential in food and pharmaceutical industry., (Copyright © 2017 Elsevier B.V. All rights reserved.)