Fisetin glycosides synthesized by cyclodextrin glycosyltransferase from Paenibacillus sp. RB01: characterization, molecular docking, and antioxidant activity

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by α-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTase-glysosyl intermediate, in which O7 and O4’ atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids

Photopatterned antibodies for selective cell attachment

We present a phototriggerable system that allows for the spatiotemporal controlled attachment of selected cell types to a biomaterial using immobilized antibodies that specifically target individual cell phenotypes.o-Nitrobenzyl caged biotin was used to functionalize chitosan membranes and mediate site-specific coupling of streptavidin and biotinylated antibodies after light activation. The ability of this system to capture and immobilize specific cells on a surface was tested using endothelial-specific biotinylated antibodies and nonspecific ones as controls. Homogeneous patterned monolayers of human umbilical vein endothelial cells were obtained on CD31-functionalized surfaces. This is a simple and generic approach that is applicable to other ligands, materials, and cell types and shows the flexibility of caged ligands to trigger and control the interaction between cells and biomaterials.We thank Martina Knecht (MPIP) for help with the synthesis of caged biotin and Dr. Ron Unger and Prof. C. J. Kirkpatrick (University Clinic Mainz, RepairLab) for providing HUVECs. C.A.C. acknowledges funding support from the Portuguese Foundation for Science and Technology (FCT) (fellowship SFRH/BD/61390/2009) and from the International Max-Planck Research School in Mainz. The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. REGPOT-CT2012-316331-POLARIS

Engineering biomolecular microenvironments for cell instructive biomaterials

Engineered cell instructive microenvironments with the ability to stimulate specific cellular responses is a topic of high interest in the fabrication and development of biomaterials for application in tissue engineering. Cells are inherently sensitive to the in vivo microenvironment that is often designed as the cell “niche”. The cell “niche” comprising the extracellular matrix and adjacent cells, influences not only cell architecture and mechanics, but also cell polarity and function. Extensive research has been performed to establish new tools to fabricate biomimetic advanced materials for tissue engineering that incorporate structural, mechanical and biochemical signals that interact with cells in a controlled manner and to recapitulate the in vivo dynamic microenvironment. Bioactive tunable microenvironments using micro and nanofabrication have been successfully developed and proven to be extremely powerful to control intracellular signaling and cell function. This review is focused in the assortment of biochemical signals that have been explored to fabricate bioactive cell microenvironments and the main technologies and chemical strategies to encode them in engineered biomaterials with biological information.The authors thank Fundacao para a Ciencia e Tecnologia for C.A.C.'s PhD grant (SFRH/BD/61390/2009). This work was carried out under the scope of the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no REGPOT-CT2012-316331-POLARIS

Rational re-design of Lactobacillus reuteri 121 inulosucrase for product chain length control

Fructooligosaccharides (FOSs) are well-known prebiotics that are widely used in the food, beverage and pharmaceutical industries. Inulosucrase (E.C. 2.4.1.9) can potentially be used to synthesise FOSs from sucrose. In this study, inulosucrase from Lactobacillus reuteri 121 was engineered by site-directed mutagenesis to change the FOS chain length. Three variants (R483F, R483Y and R483W) were designed, and their binding free energies with 1,1,1-kestopentaose (GF4) were calculated with the Rosetta software. R483F and R483Y were predicted to bind with GF4 better than the wild type, suggesting that these engineered enzymes should be able to effectively extend GF4 by one residue and produce a greater quantity of GF5 than the wild type. MALDI-TOF MS analysis showed that R483F, R483Y and R483W variants could synthesise shorter chain FOSs with a degree of polymerization (DP) up to 11, 10, and 10, respectively, while wild type produced longer FOSs and in polymeric form. Although the decrease in catalytic activity and the increase of hydrolysis/transglycosylation activity ratio was observed, the variants could effectively synthesise FOSs with the yield up to 73% of substrate. Quantitative analysis demonstrated that these variants produced a larger quantity of GF5 than wild type, which was in good agreement with the predicted binding free energy results. Our findings demonstrate the success of using aromatic amino acid residues, at position D418, to block the oligosaccharide binding track of inulosucrase in controlling product chain length

A Versatile Toolbox for Multiplexed Protein Micropatterning by Laser Lithography

Photocleavable oligohistidine peptides (POHP) allow in situ spatial organization of multiple His-tagged proteins onto surfaces functionalized with tris(nitrilotriacetic acid) (tris-NTA). Here, a second generation of POHPs is presented with improved photoresponse and site-specifi c covalent coupling is introduced for generating stable protein assemblies. POHPs with different numbers of histidine residues and a photocleavable linker based on the 4,5-dimethoxy-o-nitrophenyl ethyl chromophore are prepared. These peptides show better photosensitivity than the previously used o-nitrophenyl ethyl derivative. Effi cient and stable caging of tris-NTAfunctionalized surfaces by POHPs comprising 12 histidine residues is demonstrated by multiparameter solid-phase detection techniques. Laser lithographic uncaging by photofragmentation of the POHPs is possible with substantially reduced photodamage as compared to previous approaches. Thus, in situ micropatterning of His-tagged proteins under physiological conditions is demonstrated for the fi rst time. In combination with a short peptide tag for a site-specifi c enzymatic coupling reaction, covalent immobilization of multiple proteins into target micropatterns is possible under physiological conditions.Gropeanu, M.; Bhagawati, M.; Gropeanu, RA.; Rodríguez Muñiz, GM.; Sundaram, S.; Piehler, J.; Campo, AD. (2013). A Versatile Toolbox for Multiplexed Protein Micropatterning by Laser Lithography. Small. 9(6):838-845. doi:10.1002/smll.201201901S8388459