Acuminosylation of Tyrosol by a Commercial Diglycosidase.

A commercial glycosidase mixture obtained from Penicillium multicolor (Aromase H2) was found to comprise a specific diglycosidase activity, ß-acuminosidase, alongside undetectable levels of ß-apiosidase. The enzyme was tested in the transglycosylation of tyrosol using 4-nitrophenyl ß-acuminoside as the diglycosyl donor. The reaction was not chemoselective, providing a mixture of Osmanthuside H and its counterpart regioisomer 4-(2-hydroxyethyl)phenyl ß-acuminoside in 58% yield. Aromase H2 is therefore the first commercial ß-acuminosidase which is also able to glycosylate phenolic acceptors.

From Hamamelitannin Synthesis to the Study of Enzymatic Acylations of D-Hamamelose.

The bioactive natural substance, hamamelitannin, was effectively synthesized in two ways. The chemical acylation of 2,3-O-isopropylidene-α,ß-D-hamamelofuranose promoted by Bu2SnO using 3,4,5-tri-O-acetylgalloyl chloride, followed by the deprotection provided hamamelitannin in 79%. Pilot enzymatic benzoylation of D-hamamelose using vinyl benzoate (4 equiv.) and Lipozyme TL IM as a biocatalyst in t-butyl methyl ether (t-BuMeO) gave mainly benzoylated furanoses (89%), of which tribenzoates reached (52%). Enzymatic galloylation of 2,3-O-isopropylidene-α,ß-D-hamamelofuranose with vinyl gallate under the catalysis of Lipozyme TL IM in t-butyl alcohol (t-BuOH) or t-BuMeO provided only the 5-O-galloylated product. The reaction in t-BuMeO proceeded in a shorter reaction time (61 h) and higher yield (82%). The more hydrophobic vinyl 3,4,5-tri-O-acetylgallate in the same reactions gave large amounts of acetylated products. Vinyl gallate and triacetylgallate in the enzymatic acylation of D-hamamelose with Lipozyme TL IM in t-BuMeO yielded 2',5-diacylated hamamelofuranoses in a yield below 20%. The use of other vinyl gallates hydrophobized by methylation or benzylation provided 2',5-diacylated hamamelofuranoses in good yields (65-84%). The reaction with silylated vinyl gallate did not proceed. The best results were obtained with vinyl 2,3,5-tri-O-benzyl gallate, and the only product, 2',5-diacylated hamamelofuranoside precipitated from the reaction mixture (84% in 96 h). After debenzylation, hamamelitannin was obtained an 82% yield from hamamelose in two steps. This synthesis is preparatively undemanding and opens the way to multigram preparations of bioactive hamamelitannin and its analogues.

Medium engineering of phenylethanoid transfructosylation catalysed by yeast ß-fructofuranosidase.

Tyrosol and hydroxytyrosol, by-products of olive oil production, are valuable substrates for enzymatic transglycosylation that can provide products with pharmaceutical potential. Phenylethanoid fructosides are produced from sucrose and phenylethanoids by the catalytic action of ß-fructofuranosidases. This work dealt with the potential of the most abundant ß-fructofuranosidase, baker's yeast invertase, for this bioconversion. The effects of sucrose and phenylethanoid concentrations were investigated with a focus on the selectivity of phenylethanoid transfructosylation and fructoside yields. For this purpose, initial rate and progress curve experiments were carried out for the initial (hydroxy)tyrosol and sucrose concentrations of 0.072-0.3 M and 1-2 M, respectively. Reaction courses exhibited either a maximum or plateau of fructoside yield in the range of about 10-18%. The addition of deep eutectic solvents was applied in the concentration range from 5 to 70% (v/v) to investigate the possibility of shifting the reaction equilibrium towards fructoside synthesis.

Synthesis of Tyrosol and Hydroxytyrosol Glycofuranosides and Their Biochemical and Biological Activities in Cell-Free and Cellular Assays.

Tyrosol (T) and hydroxytyrosol (HOT) and their glycosides are promising candidates for applications in functional food products or in complementary therapy. A series of phenylethanoid glycofuranosides (PEGFs) were synthesized to compare some of their biochemical and biological activities with T and HOT. The optimization of glycosylation promoted by environmentally benign basic zinc carbonate was performed to prepare HOT α-L-arabino-, ß-D-apio-, and ß-D-ribofuranosides. T and HOT ß-D-fructofuranosides, prepared by enzymatic transfructosylation of T and HOT, were also included in the comparative study. The antioxidant capacity and DNA-protective potential of T, HOT, and PEGFs on plasmid DNA were determined using cell-free assays. The DNA-damaging potential of the studied compounds for human hepatoma HepG2 cells and their DNA-protective potential on HepG2 cells against hydrogen peroxide were evaluated using the comet assay. Experiments revealed a spectrum of different activities of the studied compounds. HOT and HOT ß-D-fructofuranoside appear to be the best-performing scavengers and protectants of plasmid DNA and HepG2 cells. T and T ß-D-fructofuranoside display almost zero or low scavenging/antioxidant activity and protective effects on plasmid DNA or HepG2 cells. The results imply that especially HOT ß-D-fructofuranoside and ß-D-apiofuranoside could be considered as prospective molecules for the subsequent design of supplements with potential in food and health protection.

Transrutinosylation of tyrosol by flower buds of Sophora japonica.

Dried flower buds of Japanese sophora (Sophora japonica) comprising rutinosidase activity were tested in rutinosylation of tyrosol via transglycosylation process from rutin. Optimal conditions for transrutinosylation of tyrosol were 49 mM rutin and 290 mM tyrosol, giving maximum conversion up to 66.4% and 24% yield of isolated and purified rutinoside. The rutinosylation proceeded exclusively on the primary hydroxyl of tyrosol, thus forming rhamnosylated derivative of salidroside. This strict regioselectivity differentiates the sophora biocatalyst from microbial rutinosidases.

Comparative study of relationship between structure of phenylethanoid glycopyranosides and their activities using cell-free assays and human cells cultured in vitro.

The study focused on protective potential of phytochemicals applicable in prevention and health protection is of great importance. Various structures of these compounds and a wide range of their biological activities have inspired organic chemists to sythesize their effective analogues in order to further increase their efficacy. The aims of our study were (i) to synthesize phenylethanoid glycopyranosides: salidroside (SALI - tyrosol ß-d-glucopyranoside), tyrosol ß-d-galactopyranoside (TYBGAL), tyrosol α-d-galactopyranoside (TYAGAL), tyrosol α-d-mannopyranoside (TYAMAN), hydroxytyrosol α-d-mannopyranoside (HOTAMA), homosyringyl ß-d-glucopyranoside (HSYGLU), hydroxytyrosol ß-d-xylopyranoside (HOTXYL) and hydroxysalidroside (HOSALI); (ii) to determine their antioxidant capacities (cell-free approaches); (iii) to evaluate their cytotoxicity (MTT test), protectivity against hydrogen peroxide (H2O2; comet assay) and effect on the intracellular glutathione level (iGSH; flow cytometry) in experimental system utilizing human hepatoma HepG2 cells. HOSALI, HOTAMA, HOTXYL and HSYGLU manifested the highest antioxidant capacity in cell-free assays and they were most active in protection of HepG2 cells against H2O2. On the other hand, pre-treatment of HepG2 cells with SALI had protective effects even though SALI displayed almost no activity in cell-free assays. Differences in the efficacy of the analogues revealed that structures of their molecules in terms of aglycone combined with sugar moiety affect their activities.

Screening of Commercial Enzymes for Transfructosylation of Tyrosol: Effect of Process Conditions and Reaction Network.

Enzymatic fructosylation of organic acceptors other than saccharides brings new possibilities to synthesize molecules that do not exist in nature. The introduction of fructosyl moiety may lead to glycosides possessing enhanced physicochemical and bioactive properties which could be useful in the pharmaceutical and cosmetic industry. In this work, the regioselective synthesis of tyrosol ß-d-fructofuranoside (TF) catalyzed by ß-fructofuranosidase is investigated. In the first step, 32 commercial enzyme preparations are screened for fructoside-hydrolyzing activity. The most active preparations are subsequently examined for fructofuranosyl transfer from sucrose to tyrosol. The best candidate, Novozym 188, is chosen to study the effect of reaction conditions on the product formation in a batch reactor. The effects of substrate concentration, temperature, pH, time, and enzyme dosage on the concentration of TF produced are studied using the design of experiments methodology. The maximal product concentration of 3.8 g L-1 is achieved for the sucrose concentration of 1.5 m, tyrosol concentration of 29 g L-1 , temperature of 41 °C, and pH 5.1. Besides the main transfructosylation reaction between sucrose and tyrosol, several side reactions take place. A reaction network includes also the formation of fructooligosaccharides and the hydrolysis of sucrose and all reaction products.

Reaction mechanism of ß-apiosidase from Aspergillus aculeatus.

Apiosidases are glycosidases relevant for aroma development during fermentation of wines and black tea. Reaction mechanism of apiosidase from Aspergillus aculeatus in commercial glycanase Viscozyme L was studied by 1H NMR technique. Study of hydrolysis of 4-nitrophenyl ß-D-apiofuranoside revealed that this reaction proceeds with inversion of hydroxyl group in the anomeric center, which confirms inverting mechanism of the enzyme and its inability to catalyze transapiosylation in syntheses of apiosides.

Salidroside, a Chemopreventive Glycoside, Diminishes Cytotoxic Effect of Cisplatin in Vitro.

Natural products represent the source or the inspiration for the majority of the active ingredients of medicines because of their structural diversity and a wide range of biological effects. Our aims in this study were (i) to synthesize enzymatically salidroside (SAL), the most effective phenylethanoid glycoside in Rhodiola species; (ii) to examine its antioxidant capacity using cell-free assays (reducing power, DPPH radicals scavenging and Fe2+ -chelating assays); (iii) to assess its DNA-protective potential on plasmid DNA (DNA topology assay) and in HepG2 cells (comet assay) damaged by Fe2+ ions and hydrogen peroxide, respectively; and (iv) to investigate the effects of SAL, cisplatin (CDDP) and combined treatments of SAL + CDDP on cell viability (MTT test), level of DNA damage (comet assay), proliferation, cell cycle (flow cytometry) and the expression of signalling molecules associated with cell growth and apoptotic pathways (Western immunoblotting). We found out that SAL manifested low antioxidant and DNA-protective capacity in all assays used. In both parental A2780 and CDDP-resistant A2780/CP human ovarian carcinoma cells, SAL itself exerted in fact no impact on the viability, while in combination with CDDP it showed antagonistic effect supporting the chemopreventive activity on the CDDP-induced cell damage. These results were confirmed by the partial reversal of the cell cycle alterations and the DNA damage level, as well as with partial restoration of cell survival/signalling pathways, when the expression of these molecules partially returned to their proper levels.

Efficient chemoenzymatic synthesis of 4-nitrophenyl ß-d-apiofuranoside and its use in screening of ß-d-apiofuranosidases.

4-Nitrophenyl ß-d-apiofuranoside as a chromogenic probe for detection of ß-d-apiofuranosidase activity was prepared in 61% yield from 2,3-isopropylidene-α,ß-d-apiofuranose through a sequence of five reactions. The synthesis involves one regioselective enzymatic step-benzoylation of primary hydroxyl of 2,3-isopropylidene-α,ß-d-apiofuranose catalysed by Lipolase 100T and stereoselective ß-d-apiofuranosylation of p-nitrophenol using BF3⋅OEt2/Et3N. The product was used for screening of ß-d-apiofuranosidase activity in 61 samples of crude commercial enzymes and plant materials. Fifteen enzyme preparations originating from different strains of genera Aspergillus display ß-d-apiofuranosidase activity. The highest activity was found in Rapidase AR 2000 (78.27 U/g) and lyophilized Viscozyme L (64,36 U/g).

Effective enzymatic caffeoylation of natural glucopyranosides.

Reaction system was developed for enzymatic caffeoylation of model saccharidic acceptor methyl ß-d-glucopyranoside to obtain exclusively methyl 6-O-caffeoyl-ß-D-glucopyranoside. Reaction with starting concentration of acceptor 0.2 M provided 73% yield of purified product within 17 days. Reactions with low acceptor concentrations (0.04 and 0.08 M) run to the completion within 7 days. Such highly effective and regioselective reaction was promoted by Lipozyme TL IM in tert-butanol, using vinyl caffeate as acylation donor. The optimized reaction conditions were used in preparative caffeoylation of natural substances-arbutin and salidroside, giving 75% of 6-O-caffeoylated arbutin (robustaside B) and 74% of 6-O-caffeoylated salidroside as the only products after 12 and 16 days, respectively.

Synthesis of 4-nitrophenyl caffeate and its use in assays of caffeoyl esterases.

We have prepared 4-nitrophenyl caffeate by a combination of standard procedures of organic synthesis and enzymatic deacetylation. Based on hydrolysis of 4-nitrophenyl caffeate, a convenient spectrophotometric assay was developed for specific monitoring of caffeoyl esterase. The method is fast and easy to perform, and it requires no expensive equipment. Its reliability was tested on eight enzyme preparations comprising various combinations of caffeoyl, feruloyl, and acetyl esterase as well as protease activities.

Donor specificity and regioselectivity in Lipolase mediated acylations of methyl α-D-glucopyranoside by vinyl esters of phenolic acids and their analogues.

Methyl α-D-glucopyranoside as a model acceptor was acylated by several phenolic and non-phenolic vinyl esters using immobilised Lipolase. Donor specificity and regioselectivity of reaction were investigated. Conversion and rate of acylation by structurally varied donors indicates that the synthetic reactivity of Lipolase corresponds to the hydrolytic activity of feruloyl esterase type A. Lipolase exhibited remarkable regioselectivity for primary position of methyl α-D-glucopyranoside. The acylation occurred exclusively at 6-O primary position when vinyl esters of phenolic acids (hydroxybenzoates, hydroxyphenylalkanoates and hydroxycinnamates) served as acyl donors (5-77%). In addition to the major 6-O-acyl products (52-79%), 2,6-di-O-acylated derivatives were isolated from reaction mixtures (2-13%) when non-phenolic donors were used (vinyl esters of fully methoxylated derivatives of phenolic acids, along with vinyl benzoates, cinnamates or some heterocyclic analogues).

Monitoring of PQQ-dependent glucose dehydrogenase substrate specificity for its potential use in biocatalysis and bioanalysis.

Substrate specificity of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase was investigated in biosensor arrangement for understanding the suitability and the limitations of its use in bioanalysis and bioproduction of chemicals. The study demonstrated a very broad substrate specificity of biosensor utilising soluble form of PQQ-dependent glucose dehydrogenase. Nineteen saccharides out of 31 were oxidised by the sensor. Investigation confirmed strong importance of hydroxyl configuration in the positions 2 and 5 of oxidised saccharides. The broad specificity suggests that the PQQ-dependent glucose dehydrogenase could be utilised for analysis of other sugars than glucose in food samples for various production processes and for biofuel cells. In addition, the results showed that the substrate specificity of enzymes can be effectively and generally studied by biosensor arrangement for research purposes. This layout utilising immobilised enzyme allowed performing comprehensive study using a small amount of enzymes and thus saving the costs and time.

Analysis of ethanol in fermentation samples by a robust nanocomposite-based microbial biosensor.

A robust microbial biosensor was constructed from a bionanocomposite prepared by a direct mixing of bacterial cells of Gluconobacter oxydans and carbon nanotubes with ferricyanide employed as a mediator for enhanced sensitivity of ethanol oxidation. A successful integration of the device into flow injection analysis mode of operation provided a high sensitivity of detection of (74 ± 2.7) µA mM(-1) cm(-2), a low detection limit of 5 µM and a linear range from 10 µM up to 1 mM. A short response time of the biosensor allowed a sample throughput of 67 h(-1) at 0.3 ml min(-1). The biosensor exhibited high operational stability with a decrease in the biosensor response of 1.7% during 43 h of continuous operation. The device was used to analyse ethanol in fermentation samples with a good agreement with a HPLC method.

A novel microbial biosensor based on cells of Gluconobacter oxydans for the selective determination of 1,3-propanediol in the presence of glycerol and its application to bioprocess monitoring.

Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards<2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h-1) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R2=0.999).

Development of enzyme flow calorimeter system for monitoring of microbial glycerol conversion.

Glycerokinase from Cellulomonas sp. was used to develop biosensor based on flow calorimetry for quantitative analysis of glycerol during bioconversion process. An automatic flow injection analysis device with the glycerol biosensor was built and tested during growth on glycerol of 1,3-propanediol-producing bacteria. The biosensor exhibited an extreme storage and operational stability enabling us to use it for more than 2 years without significant loss of sensitivity. No interference with 1,3-propanediol and fermentation medium was observed. The linear range of glycerol concentration up to 70 mM was extended by developed automatic dilution technique with the aim of automatic online monitoring of microbial process. The analytical system was able to monitor the bioconversion process in a fully automatic way during the whole run with sampling frequency of one sample per 10 min.

An efficient chemoenzymatic route to methyl 4-O-benzyl-2,3-anhydro-beta-D-lyxopyranoside from methyl beta-D-xylopyranoside.

Methyl 4-O-benzyl-2,3-anhydro-beta-D-lyxopyranoside, an intermediate for the preparation of methyl beta-D-xylopyranoside derivatives modified at C-2, was obtained in five steps in 58% yield. The synthetic sequence starts from methyl beta-D-xylopyranoside through two main steps involving regioselective enzymatic acetylation and deacetylation catalyzed by lipase PS.

A spectrophotometric assay for feruloyl esterases.

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.