7296-64-2

The hydrolysis of o-nitrophenyl-β-d-galactopyranoside (ONPG) by β-galactosidase immobilized on Sepharose CL-4B was investigated in five different ionic liquids (ILs), 1-butyl-3-methylimidazolium X-; [X = CF3SO3-, BF4-, PF6-, CH3SO4-and N(CN)2-]. Michaelis-Menten kinetic studies were conducted in phosphate buffer and in the five ionic liquids. For the immobilized enzyme in the ILs, the Km values were lower (0.36-1.2 mmol ONPG) while the Vmax values were higher (0.04-0.008 min- 1) compared to those in aqueous phosphate buffer suggesting a marked increase in the efficiency of the immobilized enzyme in the ionic liquid. For the free enzyme in the ionic liquids, the Km values, in general, were larger (0.45-4.96 mmol ONPG) than those of the immobilized enzyme in the ionic liquid. A postulated mechanism for the hydrolysis is suggested, involving interception of the intermediate oxonium ion species by the counter ion of the ionic liquid, thereby enabling the hydrolysis to occur at a faster rate.

An exopolysaccharide (EPS) was purified from the probiotic bacterium Bacillus albus DM-15, isolated from the Indian Ayurvedic traditional medicine Dasamoolarishta. Gas chromatography-mass spectrophotometry and nuclear magnetic resonance (NMR) analyses revealed the heteropolymeric nature of the purified EPS with monosaccharide units of glucose, galactose, xylose, and rhamnose. Size-exclusion chromatography had shown the molecular weight of the purified EPS as around 240 kDa. X-ray powder diffraction analysis confirmed the non-crystalline amorphous nature of the EPS. Furthermore, the purified EPS showed the maximum flocculation activity (72.80%) with kaolin clay and emulsification activity (67.04%) with xylene. In addition, the EPS exhibits significant antioxidant activities on DPPH (58.17 ± 0.054%), ABTS (70.47 ± 0.854%) and nitric oxide (58.92 ± 0.744%) radicals in a concentration-dependent way. Moreover, the EPS showed promising cytotoxic activity (20 ± 0.97 μg mL–1) against the lung carcinoma cells (A549), and subsequent cellular staining revealed apoptotic necrotic characters in damaged A549 cells. The EPS purified from the probiotic strain B. albus DM-15 can be further studied and exploited as a potential carbohydrate polymer in food, cosmetic, pharmaceutical, and biomedical applications.

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Six undescribed oleanane-type saponins, named as Hylomeconosides L-Q, were isolated from the whole herb of Hylomecon Japonica, their structures were determined by analysis of 1D and 2D-NMR (1H–1H COSY, HSQC, and HMBC) spectroscopic data, mass spectrometry (HRESI-MS) and chromatographic data (GC and LC). Their structures were identified as 3-O-β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-galactopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-β-L-arabinopyranoside; 3-O-β-D-galactopyranosyl-(1 → 2)-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-β-D-xylopyranosyl-(1 → 3)-β-D-glucuronopyranosyl gypsogenin 28-O-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-β-D-galactopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl quillaic acid 28-O-β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-quinovopyranoside; 3-O-β-D-galactopyranosyl-(1 → 2)-[α-L-rhamnopyranosyl-(1 → 3)]-β-D-glucuronopyranosyl quillaic acid 28-O-β-D-xylopyranosyl-(1 → 3)-β-D-xylopyranosyl-(1 → 4)-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside. Hylomeconosides L-Q showed selective cytotoxicities against human cancer cell lines A549, AGS, HeLa, Huh 7, HT29 and K562. These results represent a contribution to the chemotaxonomy of the saponins of Hylomecon Japonica and their bioactivities.

The enzyme β-galactosidase can synthesise novel prebiotics such as oligosaccharides derived from lactulose (OsLu) which can be added as a supplement in infant food formula. In this study, the intracellular β-galactosidase produced by the alkaliphilic bacterium Paracoccus marcusii was extracted and purified to homogeneity using hydrophobic and metal affinity chromatography. The purification resulted in 18 U/mg specific activity, with a yield of 8.86% and an 18-fold increase in purity. The purified enzyme was a monomer with an 86 kDa molecular weight as determined by SDS PAGE and Q-TOF-LC/MS. β-Galactosidase was highly active at 50 °C and pH 6–8. The enzyme displayed an alkali tolerant nature by maintaining more than 90% of its initial activity over a pH range of 5–9 after 3 h of incubation. Furthermore, the enzyme activity was enhanced by 37% in the presence of 5 M NaCl and 3 M KCl, indicating its halophilic nature. The effects of metal ions, solvents, and other chemicals on enzyme activity were also studied. The kinetic parameters KM and Vmax of β-galactosidase were 1 mM and 8.56 μmoles/ml/min and 72.72 mM and 11.81 μmoles/ml/min on using oNPG and lactose as substrates. P. marcusii β-galactosidase efficiently catalysed the transgalactosylation reaction and synthesised 57 g/L OsLu from 300 g/L lactulose at 40 °C. Thus, in this study we identified a new β-galactosidase from P. marcusii that can be used for the industrial production of prebiotic oligosaccharides.

In recent years, the scientific interest and particularly the economic significance of halophytic plants has been highly demanding due to the medicinal and nutraceutical potential of its bioactive compounds. A xero-halophyte Bassia indica is deemed to be

Enzyme inhibitors play a crucial role in diagnosis of a wide spectrum of diseases related to bacterial infections. We report here the effect of a water-soluble self-assembled PdII8 molecular cage towards β-galactosidase enzyme activity. The molecular cage is composed of a tetrapyridyl donor (L) and cis-[(en)Pd(NO3)2] (en=ethane-1,2-diamine) acceptor and it has a hydrophobic internal cavity. We have observed that the acceptor moiety mainly possesses the ability to inactivate the β-galactosidase enzyme activity. Kinetic investigation revealed the mixed mode of inhibition. This inhibition strategy was extended to control the growth of methicillin-resistant Staphylococcus aureus. The internalization of the Pd(II) cage inside the bacteria was confirmed when bacterial solutions were incubated with curcumin loaded cage. The intrinsic green fluorescence of curcumin made the bacteria glow when put under an optical microscope. Furthermore, this curcumin loaded molecular cage shows an enhanced antibacterial activity. Thus, PdII8 molecular cage is quite attractive due to its dual role as enzyme inhibitor and drug carrier.

Eleven previously undescribed triterpene saponins, named silenegallisaponin A-K (1–11), were isolated from the aerial parts of Silene gallica L. Their structures were elucidated by analysis of 1D and 2D-NMR spectroscopic data and mass spectrometry (HR-ESI-MS). The saponins comprised caulophyllogenin, echinocystic acid, or quillaic acid substituted at C-3 by a β-D-glucuronic acid or β-D-galactopyranosyl-(1 → 3)-β-D-glucuronopyranoside and at C-28 by a β-D-fucopyranose substituted at C-2 by a β-D-glucose and at C-3 by a β-D-glucose or a β-D-quinovose.

Previously, various steroidal glycosides were reported from plants of Cestrum species. However, phytochemical investigation has not been conducted on Cestrum newellii. A systematic phytochemical investigation of the leaves of C. newellii resulted in the isolation of eight novel steroidal glycosides (1-8), which were classified into three spirostanol glycosides (1-3), two furostanol glycosides (4 and 5), two pseudofurostanol glycosides (6 and 7), and one cholestane glycoside (8). In addition, three known cholestane glycosides (9-11) were isolated and identified. The structures of the new compounds were determined based on spectroscopic data and chemical transformations. Compounds 1 and 2 are spirostanol glycosides having hydroxy groups at C-2, C-3, C-12, and C-24 of the aglycone moiety. Although C. newellii is known to be a poisonous plant, the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazoliumbromide assay exhibited that none of the isolated compoundswere cytotoxic to HL-60 human promyelocytic leukemia cells.

Free oligosaccharides are the third most abundant solid component in milk after lactose and lipids. The study of milk oligosaccharides indicate that nutrients are not only benefits the infant's gut but also perform a number of other functions which include stimulation of growth, receptor analogues to inhibit binding of pathogens and substances that promote postnatal brain development. Surveys reveal that camel milk oligosaccharides possess varied biological activities that help in the treatment of diabetes, asthma, anaemia, piles and also a food supplement to milking mothers. In this research, camel milk was selected for its oligosaccharide contents, which was then processed by Kobata and Ginsburg method followed by the HPLC and CC techniques. Structure elucidation of isolated compound was done by the chemical degradation, chemical transformation and comparison of chemical shift of NMR data of natural and acetylated oligosaccharide structure reporter group theory, the 1H, 13C NMR, 2D-NMR (COSY, TOCSY and HSQC) techniques, and mass spectrometry. The structure was elucidated as under: MEDALOSE[Figure presented]

Mass cytometry (MC) is a powerful method that combines the cellular resolution of flow cytometry with the isotopic resolution of inductively coupled plasma mass spectrometry (ICP-MS). This combination theoretically allows for the simultaneous quantificati

In this study, an enzyme-based electrochemical method was developed for the detection of Escherichia coli (E. coli) using the T7 bacteriophages engineered with lacZ operon encoding for beta-galactosidase (β-gal). The T7lacZ phages can infect E. coli, and have the ability to trigger the overexpression of β-gal during the infection of E. coli. The use of the engineered phages resulted in a more sensitive detection of E. coli by (1) overexpression of β-gal in E. coli during the specific infection and (2) release of the endogenous intracellular β-gal from E. coli following infection. The endogenous and phage-induced β-gal was detected using the electrochemical method with 4-aminophenyl-β-galactopyranoside (PAPG) as a substrate. The β-gal catalyzed PAPG to an electroactive species p-aminophenol (PAP) which could be monitored on an electrode. The electrochemical signal was proportional to the concentration of E. coli in the original sample. We demonstrated the application of our strategy in aqueous samples (drinking water, apple juice, and skim milk). Using this method, we were able to detect E. coli at the concentration of approximately 105 CFU/mL in these aqueous samples in 3 h and 102 CFU/mL after 7 h. This strategy has the potential to be extended to detect different bacteria using specific bacteriophages engineered with gene encoding for appropriate enzymes.

Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis-Menten constants Km for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine Km and turnover number kcat for trypsin digestion of angiotensin II (Km and kcat are determined to be 6.4 mM and 1.3 s-1, respectively).

In this study, unmodified, amino modified and cyanuric chloride functionalized amino modified nonporous fumed nano-silica particles (FNS, AFNS and CCAFNS, respectively) were used for the development of efficient nanobiocatalysts for application in the biosynthesis of bioactive galactosides, galacto-oligosaccharides (GOS). Hence, in an attempt to elucidate the mechanism of immobilization, based on available enzyme conformation, the effects of immobilization parameters (initial enzyme concentration, immobilization time and pH) were analyzed. Among all three used nano-sized supports, the one with amino groups (AFNS) exhibited the best β-galactosidase binding capacity of 220 mg g-1 support with the efficiency of ～90% at pH 4.5 and immobilization time 3 h. The highest hydrolytic activity of ～2200 IU g-1 was achieved, which is far higher than previously reported. Meanwhile, β-galactosidases covalently immobilized on CCAFNS and adsorbed on FNS nanoparticles were found to have similar trends with respect to immobilization efficiency (58-71%) and hydrolytic activity (～600 IU g-1). Furthermore, thermal stability at 60 °C was increased by β-galactosidases immobilized on AFNS and CCAFNS (4 and 1.4-fold, respectively) due to the protective effect of interactions formed between enzyme molecules and the surfaces of nanoparticles. Since β-galactosidase immobilized on AFNS was the nanobiocatalyst with the highest activity and stability, it was applied in GOS production. With AFNS-β-galactosidase GOS production of 90 g L-1 h-1 was achieved as compared to 30 g L-1 h-1 by free β-galactosidase, meaning that AFNS enhanced the selectivity of β-galactosidase for transgalactosylation, which is a crucial advantage for its application in GOS production.

Porcine epidemic diarrhea virus (PEDV) infections have resulted in a severe economic loss in the swine industry in many countries due to no effective treatment approach. Fifteen oleanane triterpenes (1-15), including nine new ones (1-4 and 10-14), were isolated from the flowers of Camellia japonica, and their molecular structures were determined by extensive spectroscopic methods. These compounds were evaluated for their antiviral activity against PEDV replication, and the structure-activity relationships (SARs) were discussed. Compounds 6, 9, 11, and 13 showed most potent inhibitory effects on PEDV replication. They were found to inhibit PEDV genes encoding GP6 nucleocapsid, GP2 spike, and GP5 membrane protein synthesis based on RT-PCR data. Western blot analysis also demonstrated their inhibitory effects on PEDV GP6 nucleocapsid and GP2 spike protein synthesis during viral replication. The present study suggested the potential of compounds 6, 9, 11, and 13 as promising scaffolds for treating PEDV infection via inhibiting viral replication.

Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG?0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG?0 = 1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG?F - ΔΔG?H), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower.

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrola

Two triterpenoid saponins with two known ones have been isolated from the roots of Gypsophila arrostii var. nebulosa, and two new ones from the roots of Gypsophila bicolor. Their structures were established by extensive NMR and mass spectroscopic techniques as 3-O-β-D-galactopyranosyl-(1->2)-[β-D-xylopyranos yl-(1 → 3)]-β-D-glucuronopyranosylquillaic acid 28-O-β-D-xylopyranosyl-(1 →4)-[β-D-glucopyranosyl-(1 →3)]-α-L-rhamnopyranosyl-(1 →2)-[β-D-glucopyranosyl-(1 →4)]-β-D-fucopyranosyl ester (1), 3-O-β-D-galactopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucuronopyranosylgypsogenin 28-O-β-D-xylop yranosyl-(1 →4)-[β-D-glucopyranosyl-(1→3)]-α-L-rhamnopyranosyl-(1 →2)-[β-D-glucopyranosyl-(1 →4)]-β-D-fucopyranosyl ester (2), 3-O-β-D-galactopyranosyl-(1 →2)-[β-D-xylopyranosyl-(1 →3)]-β-D-glucurono pyranosylgypsogenin 28-O-β-D-xylopyranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-[(4-O-acetyl)-β-D-quinovopyranosyl-(1 →4)]-β-D-fucopyranosyl ester (3), gypsogenic acid 28-O-β-D-glucopyranosyl-(1 →3)-{6-O-[3-hydroxy-3-methylglutaryl]-β-D-glucopyranosyl-(1→6)}-β-D-galactopyranosyl ester (4). Three compounds were evaluated against one human colon cancer cell line SW480 and one rat cardiomyoblast cell line H9c2.

Seven bisdesmosidic triterpenoid saponins named caspicaosides E-K, were isolated from the methanolic fruit extract of Gleditsia caspica Desf. Their structures were determined by 1D and 2D NMR spectroscopy as well as high resolution mass spectrometry and acid hydrolysis. The saponins comprised echinocystic acid or oleanolic acid as the aglycone and saccharide moieties at C-3 and C-28. Like most Gleditsia saponins, the oligosaccharide moiety at C-3 was identified as β-d-xylopyranosyl-(1→2)-α-l-arabinopyranosyl- (1→6)-β-d-glucopyranosyl. The common oligosaccharide moiety linked to C-28 was determined as β-d-xylopyranosyl-(1→3)-β-d-xylopyranosyl- (1→4)-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl with the presence of additional β-d-galactopyranose unit and/or another α-l-rhamnopyranose moiety. The C-28 saccharide moiety was acylated with a monoterpenic acid unit or a monoterpenic acid linked to a monoterpene- arabinoside unit at the ester-β-d-glucopyranose C-6. Esterification of C-2 and C-3 hydroxyl groups of the terminal α-l-rhamnopyranose unit with a monoterpenic acid and epoxy-monoterpenic acid units, respectively, or with two identical monoterpenic acid units, was also shown. The acylated saponins caspicaosides G-K were assayed for their in vitro cytotoxicities against the three cell lines HCT116, HepG2 and MCF7. The tested saponins showed moderate to strong activities.

Chemical investigation of the Australian plant Haemodorum simulans (Haemodoraceae) resulted in the isolation of two new phenylphenalenones, haemoxiphidone and haemodordioxolane from the bulbs together with the first report of an oxabenzochrysenone glycoside, haemodoroxychrysenose from the aerial parts of the plant. Also isolated were two previously described phenylphenalenones 5,6-dimethoxy-7-phenyl-1H,3H-naphtho[1,8-cd]pyran-1,3-dione and haemodorone and two oxabenzochrysenones 5-hydroxyl-2-methoxy-1H-naphtho[2,1, 8-mna]xanthen-1-one and 5-methoxy-1H-naphtho[2,1,8-mna]xanthen-1-one. The X-ray structure of the phenylphenalenone 5,6-dimethoxy-7-phenyl-1H,3H-naphtho[1,8-cd] pyran-1,3-dione was secured for the first time. All compounds were deduced by detailed spectroscopic analyses. HPLC-NMR chemical profiling of an enriched fraction containing a mixture of haemodordioxolane and 5,6-dimethoxy-7-phenyl- 1H,3H-naphtho[1,8-cd]pyran-1,3-dione facilitated the partial identification of these secondary metabolites. The structure previously assigned as xiphidone in our initial studies of this plant was re-assigned as the new isomer haemoxiphidone.

Glycoconjugates containing either terminal α-2,3- or α-2,6-Neu5Ac-Gal disaccharides are found on cell surfaces of many animal glycans. Each linkage can be specifically recognized by lectins and enzymes such as neuraminidases. Here we describe a one-step enzymatic synthesis of two colorimetric substrates that allow for fast distinction of specific neuraminidase activity. The Royal Society of Chemistry 2013.

The present work reports a standalone integrated scheme for the production of 5-hydroxymethyl furfural (HMF) and potassium sulphate (K2SO 4) from granular biomass rich in the sulphated polysaccharide, κ-carrageenan. Fresh Kappaphycus alvarezii seaweed was crushed to expel the juice rich in KCl (0.7 m3 t-1 of fresh seaweed) and granular biomass (0.04 t dry weight per t of fresh seaweed). The latter yielded κ-carrageenan through seawater extraction. HMF was derived from this phycocolloid through reaction with Mg(HSO4)2 acid catalyst and isolated in pure form. Galactose was a co-product which remained in the aqueous phase. The aqueous phase was recycled up to 10 times by maintaining a constant acid strength, and utilized thereafter for the recovery of K 2SO4. Selective crystallization of K2SO 4 was guided by the phase diagram and use was made of a part of the seaweed juice in this process. The spent aqueous phase rich in galactose was subjected to further reaction with HCl obtainable through bipolar electro-dialysis (ED) of seaweed juice. The reaction yielded levulinic acid (LA) and formic acid (FA) in nearly equal proportions. The processing of 1 t of granular biomass was computed to require 30.15 GJ of energy and would yield 0.18 t HMF, 0.056 t LA, 0.020 t FA, 0.27 t K2SO4, and 5.77 m3 pure water. The process energy requirement for the scheme can be met from additional supplies of granule (3.35 t). Combustion/gasification of this biomass would yield additionally 0.74 t glaserite fertilizer and the required amount of H2SO4 for Mg(HSO4) 2 preparation. The Royal Society of Chemistry.

Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (Km), the turnover number (kcat) and the enzyme inhibitor dissociation constants (ki, k i′). Using this device we determine Km and k cat for β-galactosidase and the fluorogenic substrate Resorufin β-d-galactopyranoside (RBG) to be 442 μM and 1070 s-1, respectively. Furthermore, we examine the inhibitory effects of isopropyl-β-d-thiogalactopyranoside (IPTG) on β-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.

Glycosidases provide a powerful resource for in vitro synthesis of novel anomerically pure glycosides. Generation of new low molecular weight galactosides is of interest since they are potential galectin inhibitors. Galectins are molecular targets for cancer therapy and thus their inhibitors are potential antitumor agents. Here we report the enzymatic synthesis and structural characterization of 2-aminoethyl β-d-galactopyranoside. Critical parameters for transgalactosylation using either soluble or immobilized enzyme were investigated and optimized for the galactoside synthesis. We found that 0.2 M lactose, and 0.5 M 2-aminoethanol at 50 °C for 30 min were the optimal conditions for synthesis. 2-Aminoethanol proved to be an enzyme inhibitor, fitting a mixed inhibition model with inhibition constants, Kic = 0.31 ± 0.04 M and Kiu = 0.604 ± 0.035 M.

The hydrolytic hydrogenation of hemicellulose arabinogalactan, into sugars, sugar alcohols and furfurals was carried out in a batch reactor using modified mesoporous MCM-48 material incorporated with ruthenium metal into the framework. The bi-functional catalytic materials, MCM-48 and Ru-MCM-48 were synthesized, characterized and investigated in the title reaction at total pressure of 20 bar hydrogen, using an initial arabinogalactan concentration of 0.4 wt%, at 458 K. The transformation of the hemicellulose consists of arabinogalactan hydrolysis to the monosaccharides, l-arabinose and d-galactose followed by the subsequent hydrogenation to sugar alcohols, arabitol and galactitol or dehydration of the monomers to furfural and 5-hydroxymethylfurfural. The yields of the main products, i.e. sugars, sugar alcohols and furfurals were varied depending on the strength of the acid sites and the presence of metal in the structure of the ruthenium modified catalyst. Ru-MCM-48 displayed high catalytic activity and the sugar alcohols were obtained selectively from the hemicellulose. The catalytic performance of the mesoporous MCM-48 catalysts with respect to the catalyst structure, acidity and presence of the metal was evaluated.

A new triterpeniod saponin 3-O-β-arabinopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (1), together with the known saponin 3-O-β-xylopyranosyl-(1 → 3)-[β-galactopyranosyl-(1 → 2)]-β-glucuronopyranosyl gypsogenin (2), and three known triterpenes gypsogenic acid (3), quillaic acid (4) and gypsogenin (5) were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae). Their structures were elucidated by chemical and spectral methods. Cytotoxic activity of compounds 1 and 2 were tested against seven human cancer cell lines. Compound 1 showed cytotoxic activity against all of them, while compound 2 only against two cell lines.

The methanolic extract and its 1-butanol-soluble fraction from the flower buds of Camellia japonica, cultivated in Yunnan Province, China, showed inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells. From the 1-butanol-soluble fraction, a new 28-nor-oleanane-type and three new oleanane-type triterpene saponins, sanchakasaponins A-D, were isolated together with four known triterpene saponins. Their chemical structures were elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects on melanogenesis in theophylline-stimulated B16 melanoma 4A5 cells and structure-activity relationships of the saponins were investigated.

The present invention provides novel methods for determining the presence or amount of a hydrolytic enzyme in a sample, based on novel substrates for the enzymes, and also provides compositions and methods that provide highly sensitive assay methods for such hydrolytic enzymes.

Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O 2-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose.

A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum var. porrum. On the basis of chemical conversions and detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques, its structure was established as 3-[(O-β-d-glucopyranosyl-(1 → 3)-β-d-glucopyranosyl-(1 → 2)-O-[O-β-d-glucopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranosyl)oxy] -2,6-dihydroxy-(2α,3β,5α,6β,25R)-spirostane. Results of the present study indicated that the steroidal saponin showed haemolytic effects in the in vitro assays and demonstrated antiinflammatory activity and gastroprotective property using in vivo models.

Two new unusual soyasaponins named 6''-O-methyldehydrosoyasaponin I (7) and desglucosylsoyasaponin A1 (10) along with eight known saponins, dehydrosoyasaponin IV (1), dehydrosoyasaponin III (= impatienoside A) (2), soyasaponin III (3), dehydrosoyasaponin II (= soyasaponin Bg) (4), soyasaponin II (5), dehydrosoyasaponin I (= soyasaponin Be) (6), soyasaponin I (8), and kudzusaponin SA3 (9), were isolated as their methyl esters and identified from the liquid culture of G. applanatum. Their structures were determined by chemical and spectroscopic analyses including 1D- and 2D-NMR as well as by comparison of their spectroscopic data with those of the reported in literatures. Although dehydrosoyasaponin IV was identified by LC-MS/MS method from soy protein isolate, this is the first report of the isolation of this compound. Dehydrosoyasaponin III (2) and kudzusaponin SA3 (9) were also isolated for the first time from soybean. The presence of soyasaponins in Ganoderma species seems to be unusual feature. Thus, we presumed that compounds 1-10 might all be derived from the defatted soybean flour which was added to the culture medium as a nitrogen source.

Green solvents made from glycerol change the classical regioselectivity of Biolacta No 5 β-galactosidase, from β(1→4) to β(1→6) linkages when a 2 M concentration was used. In order to explain these results, the non-proteic compounds present in

The 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) inhibitors from hawthorn fruit (Crataegus pinnatifida Bge.) were isolated and evaluated for their antihyperlipidemic effect induced by high-fat diet in mice. After being further purified with silica and polyamide column chromatography from the fractions (fractions A, F, H, and G) with a high inhibitory rate (IR) to HMGR, 24 chromatographic fractions were obtained, including 8 active fractions with a high IR to HMGR. However, the total inhibitory activity of 24 fractions was decreased by about 70%. From eight active fractions, four compounds were obtained by recrystallization and identified as quercetin (a), hyperoside (b), rutin (c), and chlorogenic acid (d), the contents of which in hawthorn EtOH extract were 0.16, 0.32, 1.45, and 0.95%, respectively. The IR values of compounds ad to HMGR were 6.28, 9.64, 23.53, and 10.56% at the corresponding concentrations of 0.16, 0.32, 1.45, and 0.95 mg/mL, respectively. It was discovered that the IR of a mixture (2.85 mg/mL) matching the original percentage of compounds a-d in hawthorn EtOH extract was up to 79.5%, much higher than that of the single compound and the total IR of these four compounds (50.01%). The in vivo results also revealed that the mixture had a more significant lipid-lowering efficacy than the monomers. Structure-activity relationship revealed the inhibitory activity and lowering-lipid ability of compounds a-c decreased with increasing glycoside numbers. It was concluded that there were synergetic effects on inhibiting HMGR and lowering lipid among compounds a-d, and the weak hydrophilic ability benefits the inhibition to HMGR and lowering-lipid efficacy.

The five new presenegenin glycosides 1-5 were isolated from Securidaca welwitschii, together with one known sucrose diester. Compounds 1-4 were obtained as pairs of inseparable (E)/(Z)-isomers of a 3,4-dimethoxycinnamoyl derivative, i.e., 1/2 and 3/4. Their structures were elucidated mainly by 2D-NMR techniques and mass spectrometry as 3-O-(β-D-glucopyranosyl)presenegenin 28-{O-β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranosyl-(1→2) -O-[β-D-glucopyranosyl-(1→3)]-4-O-[(E)-3,4-dimethoxycinnamoyl] -β-D-fucopyranosyl} ester (1) and its (Z)-isomer 2, 3-O-(β-D- glucopyranosyl)presenegenin 28-{O-β-D-galactopyranosyl-(1→4)-O-β- D-xylopyranosyl-(1→4)-O-3-O-acetyl-α-L-rhamnopyranosyl-(1→2) -O-[β-D-glucopyranosyl-(1→3)]-4-O-[(E)-3,4-dimethoxycinnamoyl] -β-D-fucopyranosyl} ester (3) and its (Z)-isomer 4, and 3-O-(β-D-glucopyranosyl)presenegenin 28-[O-β-D-galactopyranosyl- (1→3)-O-β-D-xylopyranosyl-(1→4)-O-α-L-rhamnopyranosyl- (1→2)-β-D-fucopyranosyl] ester (5) (presenegenin=(2β,3β, 4α)-2,3,27-trihydroxyolean-12-ene-23,28-dioic acid). Copyright 2010 Verlag Helvetica Chimica Acta AG, Zuerich, Switzerland.

Osteospermum vaillantii (Decne) T. Norl., collected from southern Saudi Arabia, yielded two new saponins characterised as 3-O-β-D-glucopyranosyl- (2′ → 1″)-β D-glucopyranosyl-(3′ → 1?)-O-β D-galactopyranosyl-oleanolic acid, designated as osteosaponin (1),

Three binuclear copper(II) complexes were characterized as solids by X-ray diffraction and in solution by UV/vis spectrophotometric titration, and subsequently evaluated for their glycosidase-like activity. The structure analysis revealed comparable intermetallic Cu ? ? ? Cu distances (~3.5 A) for the complexes 2 and 3. Despite this similarity, the composition of the complexes differs significantly in aqueous solution as revealed by spectrophotometric titrations. The hydrolysis of selected nitrophenylglycopyranosides is up to 11,000-fold accelerated over background in the presence of the copper(II) complexes in 3-(cyclohexylamino)-1- propanesulfonic acid (CAPS) buffer at pH 10.5 and 30 °C.

A genomic library of bacterial strain Paenibacillus thiaminolyticus was constructed and the plasmid DNA of the clone, containing the gene encoding β-D-galactosidase with β-D-fucosidase activity, detected by 5-bromo-4-chloro-3-indoxyl β-D-galactopyranoside, was sequenced. Cells of Escherichia coli BL21 (DE3) were used for production of the enzyme in the form of a histidine-tagged protein. This recombinant fusion protein was purified using Ni-NTA agarose affinity chromatography and characterized by using p-nitrophenyl β-D-fucopyranoside (Km value of 1.18 ± 0.06 mmol/L), p-nitrophenyl β-D-galactopyranoside (Km value of 250 ± 40 mmol/L), p-nitrophenyl β-D-glucopyranoside (Km value of 77 ± 6 mmol/L), and lactose (Km value of 206 ± 5 mmol/L) as substrates. Optimal pH and temperature were estimated as 5.5 and 65°C, respectively. According to the amino acid sequence, the molecular weight of the fusion protein was calculated to be 68.6 kDa and gel filtration chromatography confirmed the presence of the enzyme in a monomeric form. In the following step, its ability to catalyze transfucosylation reactions was tested. The enzyme was able to catalyze the transfer of fucosyl moiety to different p-nitrophenyl glycopyranosides (producing p-nitrophenyl β-D-fucopyranosyl-(1,3)-β-D-fucopyranoside, p-nitrophenyl β-D-fucopyranosyl-(1,3)-α-D-glucopyranoside, p-nitrophenyl β-D-fucopyranosyl-(1,3)-α-D-mannopyranoside, and p-nitrophenyl β-D-fucopyranosyl-(1,6)-α-D-galactopyranoside) and alcohols (producing methyl β-D-fucopyranoside, ethyl β-D-fucopyranoside, 1-propyl β-D-fucopyranoside, 2-propyl β-D-fucopyranoside, 1-octyl β-D-fucopyranoside, and 2-octyl β-D-fucopyranoside). These results indicate the possibility of utilizing this enzyme as a promising tool for enzymatic synthesis of β-D-fucosylated molecules. The Author 2009. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

The reinvestigation of saponin composition from Medicago arabica from Italy allowed the detection of nineteen (1-19) saponins. All of them were purified by reverse-phase chromatography and their structures elucidated by spectroscopic and spectrometric (1D and 2D NMR; ESI-MS/MS) and chemical methods. Fourteen were known saponins, previously found in other plants including other Medicago species. They have been identified as glycosides of oleanolic acid, 2β,3β-dihydroxyolean-12-en-28-oic acid, hederagenin, bayogenin and soyasapogenol B. Five saponins, identified as 3-O-[-α-L- arabinopyranosyl(1→2)-β-D-glucuronopyranosyl]-30-O-β-D- glucopyranosyl 2β,3β,30-trihydroxyolean-12-en-28-oic acid (1), 3-O-[α-L-arabinopyranosyl(1→2)-β-D-glucuronopyranosyl] -30-O-[β-D-glucopyranosyl]3β,30-dihydroxyolean-12-en-28-oic acid (2), 3-O-|β-D-glucuronopyranosyl]-30-O-[α-L-arabinopyranosyl(1→2) -β-D-glucopyranosyl] 2β,3β,30-trihydroxyolean-12-en-28-oic acid (3), 3-O-[β-D-glucuronopyranosyl]-30-O-[α-L- arabinopyranosyl(1→2)-β-D-glucopyranosyl] 3β,30-dihydroxyolean- 12-en-28-oic acid (4) and 3-O-[β-D-glucuronopyranosyl]-30-O-[β-D- glucopyranosyl] 2β,3β,30-trihydroxyolean-12-en-28-oic acid (5), are reported here as new natural compounds. These new saponins, possessing a hydroxy group at the 30-methyl position of the triterpenic skeleton, have never been previously found in the genus Medicago.

Seven new triterpenoid saponins (1-7), have been isolated and elucidated from the roots of Gypsophila oldhamiana together with five known triterpenoid saponins (8-12). These saponins which could be classified into three series: 3-O-monoglucosides (1, 8, 9), 28-O-monoglucosides (2-4, 12) and 3, 28-O-bidesmosides (5-7, 10, 11), have been evaluated for their α-glucosidase inhibition activity. As a result, the preliminary structure-activity relationships were discussed based on the position of sugar linkage attached to the aglycone, and 28-O-monoglucosides 2-4 and 12 showed significant inhibitory activities on α-glucosidase.

Electrochemical responses (e.g., chronoamperometric) obtained with an immobilized enzyme that produces an electroactive species may be used to quantitate the amount of enzyme or the concentration of its substrate. It is shown, on theoretical and experimental bases, that product-to-substrate coupling with a second enzyme co-immobilized with the first within one or within a small number of monolayers, allows high amplification rates (higher than 1000), avoids membrane transport limitations, and lends itself to precise kinetic analyses that provide guidelines for optimization of the analytical sensitivity. Very large amplification factors, as large as several thousands, can be reached experimentally, in agreement with appropriately derived theoretical predictions, thus opening the route to the rational design of high-performance substrate sensing or affinity assays applications. Copyright

Assays of hydrolytic enzyme activity, such as of glycosidases and phosphatase, as well as several proteases, using a semi-wet supramolecular hydrogel array composed of a glycosylated amino acetate are described. It has been demonstrated that the microcavity formed by gel fibrils is suitable to immobilize native enzymes without denaturation under semi-wet conditions, and thus the nanofiber has been rationally used as a sensing domain to monitor enzymatic reactions. By using a fluorogenic substrate, reducing the size of the hydrogel can significantly improve the problem of suppressed diffusion within the gel matrix thus making the hydrogel a promising semi-wet matrix for evaluating enzyme activity. Confocal laser scanning microscopy observa lions have shown that an environmentally sensitive fluorescent probe accumulates in the hydrophobic domain of the gel fiber and emits fluorescence more strongly upon hydrolytic cleavage of the substrate peptides. Not only a simple environmentally sensitive probe but also a FRET (fluorescence resonance energy transfer)-type read-out mode can be devised to analyze the enzymatic hydrolysis-triggered redistribution of the probe between the nano-space and the nanofiber to accomplish a more clearly distinguished enzyme assay. Thus, it is clear that three distinct read-out modes, that is, 1) fluorogenic substrates, 2) substrates bearing an environmentally sensitive probe, or 3) a substrate exhibiting FRET, can operate under the semi-wet hydrogel conditions used in these investigations. In addition, owing to the unique properties of the present supramolecular hydrogel in semi-wet conditions, that is, its phase-segregation properties and dynamics, the supramolecular substrate/enzyme array has successfully been used for high-throughput screening of single and multiple enzymes based on their activity, lysate analysis, and quantitative evaluation of inhibitor potency and selectivity.

The thermal stability of Kluyveromyces fragilis β-galactosidase was studied in a phosphate buffer (pH 7.5) over a range of temperatures of 10 to 42°C. The shape of the kinetic curves changed with temperature, revealing two stages of the process: the melting of the conformational lock and the dissociation of the labile dimer (25 and 30°C) and the dissociation of the labile dimer and the denaturation of the monomer (35°C). At higher temperatures, these stages are indistinguishable. A comparative kinetic analysis of the thermal stability of yeast β-galactosidases was performed, and the relevant kinetic parameters were calculated. The data reported in the literature were processed within the framework of a model of dissociative thermal inactivation.

The polyphenolic compound 1-O-galloyl-3,6-hexahydroxybiphenyl-D- galactopyranoside (pusilagin) was isolated from the aerial part. The tannin structure was established using acid hydrolysis and IR, PMR, and 13C NMR spectral methods.

This paper describes a microfabricated enzyme assay system including a micromixer that can be used to perform stopped-flow reactions. Samples and reagents were transported into the system by electroosmotic flow (EOF). Streams of reagents were merged and passed through the 100-pL micromixer in a system of roughly 6 nL volume. β-Galactosidase (β-Gal) was chosen as a model enzyme for these studies and was used to convert the substrate fluorescein mono-β-D-galactopyranoside (FMG) into fluorescein. Results obtained with microfabricated systems using the micromixer compared well to those obtained with an external T mixing device. In contrast, assays performed in a microfabricated device by merging two streams and allowing mixing to occur by lateral diffusion did not compare well. Using the microfabricated mixer, Km and kcat values of 75 ± 13 μM and 44 ± 3 s-1 were determined. These values compare well to those obtained with the conventional stopped-flow apparatus for which Km was determined to be 60 ± 6 μM and kcat was 47 ± 4 s-1. Enzyme inhibition assays with phenylethyl-β-D-thiogalactoside (PETG) were also comparable. It was concluded that kinetically based, stopped-flow enzyme assays can be performed in 60 s or less with a miniaturized system of roughly 6 nL liquid volume when mixing is assisted with the described device.

We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans β-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo β-D-galactopyranoside (S6Gal β-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6′-sulfo N-acetyllactosamine (S6Gal β1-4GlcNAc) and its positional isomer 6′-sulfo N-acetylisolactosamine (S6Gal β1-6GlcNAc) were observed by HPAEC-PAD, in 49% total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Gal β1-2Glc was mainly produced along with β-(1-1)α, β-(1-3), β-(1-6) isomers in 74% total yield. When methyl α-D-glucopyranoside (Glcα-OMe) was an acceptor, the enzyme also formed mainly S6Gal β1-2Glcβ-OMe with its β-(1-6)-linked isomer in 41% total yield based on the donor added. In both cases, it led to the predominant formation of β-(1-2)-linked disaccharides. In contrast, with the corresponding methyl β-D-glucopyranoside (Glc β-OMe) acceptor, S6Gal β1-3Glc β-OMe and S6Gal β1-6Glc β-OMe were formed in a low total yield of 12%. These results indicate that the regioselectivity and efficiency on the β-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.

This paper describes the conditions in which in situ NMR spectroscopy is a suitable technique to use when following the course of enzymatic transglycosylation reactions. Using this methodology, the reactions must be carried out in D2O. Our experiments indicate that the rate of the transglycosylation reaction is reduced in this solvent while the rate of the hydrolysis of the disaccharides produced is enhanced depending on the nature of the anomeric substituent. However, this undesirable effect is generally weak because the rates of the transglycosylation reactions are always faster than the rates of the hydrolysis whatever the solvent. The great advantage of NMR spectroscopy lies in its potential to detect, in a single experiment, all the components of the reaction without any disturbance of the reaction medium.

Chloroethyl 1-thio-β-D-glycopyranosides of the D-galacto and D-gluco configurations 5a-5c were prepared by alkylation of the corresponding 1-thio-β-D-hexopyranoses 3a-3c with 1-bromo-2-chloroethane followed by deacetylation. The starting 1-thio-β-D-hexopyranoses were obtained from the acetylated glycopyranosyl halides via isothiouronium salts. It was demonstrated that the chloroethyl thioglycosides 5a-5c undergo hydrolysis in aqueous solutions to give the 2-hydroxyethyl thioglycosides 6a-6c and reducing hexoses and that this hydrolysis proceeds via episulfonium salts. The hydrolysis was monitored by 1H and 13C NMR spectroscopy. In 1% aqueous solutions of sodium carbonate containing phenol or aniline, the thioglycosides 5a-5c provide, in addition to the above hydrolysis products, also the phenoxyethyl and phenylaminoethyl thioglycosides 9a, 10a and 9b, 10b, respectively.

The oxidation of several sugars by NaBrO3 catalyzed by [Ru(azpy)2(H2O)2]2+, is reported and compared with earlier oxidation results with octyl α-D-glucoside (α-OGP). The sugars studied were 1-octyl β-D-glucoside (β-OGP), 1-octyl β-D-galactoside (β-OGaP), 1-phenyl β-D-glucoside (β-PGP), 1-methyl α-D-glucoside, 1-methyl β-D-glucoside and 1-decyl β-D-maltoside (β-DMP). The results with β-DMP are compared with previously reported oxidation results with β-DMP heterogeneously catalyzed over platinum. The structure of the sugar substrate appears to have a significant effect on the activity and selectivity of the oxidation reaction. The main conclusions from this study are that β-isomers are in general less reactive than α-isomers; α-PGP in particular is very unreactive. The kind of substitution (pnenyl versus alkyl, octyl versus methyl) on the anomeric centre has considerable effect on the reactivity of the sugar. The oxidation of β-DMP catalyzed by [Ru(azpy)2(H2O)2]2+ results in a number of different oxidation products. The C6-primary hydroxy groups of both glucose units of β-DMP can be oxidized to carboxylic groups, in contrast to the previously reported heterogeneous catalyzed oxidation of β-DMP by dioxygen. However, the main reaction is the splitting of the decyl chain from the maltose unit The α-1,4-O link between the glucose units appears to be quite stable under the catalytic oxidation conditions used. Based on the results obtained, possible reaction mechanisms are discussed.

Proofs are given of the structures of two new glycosides of the furostan series isolated from the leaves of the plant Nolina microcarpa S.Wats. (family Dracaenaceae).Nolinofuroside D is (25S)-furost-5-ene-1β, 3β,22 α,26-tetraol 1-O-β-D-galactopyranoside 26-O-β-D-glucopyranoside (I), and nolinofuranoside F is (25S)-furost-5-ene-1β,3β,22α,26-tetraol 1-O-β-D-fucopyranoside 26-O-β-D-glucopyranoside 3-O-α-L-rhamnopyranoside (VII).The latter was characterized as its 22-O-methyl ether (VIII).Nolinofuranoside E (IV) has the structure of (25S)-furost-5-ene-1β,3β,22α,26-tetraol 26-O-β-glucopyranoside 1-O- 2)-β-D-fucopyranoside>, which followed from the structure of the fermentation product (VI).The products of the fermentation of the above-named compounds were present in the plant in only trace amounts.Only one of them - nolinospiroside D (III) - has not been described previously.This monoside of the spirostan series is (25S)-spirost-5-ene-1β,3β-diol 1-O-β-D-galactopyranoside.

A temperature-controlled reaction vessel was closely coupled to the ion-spray LC/MS interface on an atmospheric pressure ionization, triple quadrupole mass spectrometer and used to study mechanistic and kinetic aspects of reactions taking place in solution.Free access to the reaction vessel and temperature control as well as minimal sample consumption make continuous-introduction ion-spray mass spectrometry a useful approach for real-time measurement of reactions.The reaction medium, reactants, and products were transferred to the ion source of the mass spectrometer from the reaction vessel in less than 1 s.A variety of model reactions have been investigated to demonstrate the feasibility and potential for continuous-introduction ion-spray mass spectrometry for real-time reaction monitoring.The half-life for the solvolysis of methandrostenolone sulfate in aqueous medium and the Michaelis-Menten constant of the enzymatic hydrolysis of O-nitrophenyl β-D-galactopyranoside by lactase were determined.The enzymatic hydrolysis of dynorphin 1-8 by α-chymotrypsin and leucine aminopeptidase and the reduction of the disulfide bridge in oxytocin with β-mercaptoethanol were obtained on-line to emphasize the technique's capability for peptide sequencing and structural elucidation.