Rapid screening of monoamine oxidase B inhibitors in natural extracts by capillary electrophoresis after enzymatic reaction at capillary inlet

A facile capillary electrophoresis (CE) method was developed for the screening of monoamine oxidase B (MAO-B) inhibitors in natural extracts. In this method, the enzymatic reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds, and detected by their UV absorbance at 280 nm. Conditions for the separation of substrates, products and enzyme were optimized. The optimal buffer composition was 50 mM N-2-hydroxyethyl-piperazine-N′-2-ethane sulphonic acid (HEPES) solution containing 10 mM SDS (pH = 7.4). Under the optimal condition, the baseline separation of substrates, products and enzyme was achieved within 2 min. The present method was used to determine MAO-B kinetic constants, K_i, K_m and IC₅₀ based on quantitative of the substrate peak area compared with the reference electropherogram obtained from without the inhibitor. A validation study shows good reproducibility for both migration time (RSD = 1.8%) and peak area (RSD = 3.9%). Finally, the screening of 16 natural extracts was performed, and 2 natural extracts from Fructus crataegi and Radix polygoni multiflori were identified to be positive for MAO-B inhibition.     

Capillary electrophoresis for screening of 20S proteasome inhibitors

A method for studying 20S proteasome inhibitors by capillary electrophoresis (CE) has been developed. Proteasome plays a fundamental role in degrading key regulatory proteins. The 20S proteasome can degrade intrinsically disordered proteins in an ATP-independent manner without additional “helper” molecules. The discovery of new proteasome inhibitors with little or no toxicity is highly desirable in anticancer therapy. In this study, the inhibitory effects of MG132 and MG115 on the 20S proteasome were evaluated by CE for the first time. The optimized CE conditions were as follows: fused-silica capillary of 30 cm effective length and 75 μm internal diameter, pressure injection of 0.5 psi for 5 s, 50 mM Hepes buffer (pH 7.6) with 2% dimethyl sulfoxide, constant voltage of 20 kV, and detection wavelength at 340 nm. Also, the new method was used to study the inhibitory effects of 30 novel peptidyl vinyl ester derivatives of MG132. The 50% inhibition concentrations (IC₅₀ values) of MG132 and MG115 were 40.0 and 84.7 nM, respectively. Two new compounds, XP32 and XP35, showed considerable inhibitory effects on the 20S proteasome. When the concentrations of them were fixed at 172 nM, their inhibition rates were 36.2% and 29.1%, respectively. The results showed that the CE method was powerful, sensitive, and fast and required little sample. It could be employed as one of the reliable drug screening methods for 20S proteasome inhibitors.     

An enzyme immobilized microassay in capillary electrophoresis for characterization and inhibition studies of alkaline phosphatases

A simple and fast dynamically coated capillary electrophoretic method was developed for the characterization and inhibition studies of alkaline phosphatases (EC 3.1.3.1). An inside capillary enzymatic reaction was performed, and hydrolysis of the substrate 4-nitrophenylphosphate to 4-nitrophenol was measured. Fused-silica capillary surface was dynamically modified with polycationic polybrene coating. By reversal of the electroosmotic flow (EOF), analysis time was reduced up to 3 min as the anionic analytes were migrated in the same direction as the EOF. Furthermore, the sensitivity of the method was increased using electroinjection through high-field amplified injection. The baseline separation of 4-nitrophenylphosphate and 4-nitrophenol was achieved by employing 50 mM sodium phosphate as the running buffer (pH 8.5), 0.0025% polybrene, and a constant voltage of −15 kV, and the products were detected at 322 nm. Under the optimized conditions, a good separation with high efficiency was achieved. The new method was applied to study enzyme kinetics and inhibitor screening. K_m and K_i values obtained with the new CE method were compared well with the standard spectrophotometric method. Dynamic coating of fused-silica capillary gave fast and reproducible separation of substrate and product. The method can be easily optimized for inhibition studies of other isozymes.     

Bioconversion using immobilized recombinant flocculent yeast cells carrying a fused enzyme gene in an `intelligent' bioreactor

Immobilized recombinant cells of the flocculent yeast Saccharomyces diastaticus carrying an expression plasmid for a fused enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 reductase were used in the bioconversion reaction from acetanilide (AA) to p-acetaminophene (p-AAP). Immobilization of the strain within reticulated polyurethane foam biomass support particles (BSPs) was effected passively in situ in a fluidized-bed bioreactor using `draw and fill' operation. In repeated batch reactions both the final product concentration and the production rate were notably improved compared with the results obtained using freely suspended cells without BSPs. Cells immobilized within BSPs exhibited a significantly high level of expression of the fused enzyme. In addition, a high proportion of plasmid-carrying cells was maintained among the immobilized cells, in contrast to a much lower proportion among freely suspended cells released from the BSPs. Since the bioreactor became packed with highly expressing cells immobilized within BSPs as a consequence of spontaneous screening, it was termed an `intelligent' bioreactor, and is believed to offer significant potential for the further development of efficient production processes.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Periplaneta americana midgut proteases differentially expressed against dietary components from different plant seeds

Insect midgut proteases are known to be regulated by plant protease inhibitors. In the present study, the antinutritional effects of a variety of seed extracts against Periplaneta americana (Linnaeus) (Dictyoptera: Blattidae) midgut proteases are assessed in vitro and in vivo. Bioassays are conducted by allowing P. americana to feed on diets incorporated with the tested seed extracts. Low survival rates are recorded on a diet incorporated with seed powders of Ricinus communis (10%), Glycine max (30%), Datura alba (50%) and Mucuna pruriens (50%). Proteolytic and residual proteolytic activities are highly inhibited by the four seed extracts. Electrophoretic analysis shows that the majority of P. americana midgut protease isoforms are inhibited by the four seed extracts in vivo as well as in vitro. The midgut physiology of P. americana is affected by the seed extracts of D. alba, M. pruriens and Sapindus laurifolius, by over synthesising or changing mobilities of existing protease isoforms. Furthermore, some key proteases in P. americana midgut are involved in the regulation of other protease isoforms. These results suggest that seed extracts from the above plants are potential sources of plant protease inhibitors for managing economically important crop pests.     

A Prospective of Multiple Biopharmaceutical Activities of Procyanidins-Rich Uapaca togoensis Pax Extracts: HPLC-ESI-TOF-MS Coupled with Bioinformatics Analysis

The article reports the chemical composition, antioxidant, six key enzymes inhibitory and antimicrobial activities of two solvent extracts (water and methanol) of leaves and stem bark of Uapaca togoensis. For chemical composition, methanol extract of stem bark exhibited significant higher total phenolic (129.86 mg GAE/g) and flavanol (10.44 mg CE/g) contents. Methanol extract of leaves and water extract of stem bark showed high flavonoids (20.94 mg RE/g) and phenolic acid (90.40 mg CAE/g) content, respectively. In addition, HPLC-ESI-TOF-MS analysis revealed that U. togoensis was rich in procyanidins. The methanol and water extracts of stem bark had overall superior antioxidant activity; however, only methanol extract of stem bark showed higher inhibition of cholinesterase (AChE: 2.57 mg GALAE/g; BChE: 4.69 mg GALAE/g), tyrosinase (69.53 mg KAE/g) and elastase (2.73 mmol CE/g). Potent metal chelating ability was showed by water extract of leaves (18.94 mg EDTAE/g), higher inhibition of amylase was detected for water extracts of leaves (0.94 mmol ACAE/g) and stem bark (0.92 mmol ACAE/g). The tested extracts have shown wide-spectrum antibacterial properties and these effects have shown to be more effective against Aspergillus ochraceus, Penicillium funiculosum, Trichoderma viride, Bacillus cereus, Escherichia coli and Pseudomonas aeruginosa. The results revealed that the antioxidant, enzyme inhibitory and antimicrobial activities depended on the extraction solvents and the parts of plant. Bioinformatics analysis on the 17 major compounds showed modulation of pathway associated with cancer. In brief, U. togoensis might be valuable as potential source of natural agents for therapeutic application.     

Inhibition study of alanine aminotransferase enzyme using sequential online capillary electrophoresis analysis

We report the study of several inhibitors on alanine aminotransferase (ALT) enzyme using sequential online capillary electrophoresis (CE) assay. Using metal ions (Na⁺ and Mg²⁺) as example inhibitors, we show that evolution of the ALT inhibition reaction can be achieved by automatically and simultaneously monitoring the substrate consumption and product formation as a function of reaction time. The inhibition mechanism and kinetic constants of ALT inhibition with succinic acid and two traditional Chinese medicines were derived from the sequential online CE assay. Our study could provide valuable information about the inhibition reactions of ALT enzyme.     

Multiplexed Capillary Electrophoresis as Analytical Tool for Fast Optimization of Multi‐Enzyme Cascade Reactions – Synthesis of Nucleotide Sugars

Nucleotide sugars are considered as bottleneck and expensive substrates for enzymatic glycan synthesis using Leloir‐glycosyltransferases. Synthesis from cheap substrates such as monosaccharides is accomplished by multi‐enzyme cascade reactions. Optimization of product yields in such enzyme modules is dependent on the interplay of multiple parameters of the individual enzymes and governed by a considerable time effort when convential analytic methods like capillary electrophoresis (CE) or HPLC are applied. We here demonstrate for the first time multiplexed CE (MP‐CE) as fast analytical tool for the optimization of nucleotide sugar synthesis with multi‐enzyme cascade reactions. We introduce a universal separation method for nucleotides and nucleotide sugars enabling us to analyze the composition of six different enzyme modules in a high‐throughput format. Optimization of parameters (T, pH, inhibitors, kinetics, cofactors and enzyme amount) employing MP‐CE analysis is demonstrated for enzyme modules for the synthesis of UDP‐α‐D‐glucuronic acid (UDP‐GlcA) and UDP‐α‐D‐galactose (UDP‐Gal). In this way we achieve high space‐time‐yields: 1.8 g/L?h for UDP‐GlcA and 17 g/L?h for UDP‐Gal. The presented MP‐CE methodology has the impact to be used as general analytical tool for fast optimization of multi‐enzyme cascade reactions.     

Construction of an effective screening system for detection of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> quorum sensing inhibitors and its application in bioautographic thin-layer chromatography

In Pseudomonas aeruginosa, quorum sensing (QS) regulates dozens of genes and proteins, many of which contribute to the virulence of this pathogen. QS inhibitory (QSI) compounds have been proposed as potential agents for treatment of bacterial infections. To search for Ps. aeruginosa QS inhibitors, we constructed an effective screening system, QSIS-lasI selector, based on the PlasI-sacB reporter, in which QS could be induced with 20 nM 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide (3-oxo-C₁₂-HSL). During screening of the crude extracts from 65 marine fungi, an isolate of Penicillium atramentosum was found to have QSI activity. Thin-layer chromatography assay of the fungal extracts for bioautographic identification of QSIS-lasI indicated that this fungus produced several QSI compounds, including QS inhibitors other than penicillic acid or patulin.     

Cytotoxic,immunosuppressive, trypanocidal and antileishmanial activities of Basidiomycota fungi present in Atlantic Rainforest in Brazil

One hundred three Basidiomycota fungi representing 84 species and 17 families were collected from different Atlantic Rainforest in Brazil. Their basidiomes and fermentation broth extracts were screened in a bioassay panel that included three human cancer cells lines, human peripheral blood mononuclear cells (PBMCs), the enzyme trypanothione reductase (TryR) from Trypanosoma cruzi, and amastigote forms of Leishmania amazonensis. Forty-two extracts representing 21 genera and 35 species presented activities higher than 60% in one or more assays employed in this study. Eighteen extracts were toxic to one or more human cancer cell lines. Extracts from Lentinus strigosus CCB 178 and Lentinus sp. UFMGCB 38 showed selectivity towards cancer cells as they showed only a minor impact on PBMCs. Six extracts suppressed PBMCs proliferation and showed low toxic effect to cancer cells. Thirty-four extracts inhibited the activity of the TryR. Of these, five showed low toxicity towards PBMCs. Extracts from Gymnopilus areolatus, Irpex lacteus, L. strigosus, Nothopanus hygrophanus, Pleurotus flabellatus, and unidentified Basidiomycetes were toxic to L. amazonensis. The results of this screening reinforce the potential of Basidiomycota fungi as sources of bioactive natural products that may be developed into new therapeutic agents for cancer and neglected diseases such as trypanosomiasis and leishmaniasis.     

A new screening method based on yeast-expressed human dipeptidyl peptidase IV and discovery of novel inhibitors

Dipeptidyl peptidase (DPP) IV inhibitors provide a new strategy for the treatment of type 2 diabetes. Human DPP-IV gene was cloned from differentiated Caco-2 cells and expressed in Pichia pastoris. The recombinant enzyme was used in a new system for screening of DPP-IV inhibitors. By high throughput screening, a novel compound (W5188) was identified from 75,000 compounds with an IC₅₀ of 6.5 μM. This method is highly reproducible and reliable for discovery of DPP-IV inhibitors as shown by Z′ value of 0.73 and S/N ratio of 6.89.     

Molecular cloning,purification, and characterization of a novel thermostable cinnamoyl esterase from Lactobacillus helveticus KCCM 11223

A gene encoding cinnamoyl esterase (CE), which breaks down chlorogenic acid (ChA) into caffeic and quinic acids, was cloned from Lactobacillus helveticus KCCM 11223. The gene with an open reading frame of 759 nucleotides was expressed in Escherichia coli, which resulted in a 51.6-fold increase in specific activity compared to L. helveticus KCCM 11223. The recombinant CE exists as a monomeric enzyme having a molecular weight of 27.4?kDa. Although the highest activity was observed at pH 7, the enzyme showed stable activity at pH 4.0–10.0. Its optimum temperature was 65°C, and it also possessed a thermophilic activity: the half-life of CE was 24.4?min at 65°C. The half-life of CE was 145.5, 80.5, and 24.4?min at 60, 62, and 65°C, respectively. The K_m and V_max values for ChA were 0.153?mM and 559.6?µM/min, respectively. Moreover, the CE showed the highest substrate specificity with methyl caffeate among other methyl esters of hydroxycinnamic acids such as methyl ferulate, methyl sinapinate, methyl p-coumarate, and methyl caffeate. Ca²⁺, Cu²⁺, and Fe²⁺ significantly reduced the relative activity on ChA up to 70%. This is the first report on a thermostable CE from lactic acid bacteria that can be useful to hydrolyze ChA from plant cell walls.     

Phenolic extracts from various Algerian plants as strong inhibitors of porcine liver carboxylesterase

Carboxylesterases (CE), expressed at high levels in human liver and intestine, are thought to detoxify xenobiotics. The goal of this study was to study the effect of phenolic compounds from several plants from the Algerian Atlas used traditionally in Arab folk medicine on the enzymatic activity of porcine liver carboxylesterase. The plants have shown a potent inhibition of carboxylesterase (CE) enzymatic activity in a concentration-dependent manner. Results indicate that the Phenolic extracts from these plants lead to the inactivation of the CE pI = 5.1 with K_i values in micromolar range (1.4–38 μM). These results encourage further biological investigation and identification the inhibitors responsible for this activity.     

A scalable membrane gradostat reactor for enzyme production using Phanerochaete chrysosporium

This is the first demonstration of process scale-up of a membrane gradostat reactor for continuous enzyme production using Phanerochaete chrysosporium ME446. The fungus was immobilised by reverse filtration on to externally unskinned, ultrafiltration capillary membranes and then nutrient gradients were induced across the biofilm. A 10-fold scale-up from a single capillary bioreactor to a 2.4 l multi-capillary unit resulted in a 7-fold increase in enzyme productivity with a peak at 209 U l^–1 d^–1. Subsequent scale effects on the spore distribution, continuous manganese peroxidase production profile and biofilm development are discussed.     

Characterization of Muscle Sarcoplasmic and Myofibrillar Protein Hydrolysis Caused by Lactobacillus plantarum

Strains of Lactobacillus plantarum originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening, L. plantarum CRL 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE. The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine. In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE.     

Ferredoxin–NADP+ Reductase from Tsuga canadensis. A Procedure for Isolation and Properties

Activity of ferredoxin-NADP⁺ reductase in leaf extracts of eastern hemlock [Tsuga canadensis (L.) Carr.] was relatively low, but could be markedly increased by use of protective agents. The best method employed polyvinylpolypyrrolidone (PVP) in the extraction medium plus removal of phenolic compounds by filtering the extracts through an insoluble PVP (Polyclar AT) column. Further purification of the enzyme was achieved by means of DEAE cellulose chromatography and DEAE Sephadex chromatography. A 94-fold purification of the enzyme with a total recovery of 43% was obtained. The eastern hemlock ferredoxin-NADP⁺ reductase was characterized by its diaphorase activity, i.e. the transfer of electrons from NADPH to an electron acceptor. 2,6-dichlorophenol indophenol. The pH optimum for the oxidation of NADPH is between 8.5 and 9.0. The enzyme is highly specific for its electron donor. NADPH, but shows low specificity for electron acceptors. The apparent Michaelis constant values of the enzyme for NADPH. NADH, and 2,6-dichlorophenol indophenol are 2.4 × 10^?5, 5.4 × 10^?3, and 4.7 × 10^?5M respectively. The molecular weight of the enzyme, as estimated by gel filtration, is about 45,000. The enzyme is inhibited by both organic and inorganic mercurials and certain cations. Comparison of properties of eastern hemlock ferredoxin-NADP⁺ reductase and spinach ferredoxin-NADP⁺ reductase shows that both enzymes are similar.     

Evaluation of the inhibitory activities of aceraceous plants on fatty acid synthase

Fatty acid synthase (FAS) is a very significant lipogenic enzyme participating in energy metabolism in vivo and has been reported as a potential new therapeutic target for cancer treatment. The extracts from sixteen Aceraceae were prepared to assay their inhibitory activities against duck liver FAS and their correlated antitumor bioactivity. Their inhibition of FAS was composed of a reversible fast-binding inhibition, by which 0.41 μg/mL of the A. campestre extract inhibits 50% FAS activity, and an irreversible slow-binding inhibition with inactivation rate constants, k_obs, ranging between 1.5 × 10^{? 3} and 10.6 × 10^{? 3} min^{? 1}. Three Aceraceae extracts were selected from their smaller IC₅₀ values to study different type of inhibitions against the three substrates in the FAS overall reaction. As compared with other reported FAS inhibitors including EGCG with regard to inhibition constant and IC₅₀ value, the extracts appeared to be more efficient inhibitors, and exhibited a considerable inhibition against the growth of five types of cancer cells (China patent application number 200610088901.6), which may be related to the inhibition of lipogenesis in these cells.     

Capillary electrophoresis-based assay of phosphofructokinase-1

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg–ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg–ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg–ATP and Mg–ADP. The separation was enhanced by the addition of Mg²⁺ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC₅₀ value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.     

On Growth-inhibition against Aspergillus oryzae No 40 by the Compound III-3 in Rice Grain

Partially purified lipoxygenase (LOX) extracts were obtained from Fusarium proliferatum, Fusarium oxysporum, Chlorella pyrenoidosa, and Saccharomyces cerevisiae; the enzymatic extract of F. proliferatum showed the highest LOX activity while those of F. oxysporum and S. cerevisiae demonstrated only 27.8 and 16.5% of the activity at pH 8.0, and 61.2 and 9.7% of the enzyme activity at pH 10.0, respectively. The lowest LOX activity was that in the C. pyrenoidosa extract. The microbial enzymatic preparations were assayed with linoleic acid as substrate, which was bioconverted into 9- and 13-hydroperoxides (HPODEs) by all four extracts; in additon, the LOX activity in the F. oxysporum extract produced the 10- and 12-HPODEs from linoleic acid while that of the C. pyrenoidosa extract produced only the 10- HPODE. When assayed with the 9- and 13-HPODEs as substrates, the selected microbial extracts had secondary enzyme activities, one of which produced hexanal. The highest hexanal-producing activity was 1.51 and 1.39 nmol hexanol/mg protein/min in the F. proliferatum and C. pyrenoidosa extracts, respectively, while those of F. oxysporum and S. cerevisiae had approximately 15% of the HPODE-cleaving enzyme activity. The C. pyrenoidosa extract had the highest proportion of pentanone, which was produced at only one-fourth the concentration by the HPODE-cleaving enzyme activity in the three other microbial enzymatic extracts.