Rational modification of substrate binding site by structure-based engineering of a cellobiose 2-epimerase in <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

Background

Lactulose, a synthetic disaccharide, has received increasing interest due to its role as a prebiotic, specifically proliferating Bifidobacilli and Lactobacilli and enhancing absorption of calcium and magnesium. The use of cellobiose 2-epimerase (CE) is considered an interesting alternative for industrial production of lactulose. CE reversibly converts d-glucose residues into d-mannose residues at the reducing end of unmodified β-1,4-linked oligosaccharides, including β-1,4-mannobiose, cellobiose, and lactose. Recently, a few CE 3D structure were reported, revealing mechanistic details. Using this information, we redesigned the substrate binding site of CE to extend its activity from epimerization to isomerization.

Results

Using superimposition with 3 known CE structure models, we identified 2 residues (Tyr114, Asn184) that appeared to play an important role in binding epilactose. We modified these residues, which interact with C2 of the mannose moiety, to prevent epimerization to epilactose. We found a Y114E mutation led to increased release of a by-product, lactulose, at 65 °C, while its activity was low at 37 °C. Notably, this phenomenon was observed only at high temperature and more reliably when the substrate was increased. Using Y114E, isomerization of lactose to lactulose was investigated under optimized conditions, resulting in 86.9 g/l of lactulose and 4.6 g/l of epilactose for 2 h when 200 g/l of lactose was used.

Conclusion

These results showed that the Y114E mutation increased isomerization of lactose, while decreasing the epimerization of lactose. Thus, a subtle modification of the active site pocket could extend its native activity from epimerization to isomerization without significantly impairing substrate binding. While additional studies are required to scale this to an industrial process, we demonstrated the potential of engineering this enzyme based on structural analysis.

     

Enzymatic properties of cellobiose 2-epimerase from <Emphasis Type="Italic">Ruminococcus albus</Emphasis> and the synthesis of rare oligosaccharides by the enzyme

The gene for cellobiose 2-epimerase (CE) from Ruminococcus albus NE1 was overexpressed in Escherichia coli cells. The recombinant CE was purified to homogeneity by a simple purification procedure with a high yield of 88%, and the molecular mass was 43.1 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and 44.0 kDa on gel chromatography. It exhibited optimal activity around at 30 degrees C and pH 7.5, and the enzyme activity was inhibited by Al3+, Fe3+, Co2+, Cu2+, Zn2+, Pb2+, Ag+, N-bromosuccinimide, iodoacetate, and 4-chloromercuribenzoate. In addition to cello-oligosaccharides, the enzyme was found to effectively 2-epimerize lactose to yield 4-O-beta-D-galactopyranosyl-D-mannose (epilactose), which occurs in cow milk as a rare oligosaccharide. The Km and kcat/Km values toward lactose were 33 mM and 1.6 s(-1) mM(-1), and those toward cellobiose were 13.8 mM and 4.6 s(-1) mM(-1), respectively. N-Acetyl-D-glucosamine, uridine 5'-diphosphate-glucose, D-glucose 6-phosphate, maltose, sophorose, laminaribiose, and gentiobiose were inert as substrates for the recombinant CE. We demonstrated that epilactose was resistant to rat intestinal enzymes, utilized by human adult bifidobacteria, and stimulated the tight junction permeability in Caco-2 cells. These results strongly suggest that this rare disaccharide is promising for use as a prebiotic.     

Cloning and sequencing of the gene for cellobiose 2-epimerase from a ruminal strain of Eubacterium cellulosolvens

Cellobiose 2-epimerase (CE; EC 5.1.3.11) is known to catalyze the reversible epimerization of cellobiose to 4-O-beta-d-glucopyranosyl-d-mannose in Ruminococcus albus cells. Here, we report a CE in a ruminal strain of Eubacterium cellulosolvens for the first time. The nucleotide sequence of the CE had an ORF of 1218 bp (405 amino acids; 46 963.3 Da). The CE from E. cellulosolvens showed 44-54% identity to N-acyl-d-glucosamine 2-epimerase-like hypothetical proteins in the genomes of Coprococcus eutactus, Faecalibacterium prausnitzii, Clostridium phytofermentans, Caldicellulosiruptor saccharolyticus, and Eubacterium siraeum. Surprisingly, it exhibited only 46% identity to a CE from R. albus. The recombinant enzyme expressed in Escherichia coli was purified by two-step chromatography. The purified enzyme had a molecular mass of 46.7 kDa and exhibited optimal activity at around 35 degrees C and pH 7.0-8.5. In addition to cello-oligosaccharides, it converted lactose to epilactose (4-O-beta-d-galactopyranosyl-d-mannose).     

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.     

Electricity generation from young landfill leachate in a microbial fuel cell with a new electrode material

This study aims at evaluating the performance of a two-chambered continuously fed microbial fuel cell with new Ti–TiO₂ electrodes for bioelectricity generation from young landfill leachate at varying strength of wastewater (1–50 COD g/L) and hydraulic retention time (HRT, 0.25–2 days). The COD removal efficiency in the MFC increased with time and reached 45 % at full-strength leachate (50 g/L COD) feeding. The current generation increased with increasing leachate strength and decreasing HRT up to organic loading rate of 100 g COD/L/day. The maximum current density throughout the study was 11 A/m² at HRT of 0.5 day and organic loading rate of 67 g COD/L/day. Coulombic efficiency (CE) decreased from 57 % at feed COD concentration of 1 g/L to less than 1 % when feed COD concentration was 50 g/L. Increase in OLR resulted in increase in power output but decrease in CE.     

Biochemical Characterization of a Thermophilic Cellobiose 2-Epimerase from a Thermohalophilic Bacterium,Rhodothermus marinus JCM9785

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2–10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.     

Endogenous and exogenous domain markers of the rat hepatocyte plasma membrane

We have used a combined biochemical and morphological approach to establish the suitability of certain endogenous and exogenous domain markers for monitoring the separation of rat hepatocyte plasma membrane domains in sucrose density gradients. As endogenous domain markers, we employed two of the integral plasma membrane protein antigens, HA 4 and CE 9, localized to the bile canalicular and sinusoidal/lateral domains, respectively, of the hepatocyte plasma membrane in rat liver tissue (Hubbard, A. L., J. R. Bartles, and L. T. Braiterman, 1985, J. Cell Biol., 100:1115-1125). We used immunoelectron microscopy with a colloidal gold probe to demonstrate that HA 4 and CE 9 retained their domain-specific localizations on isolated hepatocyte plasma membrane sheets. When the plasma membrane sheets were vesiculated by sonication and the resulting vesicles were centrifuged to equilibrium in sucrose density gradients, quantitative immunoblotting revealed that the vesicles containing HA 4 and those containing CE 9 exhibited distinct density profiles. The density profile for the bile canalicular vesicles (marked by HA 4) was characterized by a single peak at a density of 1.10 g/cm3. The density profile for the sinusoidal/lateral vesicles (marked by CE 9) was bimodal, with a peak in the body of the gradient at a density of 1.14 g/cm3 and a smaller amount in the pellet (density greater than or equal to 1.17 g/cm3). We used this sucrose gradient fractionation as a diagnostic procedure to assign domain localizations for several other hepatocyte plasma membrane antigens and enzyme activities. In addition, we used the technique to demonstrate that 125I-wheat germ agglutinin, introduced during isolated liver perfusion at 4 degrees C, can serve as an exogenous domain marker for the sinusoidal domain of the rat hepatocyte plasma membrane.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group.

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.     

Comparison of a capillary electrophoresis method with high-performance liquid chromatography for the determination of biogenic amines in various food samples

A capillary electrophoresis (CE) and a high-performance liquid chromatography (HPLC) method to analyze biogenic amines in food were compared. An automated precolumn derivatization with o-phthaldialdehyde (OPA) allows for the determination of aliphatic amines and amino acids by HPLC. In contrast, for the measurement of histamine and tyramine by CE, no laborious sample pretreatment was necessary. The biogenic amines were separated by CE or HPLC in less than 9 or 20 min, respectively. The calibration curves were linear to at least 100 mg/kg (r=0.999) and 1,000 mg/kg for HPLC and CE, respectively, with detection limits for histamine of 0.5 mg/kg (fluorescence detector) or 1 mg/kg (diode array detector) with HPLC and 2 mg/kg with CE. The detection limits for tyramine were 1.5 mg/kg with HPLC and 6 mg/kg with CE and for further amines (e.g., putrescine, spermidine, cadaverine, agmatine) ranging from 1.0 to 8.5 mg/kg with HPLC. There was a good correlation between CE and HPLC (correlation coefficient for histamine: 0.994).     

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.     

Differential proteome analysis and mass spectrometric characterization of germ line development-related proteins of Caenorhabditis elegans

Proteome maps and differences of protein patterns of the synchronized larval stage L4 of the temperature-sensitive Caenorhabditis elegans (C. elegans) glp-1 mutant (e2144ts) were investigated after cultivation at 15 degrees C (developing a normal phenotype) or 25 degrees C (developing a mutated phenotype) by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. From the 183 identified protein spots six proteins were found differently expressed. The Vit-6 vitellogenin (CE28594), the hypothetical 17.2 protein (CE25224), the hypothetical 17.4 protein (CE16999), and the heat shock protein 16 kDa (CE14249) were more abundant when growing worm cultures at 25 degrees C. By contrast, the nucleoside diphosphate kinase (CE09650) was found increased at 15 degrees C. Most notably, the eukariotic initiation factor 5A-1 (CE00503), highly abundant at 15 degrees C, was not present in cultures grown at 25 degrees C. Its absence at 25 degrees C can not be attributed to lack of the enzymatic machinery that is necessary for hypusinylation. Instead, a direct downstream effect of the lack of functionality of GLP-1 may cause the expression of this protein. The yolk proteins 115 kDa and 88 kDa were attributed by mass spectrometric protein structure analysis as C-terminal and N-terminal fragments of the Vit-6 vitellogin protein (CE28594), respectively. The cleavage site between both derivatives was located between R764 and A768. A conflict in the database sequences at amino acid positions 1622 and 1623 of vitellogenin-6 was solved by mass spectrometric sequence analysis. The combination of 2-DE with mass spectrometry enabled the identification of mutation-associated differences on somatic gonadal cell and germ line cell development-associated proteins.     

Fish oil decreases hepatic cholesteryl ester secretion but not apoB secretion in African green monkeys

Two groups of African green monkeys were fed diets containing 40% of calories as fat with half of the fat calories as either fish oil or lard. The fish oil-fed animals had lower cholesterol concentrations in blood plasma (33%) and low density lipoproteins (LDL) (34%) than did animals fed lard. Size and cholesteryl ester (CE) content of LDL, strong predictors of coronary artery atherosclerosis in monkeys, were significantly less for the fish oil-fed animals although the apoB and LDL particle concentrations in plasma were similar for both diet groups. We hypothesized that decreased hepatic CE secretion led to the smaller size and reduced CE content of LDL in the fish oil-fed animals. Hepatic CE secretion was studied using recirculating perfusion of monkey livers that were infused during perfusion with fatty acids (85% 18:1 and 15% n-3) at a rate of 0.1 mumol/min per g liver. The rate of cholesterol secretion was less (P = 0.055) for the livers of fish oil versus lard-fed animals (3.3 +/- 0.5 vs. 6.0 +/- 1.2 mg/h per 100 g, mean +/- SEM) but the rate of apoB secretion was similar for both groups (0.92 +/- 0.15 vs. 1.01 +/- 0.13 mg/h per 100 g, respectively). The hepatic triglyceride secretion rate was also less (P less than 0.05) for the fish oil-fed animals (8.3 +/- 2.5 vs. 18.3 +/- 4.4 mg/h per 100 g). Liver CE content was lower (P less than 0.006) in fish oil-fed animals (4.1 +/- 0.8 vs. 7.4 +/- 0.7 mg/g) and this was reflected in a lower (P less than 0.04) esterified to total cholesterol ratio of perfusate VLDL (0.21 +/- 0.045 vs. 0.41 +/- 0.06). The hepatic VLDL of animals fed fish oil had 40-50% lower ratios of triglyceride to protein and total cholesterol to protein. From these data we conclude that livers from monkeys fed fish oil secreted similar numbers of VLDL particles as those of lard-fed animals although the hepatic VLDL of fish oil-fed animals were smaller in size and relatively enriched in surface material and depleted of core constituents. Positive correlations between plasma LDL size and both hepatic CE content (r = 0.87) and hepatic VLDL cholesterol secretion rate (r = 0.84) were also found.(ABSTRACT TRUNCATED AT 400 WORDS)     

Practical Preparation of Epilactose Produced with Cellobiose 2-Epimerase from Ruminococcus albus NE1

A practical purification method for a non-digestible disaccharide, epilactose (4-O-β-galactosyl-D-mannose), was established. Epilactose was synthesized from lactose with cellobiose 2-epimerase and purified by the following procedure: (i) removal of lactose by crystallization, (ii) hydrolysis of lactose by β-galactosidase, (iii) digestion of monosaccharides by yeast, and (iv) column chromatography with Na-form cation exchange resin. Epilactose of 91.1% purity was recovered at 42.5% yield.     

Simultaneous saccharification and co-fermentation of crystalline cellulose and sugar cane bagasse hemicellulose hydrolysate to lactate by a thermotolerant acidophilic Bacillus sp

Polylactides produced from renewable feedstocks, such as corn starch, are being developed as alternatives to plastics derived from petroleum. In addition to corn, other less expensive biomass resources can be readily converted to component sugars (glucose, xylose, etc.) by enzyme and/or chemical treatment for fermentation to optically pure lactic acid to reduce the cost of lactic acid. Lactic acid bacteria used by the industry lack the ability to ferment pentoses (hemicellulose-derived xylose and arabinose), and their growth and fermentation optima also differ from the optimal conditions for the activity of fungal cellulases required for depolymerization of cellulose. To reduce the overall cost of simultaneous saccharification and fermentation (SSF) of cellulose, we have isolated bacterial biocatalysts that can grow and ferment all sugars in the biomass at conditions that are also optimal for fungal cellulases. SSF of Solka Floc cellulose by one such isolate, Bacillus sp. strain 36D1, yielded l(+)-lactic acid at an optical purity higher than 95% with cellulase (Spezyme CE; Genencor International) added at about 10 FPU/g cellulose, with a product yield of about 90% of the expected maximum. Volumetric productivity of SSF to lactic acid was optimal between culture pH values of 4.5 and 5.5 at 50 degrees C. At a constant pH of 5.0, volumetric productivity of lactic acid was maximal at 55 degrees C. Strain 36D1 also co-fermented cellulose-derived glucose and sugar cane bagasse hemicellulose-derived xylose simultaneously (SSCF). In a batch SSCF of 40% acid-treated hemicellulose hydrolysate (over-limed) and 20 g/L Solka Floc cellulose, strain 36D1 produced about 35 g/L lactic acid in about 144 h with 15 FPU of Spezyme CE/g cellulose. The maximum volumetric productivity of lactic acid in this SSCF was 6.7 mmol/L (h). Cellulose-derived lactic acid contributed to about 30% of this total lactic acid. These results show that Bacillus sp. strain 36D1 is well-suited for simultaneous saccharification and co-fermentation of all of the biomass-derived sugars to lactic acid.     

Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells.

A comparison of the ability of vesicular stomatitis virus (VSV) to generate and replicate defective interfering (DI) particles in primary chick embryo (CE) and mouse L cells was investigated as a means of analyzing host control over DI-particle synthesis and interfering capacity. Serial undiluted passage of VSV in CE and L cells indicate that VSV-DI particles are generated and (or) replicate with greater efficiency in CE than in L cells. When DI particles accumulate in L cells, they are able to interfere with infectious particle replication. The DI particles from CE cells interfered to the same extent with infectious particle replication in both CE and L cells. L cells, therefore, are not considered 'low-interference' hosts in which DI particles are produced and do not interfere with infectious virus replication, but rather hosts which restrict the production of DI particles.     

Determination of metformin hydrochloride using precolumn derivatization with acetaldehyde and capillary electrophoresis coupled with electrochemiluminescence

A novel method was developed using capillary electrophoresis (CE) coupled with tris(2,2′‐bipyridyl)ruthenium(II) electrogenerated chemiluminescence (ECL) for highly sensitive detection of metformin hydrochloride (MH) derivatizatized with acetaldehyde. The precolumn derivatization of MH with acetaldehyde was performed in phosphate buffer solution (0.3 mol/L, pH 7.5) at room temperature for 120 min. The effects of acetaldehyde concentration, buffer pH, electrokinetic voltage and injection time were investigated. Under optimized detection conditions, the MH ECL detection sensitivity was more than 120 times that without derivatization. The linear concentration range for MH was 0.001–15.00 μg/mL (with a correlation coefficient of 0.9992). The detection limit was 0.31 ng/mL with a signal:noise ratio of 3. The recoveries of MH in human urine were in the range 98.50–99.72%. Copyright © 2011 John Wiley & Sons, Ltd.     

Cells Persistently Infected with Newcastle Disease Virus: II. Ribonucleic Acid and Protein Synthesis in Cells Infected with Mutants Isolated from Persistently Infected L Cells

A comparison of the replication patterns in L cells and in chick embryo (CE) cell cultures was carried out with the Herts strain of Newcastle disease virus (NDV(o)) and with a mutant (NDV(pi)) isolated from persistently infected L cells. A significant amount of virus progeny, 11 plaque-forming units (PFU)/cell, was synthesized in L cells infected with NDV(o), but the infectivity remained cell-associated and disappeared without being detectable in the medium. In contrast, in L cells infected with NDV(pi), progeny virus (30 PFU/cell) was released efficiently upon maturation. It is suggested that the term "covert" rather than "abortive" be used to describe the infection of L cells with NDV(o). In both L and CE cells, the latent period of NDV(pi) was 2 to 4 hr longer than for NDV(o). The delay in synthesis of viral ribonucleic acid (RNA) in the case of NDV(pi) coincided with the delay in the inhibition of host RNA and protein synthesis. Although both NDV(o) and NDV(pi) produced more progeny and more severe cell damage in CE cells than in L cells, the shut-off of host functions was significantly less efficient in CE cells than in L cells. Paradoxically, no detectable interferon was produced in CE cells by either of the viruses, whereas in L cells most of the interferon appeared in the medium after more than 90% of host protein synthesis was inhibited. These results suggest that the absence of induction of interferon synthesis in CE cells infected with NDV is not related to the general shut-off of host cell synthetic mechanisms but rather to the failure of some more specific event to occur. In spite of the fact that NDV(pi) RNA synthesis commenced 2 to 4 hr later than that of NDV(o), interferon was first detected in the medium 8 hr after infection with both viruses. This finding suggests that there is no relation between viral RNA synthesis and the induction of interferon synthesis.     

Determination of agmatine in biological samples by capillary electrophoresis with chemiluminescence detection

A fast and simple method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of agmatine, a recently identified neurotransmitter/modulator. The CE run time was approximately 2 min for each sample injected. CL detection employed a lab-built reaction flow cell and a photon counter. The CL reagents used were luminol and NaBrO. The optimized conditions for the CL detection were 5 x 10(-4)M luminol added to the CE running buffer and 5.0 x 10(-4)M NaBrO in 100 mM NaCO3-NaOH buffer solution at pH 12.5 introduced post column. Detection limit for agmatine was 4.3 x 10(-6)M (S/N=3). The precision (R.S.D.) on peak height (at 1 x 10(-5)M agmatine) and migration time were 3.7 and 2.5%, respectively. The present CE-CL method was evaluated with the determination of agmatine in tissue samples taken from rat brain, and rat and monkey stomachs. Samples were directly injected into the CE-CL system after the removal of proteins. A higher level of agmatine was detected in the stomach samples. Agmatine concentrations in the tissue samples taken from rat and monkey stomachs were similar at approximately 1950 ng/g wet tissue.     

Determination of alkaloids in Sinomenium acutum by field‐amplified sample stacking in capillary electrophoresis with chemiluminescene detection

A simple and rapid capillary electrophoresis (CE) with an acidic potassium permanganate chemiluminescence (CL) detection method was developed to determine three alkaloids (curine, sinomenine and magnoflorine) simultaneously. A laboratory‐built CE–CL detection interface was used. The field‐amplified sample stacking technique was applied to the online concentration of alkaloids. Experimental conditions for CE separation and CL detection were investigated in detail to acquire optimum conditions. Under optimal conditions, the three alkaloids were baseline separated within 6 min, and the detection limits (S/N = 3) ranged from 0.03 µg/mL to 0.49 µg/mL. This method was successfully applied to determine the above three alkaloids in Sinomenium acutum, and the result of the determination of sinomenine was in good agreement with those given by high‐performance liquid chromatography and CE methods. In addition, a possible CL reaction mechanism of sinomenine–KMnO₄–H₂SO₄ was proposed. Copyright © 2013 John Wiley & Sons, Ltd.