A fast, sensitive HPLC method for the determination of esterase activity on alpha-tocopheryl acetate

Although data on the behavior of tocopheryl acetate (Tac) in the gut is rare, some studies show that this ester is hydrolyzed in the intestine and the released tocopherol (Tol) is absorbed. An HPLC method is published for the simultaneous analysis of Tol and Tac in mixed micelles. This study shows how this method is used for the measurement of esterase activity on Tac using commercial cholesterol esterase. The rate of hydrolysis is determined in less than 20 min on the basis of both Tac disappearance and Tol appearance. The present method is useful for investigating esterase activities on Tac in model systems that mimick the absorption environment or in any biological or nonbiological medium.     

Effect of Calcium Cation on Thermophilic Acylamino Acid-releasing Enzyme Ape1547 from Aeropyrum pernix K1

The gene of enzyme(Ape1547) was cloned from hyperthermophilic archaeon Aeropyrum pernix K1 and expressed in Escherichia coil.The effect of calcium cation on the properties of Ape1547 was studied.Ape1547 exhibits both peptidase activity and esterase activity.The fluorescence spectrum shows that calcium cation quenches the fluorescence of the enzyme through static quenching mechanism,indicating that calcium cation was bound to the enzyme.Based on the study of calcium cation on CD ellipticity of Ape1547 by cir...     

Peptidase Activities of Tripeptidyl Peptidase Ⅰ(TPP Ⅰ): Exopeptidase and Endopeptidase

The defect of TPP Ⅰ causes a disease, late infantile neuronal ceroid lipofuscinosis(LINCL, CLN2). To investigate the bio-activity of tripeptidyl peptidase Ⅰ(TPP Ⅰ) from rat kidneys, the effects of digestion of angiotensin Ⅱ(Ang Ⅱ) and a synthetic endo-type substrate(Gly1-Lys-Pro-Iie-Pro5-Phe-Phe-Arg-Leu-Lys10) via TPP Ⅰ were analyzed by HPLC and TOF-MS. The data suggest that the degradation rate of Ang Ⅱ can reach 18.2% by the rat TPP Ⅰ and DRV(Asp-Arg-Val) can be released from N-termini of Ang Ⅱ within 16 h. In addition, the synthetic endo-type substrate is cleaved at the same position between Phe6 and Phe7. Accordingly, TPP Ⅰ shows two kinds of peptidase activities. One is a tripeptidyl peptidase activity and the other is a pepstatin insensitive carboxyl endopeptidase activity. Tripeptidyl peptidase activity and pepstatin insensitive carboxyl endopeptidase activity seem to be dual phases of one enzyme, TPP Ⅰ.     

Expression and Characterization of a Thermostable Acyl-peptide Releasing Enzyme ST0779 from Sulfolobus tokodaii

Acyl-peptide releasing enzyme(AARE) belongs to a serine peptidase family and catalyzes the NH₂-terminal hydrolysis of N^α-acylpeptides to release N^α-acylated amino acids. ORF0779(ORF=open reading frame) from thermophilic archaea Sulfolobus tokodaii(ST0779) was cloned and expressed in E. coli BL21 and the expressed protein was identified as a thermostable AARE. The target protein could be optimally overexpressed in E. coli at 30 ℃ for 8 h with 0.1 mmol/L isopropyl β-dthiogalactoside(IPTG). The crude enzyme was heated at 70 ℃ for 30 min, and then the target protein could account for above 40% of the total protein. The purification fold was 27 and the enzyme showed both esterase activity and peptidase activity. The optimal temperature and pH for ST0779 were 70 ℃ and 8.0 when Ac-Ala₃ was used as substrate. The half-life of the enzyme(0.2 mg/mL) at 90 ℃ was about 16 h, indicating that the enzyme exhibits a favorable thermostability. The activity of ST0779 could still remain over 85% after being treated at 25 ℃ in different buffers with pH range from 6.0 to10.0 for 24 h, which indicates ST0779 is stable in neutral or slight alkali environment. Under neutral or slightly alkali conditions, the enzyme exhibits really high catalytic efficiency against acyl-peptide, and the optimal substrate is Ac-Ala₃. Most metal ions have no inhibition effect on the activity of ST0779, while 4% activity of ST0779 is inhibited in the presence of K⁺. This enzyme was supposed to be applied in the analysis of protein sequencing and the synthesis of small peptides.     

胰高血糖素样肽-1类似物的合成及其生物学活性研究

通过改变胰高血糖素样肽-1 (GLP-1)易被酶水解部位, 采用Fmoc/t-Bu正交保护固相合成策略, 并运用微波照射促进高效快速地合成了抗二肽基肽酶IV (DPP IV)的GLP-1类似物, 最后经过反相制备高效液相系统纯化得到目标多肽纯品. 生物学活性研究结果显示, 改造后的GLP-1类似物能有效抵抗DPP IV的水解, 并且有很好的降血糖活性. 所合成的GLP-1及其类似物的分子量和纯度均经过电喷雾质谱和高效液相确证.     

Production and Study of Monoclonal Antiidiotypic Antibody with a Catalytic activity similar to Carboxypeptidase A

Catalytic antibody(abzynie) is a new class of biocatalysts. Combining the specificity and the enormous molecular diversity of the antibody with high catalytic efficiency of enzyme to produce catalytic antibody, that is a new method of designing protein. A method of producing catalytic antibody developped recently is to produce antiidiotypic antibody. As the "internal images" of the antigen structure, the antiidiotypic antibody can exhibit the properties similar to the antigen¹. To produce the antiidiotypic antibody of the natural enzyme, we may obtain the catalytic antibody whose structure and catalytic activity are similar to the natural euzyme. A. Friboulet et al successfully produced the antiidiotypic catalytic antibody of acetylcholinesterase, whose catalytic activity approaches the natural acetylcholinesterase². Carboxypeptidase (CPA) has peptidase and esterase activities, to produce the antiidiotypic antibody of CPA, we may obtain the catalytic antibody which catalyze the hydrolysis of amide bond and highly efficiently catalyze the hydrolysis of ester bond. Therefore, this research work is very significant. In this report, we produced a monoclonal antiidiotypic antibody with a catalytic activity similar to the esterase activity of CPA and studied it's kinetic property.     

Micropeptins from Microcystis aeruginosa collected in Dalton reservoir, Israel

Three new micropeptins, micropeptin DR1056, DR1060 and DR1006 and three known metabolites, micropeptin SF909, aeruginosins 298A and B were isolated from the extracts of a Microcystis aeruginosa bloom material collected in Dalton reservoir, Israel. The planar structure of the compounds was determined by homonuclear and inverse-heteronuclear 2D NMR techniques as well as high-resolution mass spectrometry. The absolute configuration of the asymmetric centers of the amino acids was studied using Marfey’s method for HPLC. The inhibitory activity of the compounds was determined for the serine proteases: trypsin, chymotrypsin, and elastase and for amino peptidase N.     

Determination of cocaine and norcocaine in plasma and cell cultures using high-performance liquid chromatography

A new simple high-performance liquid chromatographic (HPLC) method was developed for the determination of cocaine and norcocaine. Cocaine and norcocaine in biological samples were buffered to pH 9.0, extracted with diethyl ether and reextracted in a 0.1% aqueous solution of tetramethylammonium hydrogen sulfate (TMAHS) with a theoretical yield of extraction of 100%. The HPLC elution of cocaine and norcocaine was performed using a Spherisorb RP-18, 100 mm x 4.6 mm I.D., 5 microns particle size column with a mobile phase containing acetonitrile-0.1% TMAHS aqueous solution (60:40). The compounds were entirely separated, and a reliable limit of quantitation was set at 20 ng/ml when extracted from 0.5 ml of plasma. No interference with 26 other drugs was found. Cocaine and norcocaine stability studies showed that their half-lives in human plasma incubated at 37 degrees C were 50.8 and 43.2 min, respectively. In contrast, plasma from dogs or rats exhibited only weak or no enzymatic esterase activity towards cocaine and norcocaine resulting in less rapid degradation. Hydrolysis could be efficiently inhibited with sodium fluoride and prevented by storage of the sample at -20 degrees C. The highly sensitive assay also allowed the assessment of the oxidative metabolism pathway of cocaine to norcocaine in primary rat hepatocyte cultures.     

Analysis of endoproteinase Arg C action on adrenocorticotrophic hormone by capillary electrophoresis and reversed-phase high-performance liquid chromatography.

The specificity and rate of cleavage of adrenocorticotrophic hormone (ACTH) peptide bonds by endoproteinase Arg C were analyzed using capillary electrophoresis (CE) and reversed-phase (C18) high-performance liquid chromatography (HPLC). Acidic cleavage products were readily resolved by CE in uncoated capillaries using low ionic strength electrolytes. However, products predicted to have a net positive charge greater than 2 or more than 4 positively charged groups per peptide did not migrate out from the capillary at low ionic strength. Addition of salts and zwitterions to the electrolyte decreased capillary-peptide interactions such that all of the ACTH peptides examined were eluted with high efficiency separation by CE. Commercially obtained endoproteinase Arg C preparations exhibited peptidase activity at Lys-15-Lys16 and at Lys16-Arg17 in addition to the expected cleavage at Arg-X bonds. ACTH peptide bond cleavage rates for Arg8-Trp9, Arg17-Arg-18, Lys15-Lys16, and Lys16-Arg17 were 1.46, 0.096, 0.57, and 0.029 mumol min-1 mg-1 respectively. CE separations generally exhibited better resolution and were accomplished in shorter times than C18 HPLC separations. These properties make CE a particularly appropriate method for kinetic analysis of proteolytic enzyme action on peptide substrates.     

Alteration of enzyme activity and enantioselectivity by biomimetic encapsulation in silica particles

Direct encapsulation of esterase or lipase fused with the silica-precipitating R5 peptide from Cylindrotheca fusiformis in silica particles afforded high yields of active entrapped protein. The hydrolytic activity of both enzymes against p-nitrophenyl butyrate was similarly affected by encapsulation and the enantioselectivity of the esterase was both improved and inverted.     

Micro-Scale Extraction and Analysis of Intact Carboxylesterase after Trapping on an Immunoaffinity Membrane Surface

Porcine liver carboxylesterase was captured using an immunoaffinity membrane, which was prepared by separating an anti-porcine esterase antibody using non-denaturing two-dimensional electrophoresis, followed by transfer to a polyvinylidene difluoride membrane and staining. The activity of this esterase was 0.008 units after it was captured in the tiny spaces (4 mm²) of this membrane and eluted by rinsing with 5 μL of aspartic acid solution. The molecular mass of the eluted esterase was m/z 61,885 according to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the purification of this enzyme from the porcine liver cytosol. The purified enzyme’s activity was inhibited by 6,9-diamino-2-ethoxyacridine, and this inhibition was retained even after extracting the enzyme from the immunoaffinity membrane. These results indicate that micro-scale extraction and analysis of a carboxylesterase are possible when the enzyme is trapped using an immunoaffinity membrane and eluted with aspartic acid.     

Detection of Saccharomyces cerevisiae carboxylesterase activity after native and sodium dodecyl sulfate electrophoresis by using fluorescein diacetate as substrate

A simple method for the visualisation of wine yeast esterase (carboxylesterase EC 3.1.1.1) activity on electrophoretic gels was developed, using the fluorescent substrate fluorescein diacetate. The zymogram system allows a sensitive detection of esterase bands in only 5 min of incubation of both native and sodium dodecyl sulfate gels.     

3-Hydroxyglutarate and β,γ-Epoxy Esters as Substrates for Pig Liver Esterase (PLE) and α-Chymotrypsin

The pH dependence of the α-chymotrypsin-catalyzed hydrolysis of dimethyl 3-hydroxyglutarate ( 3 ) has been studied. The e.e. was determined by HPLC analysis of diastereoisomeric camphanoic-acid derivatives. Kinetic resolution of the β,α-epoxy esters 10 and 24 by pig liver esterase has been shown to provide an alternative access to chiral β-hydroxy esters and acids of high optical purity. By this latter method, the unnatural enantiomer of γ-amino-β-hydroxybutyric acid (GABOB) has been synthesized. Finally, dimethyl meso-3,4-epoxyadipate ( 19 ) was hydrolyzed by pig liver esterase with almost 100% selectivity.     

用于农药残留快速检测的两种酶的比较

以商品乙酰胆碱酯酶(C3389,Ⅵ-S,从电鳗中提取)和自制鸡肝酶为农药检测用酶,比较了不同浓度的敌敌畏、敌百虫、马拉硫磷和西维因对这两种酶的乙酰胆碱酯酶活力和总酯酶活力的抑制情况。结果表明,鸡肝酶的乙酰胆碱酯酶活力较低,而其总酯酶活力对4种农药的灵敏度与商品乙酰胆碱酯酶活力的灵敏度相近,且总酯酶活力对除马拉硫磷外的其余3种农药的检出限都较乙酰胆碱酯酶低。同时对总酯酶活力测定的pH进行了优化,发现pH6．5(40mmol／L柠檬酸盐缓冲液)时总酯酶活力较高。     

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells

Background: Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities.Results: Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aidehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called ‘branched chain amino acid preferring’ (BrAAP) and ‘small neutral amino acid preferring’ (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity.Conclusions: The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate,     

Atomic force microscopy and enzymatic degradation of oyster shell protein and poly(aspartate)

Oyster shell protein (OSP), an aspartate-enriched regulator of crystallization, was readily observed in its natural condition by atomic force microscopy (AFM) of fragments of oyster shell. The fragments of shell consisted of layers of calcite mineral, termed folia, to which arrays of protein molecules are attached. Modification and removal of the OSP following treatment with several proteolytic enzymes such as subtilisin, carboxypeptidase B, and endoproteinase Glu-C were also observed by AFM. Similarly, poly (aspartate), a polypeptide analog of the OSP, was visualized by AFM on both calcite and mica. Images of poly(aspartate) before and after treatment with lipase demonstrated the potential utility of AFM in degradation studies. The mechanism of hydrolysis is not clear in that lipase normally is considered to be an esterase and not a peptidase.     

Cloning,Purification, and Characterization of Tripeptidyl Peptidase from <Emphasis Type="Italic">Streptomyces herbaricolor</Emphasis> TY-21

Tripeptidyl peptidase (TPP) is an exopeptidase that sequentially hydrolyzes tripeptides from the N-terminus of oligopeptides or polypeptides. We performed screening for isolating novel TPP-producing microorganisms from soil samples. TPP activity was observed in the culture supernatant of Streptomyces herbaricolor TY-21 by using Ala-Ala-Phe-p-nitroanilide (pNA) as the substrate. TPP from the culture supernatant was purified to approximately 790-fold. It was shown to cleave oxidized insulin B-chain, thereby with releasing tripeptide units, but not the N-terminal-protected peptide, Cbz-Ala-Ala-Phe-pNA. The TPP gene, designated tpp, was isolated from a partial genomic DNA library of S. herbaricolor TY-21. The TPP gene consisted of 1488 bp, and encoded a 133-amino acid pre-pro-peptide and a 362-amino acid mature enzyme containing conserved amino acid residues (Asp-36, His-77, and Ser-282) similar to the catalytic residues in subtilisin. TY-21 TPP belonged to the peptidase S8A family in the MEROPS database. The mature TY-21 TPP showed approximately 49% identity with tripeptidyl peptidase subtilisin-like (TPP S) from Streptomyces lividans strain 66.     

Reversible Inhibition of Esterase Activity After Separation and Immobilization

An inhibitor, 9-amino-1,2,3,4-tetra hydroacridine (tacrine), is a reversible inhibitor of esterases. The reversible inhibition of the enzyme activity is thought to be examined after separation and immobilization of the enzyme under non-denaturing conditions. Hydrolytic changes of phosphatidylcholine by carboxylesterase were obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after the esterase was separated by non-denaturing two-dimensional electrophoresis, was immobilized to membranes and was stained by Ponceau S. The changes were inhibited after the enzyme on the membrane was treated by tacrine. Furthermore, the hydrolytic activity of the esterase was recovered after the inhibitor was washed with aspartic acid solution. These results indicate that the phosphatidylcholine hydrolysis activity of the isolated and immobilized enzyme is reversibly inhibited under non-denaturing conditions. Furthermore, this method can be developed to the production of an enzyme reactor able to regulate amounts of lipids.     

New chromogenic substrates for feruloyl esterases

Indolyl and nitrophenyl 5-O-hydroxycinnamoyl-alpha-L-arabinofuranosides were prepared by chemo-enzymatic syntheses. These probes were designed as substrates to be used in assays of feruloyl esterase activity (EC 3.1.1.77). Color development in the assays only occurs when feruloyl esterase activity releases an intermediate chromogenic arabinoside that is a suitable substrate for alpha-L-arabinofuranosidase (EC 3.2.1.55), which in turn releases the free chromogenic group. The usefulness of these compounds was evaluated in both qualitative solid media-based assays and quantitative liquid assays that can be performed in microtiter plates using feruloyl esterases and arabinofuranosidases from various origins.     

Array-based split-pool combinatorial screening of potential catalysts

A new method for screening split-pool combinatorial libraries for catalytic activity is described. Site-selective detection of catalytic activity for solution-based reactions was made possible without cofunctionalizing beads or adding diffusion-limiting matrixes. This was done by spatially separating resin-bound catalysts on an adhesive array on a microscope slide and introducing the reacting liquid to the top of the slide. Convective mixing and evaporation was controlled using a cover slide and imaging both the formation of products within active beads and the diffusion of products out of the beads. Colored reaction products and pH-sensitive indicators were used to visually detect catalytically active beads in the presence of inactive ones. Quantitative analyses of the images support the assumption that color intensities can be used to assess the quality of hits from a combinatorial screen. The Knoevenagel condensation reaction catalysis as well as esterase screening using methyl red were used to validate the approach. Using the esterase data, it was shown that some information on activity could also be extracted from the colored plume surrounding individual beads although the precision is not as good as that from direct measurement of absorbance through the bead. It was also found that the distribution of products within a single bead can also be gleaned from the absorbance data for different-sized beads.