古细菌Aeropyrum pernix K1超嗜热酯酶APE1547的稳定性

研究了纯化的超嗜热酯酶APE1547的稳定性. 结果表明, 该酶的稳定性非常好, 蛋白的质量浓度为0.4 mg/mL时, 90 ℃的半衰期为20 h, 0.2 mg/mL时的半衰期为12 h; 而蛋白的质量浓度为0.04 mg/mL时, 保温2.5 h时残余活力仍在50%以上. 同时还研究了热变性时该酶表面疏水氨基酸的变化. 该酶的pH稳定性也很好, pH在6.5-9.0范围内作用24 h, 酶依然很稳定, 残余酶活力大于93％; 同时该酶还具有很强的耐有机溶剂的特性.     

Denaturing Effects of Urea and Guanidine Hydrochloride on Hyperthermophilic Esterase from Aeropyrum pernix K1

Introduction Esterases(EC3.1.1.x)representadiversegroup ofhydrolasescatalyzingthecleavageandformationof esterbonds.Theyarewidelydistributedinanimals,plantsandmicroorganisms.Becauseoftheiractivities inbothaqueousandnonaqueoussolventsystems,ester aseshavebe…     

离子液体中酶促Michael加成反应的研究

以离子液体作为反应介质,研究了嗜热酯酶APE1547在不同离子液体中催化咪唑与丙烯酸乙酯的Mi-clasel加成反应,并对反应条件进行了优化.在最适反应条件下,Michael加成反应产率可达98.8%,另外发现嗜热酯酶APE1547在该反应体系中可重复使用8次,其催化性能没有明显下降.     

Enzyme catalytic promiscuity: asymmetric aldol addition reaction catalyzed by a novel thermophilic esterase in organic solvent

The asymmetric aldol addition of 2-butanone and 4-nitrobenzaldehyde catalyzed by a novel thermophilic esterase (APE1547) from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents. APE1547 exhibited a good enzyme activity and enantioselectivity in the reaction. The effects of organic solvent, temperature, water content, and substrate concentration were investigated. The reaction provided optically active secondary alcohol with satisfying enantioselectivity (71.2 %ee) and enzyme activity (38.1 µmol/g/h) under the optimum conditions. A high yield (68.7%) could be obtained when the reaction time was approximately 120 h.     

超嗜热酯酶APE1547中特殊位置氢键对酶活力和热稳定性的影响

探讨了APE1547蛋白的β-推进器结构中第3和第4“叶片”间的侧链氢键(Thr127-Gly154, Leu182-Arg145-Glu122)对蛋白质的作用.     

Enhanced Activity and Enantioselectivity of a Hyperthermophilic Esterase from Archaeon Aeropyrum pernix K1 by Acetone Treatment

To improve the activity and enantioselectivity of hyperthermophilic archaeon Aeropyrum pernix K1 esterase (APE1547) and its mutants, they were purified by acetone-treated method. It was found that the acetone treatment not only caused APE1547 and its mutants to display higher activity and enantioselectivity but also saved more than 90% of time spent in purifying them by Ni-chelating column. In hydrolysis of p-nitrophenyl caprylate, the acetone-treated APE1547 and mutant A containing the following substitutions R11G, L36P, V225A, I551L, and A564T showed 5.7- and 6.9-fold active increase, respectively. In the resolution of 2-octanol acetate, the acetone-treated mutant A had a 9-fold enantioselective increase relative to that purified by Ni-chelating column. In addition, the impact of pH, temperature, and chemical reagents on activity of APE1547 and mutant A was discussed in this paper.     

Acylation of Quercetin with a Novel Thermophilic Esterase as Biocatalyst

The regioselective acylation of quercetin catalyzed by a novel thermophilic esterase(APE1547)from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents.The effects of acyl don...     

Study on a Specific Site Tyr~(444) on a Hyperthermophilic Enzyme APE1547

Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrum pernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547,site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme,the thermostability of mutants T444S and T444G decreased by 10...     

水解酶在二乙基锌与芳香醛加成反应中的应用(英文)

报道了水解酶催化二乙基锌与芳香醛的加成反应,对反应条件进行了优化.在最适条件(嗜热酯酶APE1547为酶源,反应温度40℃,氯仿为溶剂,4-Cl-苯甲醛为底物)下,加成反应生成的光学活性醇产率最高达78%,ee值最高可达56%.通过实验结果和分子动力学分析对可能的反应机理进行了推测.本研究进一步拓展了酶的非专一性.     

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...     

Interaction of Functionalized Naphthalenophanes with Abasic Sites in DNA: DNA Cleavage,DNA Cleavage Inhibition,and Formation of Ligand–DNA Adducts

Ligands interacting with abasic (AP) sites in DNA may generate roadblocks in base-excision DNA repair (BER) due to indirect inhibition of DNA repair enzymes (e.g., APE1) and/or formation of toxic byproducts, resulting from ligand-induced strand cleavage or covalent cross-links. Herein, a series of 12 putative AP-site ligands, sharing the common naphthalenophane scaffold, but endowed with a variety of substituents, have been prepared and systematically studied. The results demonstrate that most naphthalenophanes bind to AP sites in DNA and inhibit the APE1-induced hydrolysis of the latter in vitro. Remarkably, their APE1 inhibitory activity, as characterized by IC₅₀ and K_I values, can be directly related to their affinity and selectivity to AP sites, as assessed by means of fluorescence melting experiments. On the other hand, the molecular design of naphthalenophanes has a crucial influence on their intrinsic AP-site cleavage activity (i.e., ligand-catalyzed β- and β,δ-elimination reactions at the AP site), as illustrated by the compounds either having an exceptionally high AP-site cleavage activity (e.g., 2,7-BisNP-S , 125-fold more efficacious than spermine) or being totally devoid of this activity (four compounds). Finally, the unprecedented formation of a stable covalent DNA adduct upon reaction of one ligand ( 2,7-BisNP-NH ) with its own product of the AP-site cleavage is revealed.     

Synthesis and solution properties of a new pH‐responsive polymer containing amino propanesulfonic acid residues

The reaction of diallyl amine with 1,3‐propane sultone led to the synthesis of the zwitterionic monomer 3‐(N,N‐diallylammonio)propanesulfonate. The sulfobetaine was cyclopolymerized in water in the presence of sodium chloride with t‐butylhydroperoxide as an initiator to afford a polysulfobetaine (PSB) in very good yield. PSB, upon treatment with sodium hydroxide, was converted into an anionic polyelectrolyte (APE). Although APE was readily soluble in salt‐free water, PSB needed the presence of low‐molecular‐weight salts (e.g., NaCl, KI, etc., in the range of 0.135–1.04 N) for its dissolution. The solution properties of PSB and APE were investigated with potentiometric and viscometric techniques. The basicity constant of the amine was apparent and followed the modified Henderson–Hasselbalch equation; as the degree of protonation (α) of the whole macromolecule increases, the protonation of the amine nitrogens becomes increasingly more difficult. The composition and phase diagram of the aqueous two‐phase systems of APE/PSB and poly(ethylene glycol) were also explored. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 172–184, 2003     

Pre-apoptotic sub-population cell sorting from diosgenin apoptosis induced 1547 cells by Sedimentation Field-Flow Fractionation. The effect of channel thickness on sorting performance

Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.     

High-throughput simultaneous determination of plasma water deuterium and 18-oxygen enrichment using a high-temperature conversion elemental analyzer with isotope ratio mass spectrometry

This paper presents a high-throughput method for the simultaneous determination of deuterium and oxygen-18 (18O) enrichment of water samples isolated from blood. This analytical method enables rapid and simple determination of these enrichments of microgram quantities of water. Water is converted into hydrogen and carbon monoxide gases by the use of a high-temperature conversion elemental analyzer (TC-EA), that are then transferred on-line into the isotope ratio mass spectrometer. Accuracy determined with the standard light Antartic precipitation (SLAP) and Greenland ice sheet precipitation (GISP) is reliable for deuterium and 18O enrichments. The range of linearity is from 0 up to 0.09 atom percent excess (APE, i.e. -78 up to 5725 delta per mil (dpm)) for deuterium enrichment and from 0 up to 0.17 APE (-11 up to 890 dpm) for 18O enrichment. Memory effects do exist but can be avoided by analyzing the biological samples in quintuplet. This method allows the determination of 1440 samples per week, i.e. 288 biological samples per week.     

Synthesis of TRITON™ X-based phosphate ester surfactants and their self-charring behavior

This paper describes the synthesis and characterization of a series of TRITON™ X-based surfactants with a predominantly alkyl phenol ethoxylate (APE) backbone and a phosphate ester chain end. Four phosphate-terminated TRITON™ X (or APE) derivatives (OPE2-OPO(OH)₂, OPE5-OPO(OH)₂, OPE10-OPO(OH)₂, and NPE10-OPO(OH)₂) were prepared from commercially available octyl phenol ethoxylate (OPE) of different oxyethylene units (n = 2, 5 and 10), nonyl phenol ethoxylate (NPE) of 10 oxyethylene units and phosphorous pentoxide via a simple condensation reaction. Depending on their composition and chain length of oxyethylene units used in the reaction, the surfactants show different self-charring behaviors. The phosphate-terminated TRITON™ X of the lowest number of oxyethylene units (i.e. OPE2-OPO(OH)₂) gives the largest amount of char (up to 23 wt%) at 600 °C under air condition. The carboxylic acid-terminated TRITON™ X derivatives (i.e. OPE-COOH) were also tested for comparison.     

Analysis of traces of surfactants in water with anion-exchange resin and polymeric adsorbent

Summary Determination of traces of anionic and non-ionic surfactants in water has been based on the quantitative separation of sodium alkylbenzene sulphonate (LAS) from polyoxyethylenealkylphenyl ether (APE) with a weak base anion-exchange resin, and adsorption of APE in filtrate with Amberlite XAD-2 resin. LAS is adsorbed on the anionexchange resin, eluted with hydrochloric acid-methanol and determined spectrophotometrically by methylene blue or UV absorption. APE in the filtrate is adsorbed on XAD-2 resin, eluted with methanol and determined spectrophotometrically by cobalt thiocyanate or UV absorption. Recoveries of surfactants were about 90100%. As little as 0.02 ppm can be determined. In a river water sample 0.06 ppm of LAS and 0.05 ppm of APE and in another case, 0.12 ppm of LAS and 0.07 ppm of APE were found.

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Spurenanalyse von oberflächenaktiven Substanzen in Wasser mit Hilfe eines Anionenaustauschers und eines Polymeradsorbens

Zusammenfassung Natriumalkylbenzolsulfonat (LAS) kann von Polyoxyethylenalkylphenylether (APE) getrennt werden, indem LAS an einem schwachbasischen Anionenaustauscher und im Filtrat APE an Amberlit XAD-2 adsorbiert wird. LAS wird mit Methanol-HCl eluiert und im Eluat spektralphotometrisch mit Methylenblau oder durch UV-Absorption bestimmt. APE wird mit Methanol eluiert und spektralphotometrisch mit Cobaltthiocyanat oder ebenfalls durch UV Absorption bestimmt. Bei Zusatzversuchen wurden 90–100% wiedergefunden. Bis zu 0.02 ppm können bestimmt werden. In einer Flußwasserprobe wurden 0,06 ppm LAS und 0,05 ppm APE bzw. 0,12 ppm LAS und 0,07 ppm APE gefunden.

     

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.     

High-throughput enzymatic method for enantiomeric excess determination of O-acetylated cyanohydrins

The reaction yield and enantiomeric excess of O-acetylated cyanohydrin reaction products from a library of chiral catalysts can be analyzed by a three-step screening method. Alcohol dehydrogenase and NADH are used to analyze unreacted substrate. A lipase with absolute specificity converts one enantiomer to a quantifiable product before the remaining enantiomer is hydrolyzed with an unspecific esterase and quantified.     

2‐[(Acetyloxy)methyl]‐4‐(acetylsulfanyl)‐2‐(ethoxycarbonyl)‐3‐oxobutyl Group: A Thermolabile Protecting Group for Phosphodiesters

The phosphodiester linkage of 3′‐O‐levulinoylthymidine 5′‐methylphosphate ( 5 ) has been protected with 2‐[(acetyloxy)methyl]‐4‐(acetylsulfanyl)‐2‐(ethoxycarbonyl)‐3‐oxobutyl group (to give 1 ) to study the potential of this group as an esterase‐ and thermolabile protecting group. The group turned out to be unexpectedly thermolabile, being removed as ethyl 3‐(acetyloxy)‐4‐(acetylsulfanyl)‐2‐methylidenebut‐3‐enoate ( 10 ) without accumulation of any intermediates. The half‐life of this reaction at pH 7.5 and 37° is 14 min. Hog liver esterase (HLE), in turn, removes the protecting group as ethyl 4‐(acetylsulfanyl)‐2‐methylidene‐3‐oxobutanoate ( 12 ). On using 2.6 units of HLE in 1 ml, the rate of the enzymatic deprotection was still only one third of that of the nonenzymatic reaction. The mechanisms of both reactions have been studied and discussed. The crucial step seems to be removal of the O‐bound Ac group, either by esterase or by migration to the neighboring 3‐oxo group (nonenzymatic removal). This triggers the removal by retro‐aldol condensation/elimination mechanism. No alkylation of glutathione (GSH) upon the deprotection of 1 could be detected.