Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions

Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible.     

Reverse micelles in protein separation: the use of silica for the back-transfer process

In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative "back extraction" procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (alpha-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a alpha-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. (c) 1993 John Wiley & Sons, Inc.     

Mammalian intestinal alkaline phosphatase acts as highly active exopolyphosphatase.

Recent results revealed that inorganic polyphosphates (polyP), being energy-rich linear polymers of orthophosphate residues known from bacteria and yeast, also exist in higher eukaryotes. However, the enzymatic basis of their metabolism especially in mammalian cells is still uncertain. Here we demonstrate for the first time that alkaline phosphatase from calf intestine (CIAP) is able to cleave polyP molecules up to a chain length of about 800. The enzyme acts as an exopolyphosphatase degrading polyP in a processive manner. The pH optimum is in the alkaline range. Divalent cations are not required for catalytic activity but inhibit the degradation of polyP. The rate of hydrolysis of short-chain polyP by CIAP is comparable to that of the standard alkaline phosphatase (AP) substrate p-nitrophenyl phosphate. The specific activity of the enzyme decreases with increasing chain length of the polymer both in the alkaline and in the neutral pH range. The K(m) of the enzyme also decreases with increasing chain length. The mammalian tissue non-specific isoform of AP was not able to hydrolyze polyP under the conditions applied while the placental-type AP and the bacterial (Escherichia coli) AP displayed polyP-degrading activity.     

Enzymatic production of L-tryptophan in a reverse micelle reactor

The enzymatic production of tryptophan from indole and serine was investigated in a micellar solution of the surfactant Brij 56 in cyclohexane. An anion exchanger was employed to facilitate the transfer of tryptophan and serine between the water pool of the reverse micelle and the bulk organic phase. The influence of potassium ion, water content, pH, and co-surfactant on enzyme activity is reported. Kinetic studies indicate that the enzyme is not inhibited by indole in the micellar system and that the enzyme is more stable in reverse micelles than in bulk water. The design of a continuous reverse micelle reactor, which accommodates both product recovery and enzyme reactivation, is discussed.     

Regulation of catalytic activity of acid phosphatase by lipids in a reverse micellar system

The influence of biomembrane lipids on the catalytic activity of a peripheral membrane enzyme, acid phosphatase (AP), was studied in a reverse micellar system. It was found that the interaction of AP with lipids led to a number of kinetic effects depending on lipid nature on enzyme function. The observed effects might be caused by the formation of lipoprotein complexes as well as by the influence of lipids on structure and properties of the micellar matrix. The results are important for clear understanding of molecular mechanisms of regulation of the catalytic activity of the membrane-associated enzyme in vivo. These data can also be used as a physicochemical basis for application of AP in medical fields as a diagnostic tool for diseases caused by changes in lipid metabolism, e.g. urinary, orthopedic, and allergic diseases.     

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.     

Inhibitory Effect of Magnesium Ion on the Human Placental Alkaline Phosphatase-Catalyzed Reaction in a Reverse Micellar System

Human placental alkaline phosphatase is a membrane-anchored protein. Entrapping the enzyme into a reverse micellar vesicle mimics the in vivo conditions and allows examination of the properties of the enzyme. Placental alkaline phosphatase is enzymatically active in Aerosol-OT/isooctane reverse micelles. Substantially different kinetic behavior of the enzyme has been observed in aqueous or reverse micellar systems. In aqueous solution, Mg²⁺ is a nonessential activator of the enzyme. In the experiments described in the present report Mg²⁺ was found to be an inhibitor for the enzyme in reverse micelles. This inhibition is presumably due to a time-dependent conformational change of the enzyme molecule, which resulted in a curvature in the recorder tracings of the enzyme assays. The Mg²⁺-induced conformational change of the enzyme was completely prevented by phosphate and partially reserved by EDTA. High concentrations of Zn²⁺ also strongly inhibited enzyme activity in both aqueous and reverse micellar solvent systems, presumably by occupying the Mg²⁺ (M3) site of the enzyme. However, binding of Zn²⁺ at the M3 site did not cause conformational change of the enzyme and the enzyme assay tracing was linear. The M3 site of the enzyme is proposed to have a modulatory role in vivo using magnesium ion as the modulator.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Immobilized enzymes in reverse micelles: studies with gel-entrapped trypsin and alpha-chymotrypsin in AOT reverse micelles

Trypsin and alpha-chymotrypsin were immobilized by gelentrapment in polyacrylamide cross-linked with N,N(1)-methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30-40% activity after 1 week. The enzymes obey Michaelis-Menten kinetics in the investigated concentration range with K(m) values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nalpha-benzoyl-L-arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., Z--Ala--Phe--Leu--NH(2)) using water-insoluble substrate has been performed with gelentrapped alpha-chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.     

Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi

This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degrees C, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degrees C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition, P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.     

活性铝对小麦根生长及酶活性的影响

利用铝形态分布与环境pH的相关性,通过改变染毒液pH条件,研究了不同浓度活性铝对小麦根生长、蛋白质含量及酸性磷酸酶活性的影响,并探讨了不同形态活性铝植物毒性的差异.本实验染毒液中总铝浓度设置为0(CK)、25(T₁)和75μmol·L^-1(T₂)3组,各组pH分别调至4.0、4.5、5.0和5.5.结果表明,微量Al_a与Al_b对小麦根生长均具有抑制作用.但随染毒液中活性铝组分的改变,小麦根蛋白质含量和酸性磷酸酶活性显现相反变化趋势:T₁和T₂组在pH4.0,活性铝主要成分为Al_a时(Al_a浓度高于活性铝浓度的90%),小麦根细胞蛋白质含量显著下降,酸性磷酸酶活性显著上升;T₁和T₂组在pH5.0,Al_a浓度降低至与Al_b浓度接近,且Al_a和Al_b浓度均低于10μmol·L^-1时,根细胞蛋白质含量显著上升,酸性磷酸酶活性显著下降.     

A water-mediated salt link in the catalytic site of Escherichia coli alkaline phosphatase may influence activity

Escherichia coli alkaline phosphatase catalyzes the hydrolysis of a wide variety of phosphomonoesters at similar rates, and the reaction proceeds through a phosphoenzyme intermediate. The active site region is highly conserved between the E. coli and mammalian alkaline phosphatases. The three-dimensional structure of the E. coli enzyme indicates that Lys-328, which is replaced by histidine in all mammalian alkaline phosphatases, is bridged to the phosphate through a water molecule. This water molecule is also hydrogen bonded to Asp-327, a bidendate ligand of the one of the two zinc atoms. Here we report the use of site-specific mutagenesis to convert Lys-328 to both histidine and alanine. Steady-state kinetic studies above pH 7.0 indicate that both mutant enzymes have altered pH versus activity profiles compared to the profile for the wild-type enzyme. At pH 10.3, in the presence of Tris, the Lys-328----Ala enzyme is approximately 14-fold more active than the wild-type enzyme. At the same pH in the absence of Tris the Lys-328----Ala enzyme is still 6-fold more active than the wild-type enzyme. Both mutant enzymes have lower phosphate affinities than the wild-type enzyme at all pH values investigated. Pre-steady-state kinetics at pH 5.5 reveal that the Lys-328----Ala enzyme behaves very similar to the phosphate-free wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatase activity in the larva of the euryhaline mosquito, Aëdes togoi Theobald,with special reference to sea-water adaptation

The alkaline and acid phosphatases in larvae of the euryhaline mosquito, Aëdes togoi Theobald, were measured and the distribution of alkaline phosphatase was examined histochemically. The optima pH for alkaline and acid phosphatases in the larvae were ≈9.0 and 3.2. respectively. The thorax region showed the highest activity of alkaline phosphatase. The enzyme activity of the thorax of seawater adapted larvae was about twice as high as that of freshwater larvae. When the larvae were transferred from fresh water to sea water, the alkaline phosphatase activity of the thorax increased greatly for 3 days, and thereafter decreased to the normal level of sea-water adapted larvae within seven days. In larvae transferred from sea water to fresh water, the activity of the thorax decreased gradually and after 7 days remained at the level of freshwater adapted larvae. No change in acid phosphatase activity was detected following transfer of the larvae from fresh water to sea water or vice versa. A strong alkaline phosphatase reaction was found only in the luminal border of the gastric caeca in the thorax region. The locality of this enzyme did not vary according to the salinity of environmental water.The activity change of alkaline phosphatase of the gastric caeca is discussed in relation to the absorption of the ingested medium from the gastric caeca.     

Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis

Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution. At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.     

氮添加对草地生态系统土壤pH、磷含量和磷酸酶活性的影响

磷是植物必需的大量营养元素之一,也是草地生态系统功能的重要限制因子。近年来,随着全球氮沉降的迅速增加,草地生态系统土壤磷及磷酸酶活性受到不同程度的影响。本研究采用整合分析方法,分析了草地的氮添加量、氮源种类、持续时间和土层深度等对土壤pH、全磷(TP)含量、有效磷(AP)含量、碱性磷酸酶(AlP)和酸性磷酸酶(AcP)活性的影响,以及土壤pH与磷酸酶活性的相关性。结果表明: 氮添加降低了土壤pH、TP含量和AlP活性,提高了土壤AcP活性,但对土壤AP含量无显著影响。从氮添加量来看,土壤pH、AlP显著降低在氮添加>5 g·m^-2·a^-1条件下即可发生;高水平氮添加(>10 g·m^-2·a^-1)导致AcP活性显著提高;土壤TP、AP含量显著降低仅发生在氮添加量为5～10 g·m^-2·a^-1条件下。硝酸铵处理显著降低了土壤TP含量,提高了AcP活性;尿素处理显著降低了土壤pH和AlP活性。在所有添加量下,当试验持续时间为3～10年时,土壤TP含量、AlP活性显著降低;持续时间大于3年时,土壤pH显著降低;>10年时,AcP活性显著提高。0～10 cm土层的AP含量显著升高,TP含量和AlP活性显著降低;大于10 cm土层中AP含量显著降低。土壤pH与土壤AcP活性呈显著负相关,表明氮添加引起的土壤pH改变可能是土壤磷酸酶活性变化的重要原因。     

Reverse micelles as life-mimicking systems

In this review, we attempt to demonstrate that reverse micelles are simple artificial systems that mimic many life systems from cell division to the creation of an enzyme catalytic mechanism. For a membranous enzyme like placental alkaline phosphatase, the kinetic properties observed in reverse micelles might represent those found under physiological conditions. The reverse micellar system, consisting of a positively charged surfactant, mimics a detoxification enzyme glutathione transferase. We propose a novel island-in-oil-lake reverse micellar model for the glutathione transferase that can account for almost all the catalytic properties of this enzyme. Reverse micelles may provide an excellent model system in investigating the reaction mechanism of other detoxification enzymes.     

Characterization of Enterococcus faecalis Alkaline Phosphatase and Use in Identifying Streptococcus agalactiae Secreted Proteins

We have identified and characterized an Enterococcus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis. Expression of phoZ in Escherichia coli, E. faecalis, Streptococcus agalactiae (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme's subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant, phoZDeltass, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E. faecalis and is expected to be useful in a variety of gram-positive bacteria.     

Cytochemical Localization of Certain Phosphatases in Escherichia coli

Cytochemical studies of Escherichia coli at the light and electron microscopic levels have revealed alkaline phosphatase, hexose monophosphatase, and cyclic phosphodiesterase reaction products in the periplasmic space and at the cell surface. In preparations for both light and electron microscopy, reaction product filled polar caplike enlargements of the periplasmic space, such as those described in plasmolyzed cells, indicating significant terminal concentrations of these enzymes; dense substance was often seen within these polar caps in morphological specimens. Staining of the bacterial surface was commonly encountered, but could represent artifactual accumulation of precipitate along the cell wall. Alkaline phosphatase was demonstrated with several substrates (ethanolamine phosphate, glycerophosphate, p-nitrophenylphosphate, and glucose-6-phosphate) over a wide pH range in a bacterial strain (C-90) known to be constitutive for this enzyme, whereas strains deficient in this enzyme (U-7, repressed K-37), showed no activity with these substrates. Hexose monophosphatase and cyclic phosphodiesterase activities were characterized by reaction-product deposition with specific substrates at acid or neutral, but not at alkaline, pH in strains of E. coli lacking alkaline phosphatase (U-7 and repressed K-37). Fixation in Formalin or the use of calcium as a capture reagent seemed to interfere with periplasmic staining in cells prepared for electron microscopy. Formalin fixation had little effect on biochemical assays of the phosphatase activity of intact cells in suspension, but partially reduced the activity evident in sonically treated extracts or in suspensions of dispersed cryostat sections. Glutaraldehyde treatment impaired enzyme activity more drastically.     

Expression and Localization of Escherichia coli Alkaline Phosphatase Synthesized in Salmonella typhimurium Cytoplasm

The Escherichia coli structural gene for alkaline phosphatase was inserted into Salmonella typhimurium by episomal transfer in order to determine whether this enzyme would continue to be localized to the periplasmic space of the bacterium even though it was formed in a cell that does not synthesize alkaline phosphatase. The S. typhimurium heterogenote synthesized alkaline phosphatase under conditions identical to that observed with E. coli. This enzyme appeared to be identical to that synthesized by E. coli, and was quantitatively released from the bacterial cell by spheroplast formation with lysozyme. These results showed that localization is not a property unique to the E. coli cell and suggested that, in E. coli, enzyme location is related to the structure of the protein. Formation of alkaline phosphatase in the S. typhimurium heterogenote was repressed in cells growing in a medium with excess inorganic phosphate, even though only one of the three regulatory genes for this enzyme is on the episome. Thus, S. typhimurium can supply the products of the other two regulatory genes essential for repression even though this bacterium seems to lack the structural gene for alkaline phosphatase.