Highly sensitive and selective amperometric microbial biosensor for direct determination of p-nitrophenyl-substituted organophosphate nerve agents

We report herein a whole cell-based amperometric biosensor for highly selective, highly sensitive, direct, single-step, rapid, and cost-effective determination of organophosphate pesticides with a p-nitrophenyl substituent. The biosensor was comprised of a p-nitrophenol degrader, Pseudomonas putida JS444, genetically engineered to express organophosphorus hydrolase (OPH) on the cell surface immobilized on the carbon paste electrode. Surface-expressed OPH catalyzed hydrolysis of the p-nitrophenyl substituent organophosphorus pesticides such as paraoxon, parathion, and methyl parathion to release p-nitrophenol, which was subsequently degraded by the enzymatic machinery of P. putida JS444. The electrooxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.086 mg dry weight of cells operating at 600 mV applied potential (vs Ag/AgCl reference) in 50 mM citrate--phosphate pH 7.5 buffer with 50 microM CoCl2 at room temperature. Under optimum operating conditions the biosensor measured as low as 0.28 ppb of paraoxon, 0.26 ppb of methyl parathion, and 0.29 ppb parathion. These detection limits are comparable to cholinesterase inhibition-based biosensors. Unlike the inhibition-based format, this biosensor manifests a selective response to organophosphate pesticides with a p-nitrophenyl substituent only, has a simplified single-step protocol with short response time, and can be used for repetitive/multiple and on-line analysis. The service life of the microbial amperometric biosensor was 5 days when stored in the operating buffer at 4 degrees C. The new biosensor offers great promise for rapid environmental monitoring of OP pesticides with nitrophenyl substituent.     

Molecular cloning and characterization of a thermostable carboxylesterase from an archaeon, Sulfolobus shibatae DSM5389: non-linear kinetic behavior of a hormone-sensitive lipase family enzyme

A gene coding for an esterase (SshEstI, 915 bp in length) of the thermoacidophilic archaeon Sulfolobus shibatae DSM5389 was cloned, sequenced, and overexpressed in Escherichia coli JM109 cells as a soluble, catalytically active protein. The deduced amino acid sequence of SshEstI was consistent with a protein containing 305 amino acid residues with a molecular mass of 33 kDa. Sequence comparison studies indicated that SshEstI could be a member of the hormone-sensitive lipase family, in that it had the highest sequence similarity to esterases from Sulfolobus solfataricus (90% identity) and Archaeoglobus fulgidus (42%) and a lipase from Pseudomonas sp. B11-1 (38%). The recombinant enzyme was highly thermostable and retained more than 70% of its initial activity after incubation at 90 degrees C and pH 7.0 for 30 min. The recombinant enzyme catalyzed the hydrolysis of p-nitrophenyl (p-NP) esters with C2-C16 acyl chains but not the hydrolysis of triacylglycerides such as tributyrin and triolein. The enzymatic hydrolysis of p-NP acetate proceeded in a linear manner with time, whereas that of p-NP esters with acyl chains of C5 or longer showed a biphasic profile, where a rapid release of p-nitrophenol ( approximately 3 min) was followed by a slow, sustained release. These non-linear kinetics may be explained in terms of a very slow, presteady-state burst phenomenon of p-nitrophenol release or a hysteretic behavior of SshEstI with these substrates.     

磷酸酯改性淀粉的制备及在肉类中的应用

以糯玉米淀粉为原料,采用复合磷酸盐进行酯化,以取代度为指标,分别考察了pH、正磷酸盐添加量、磷酸盐配比、反应温度、反应时间对酯化的影响,并对其进行了优化,得到最佳工艺条件:糯玉米淀粉乳浓度40%,反应pH=5.5,正磷酸盐添加量37.5g,磷酸二氢钠与磷酸氢二钠的配比0.4,反应温度150℃,反应时间2h。将磷酸酯淀粉添加在火腿肠中时,其弹性、粘聚性、胶着性、咀嚼度等明显优于添加原淀粉的产品,其中取代度为0.1257,添加8%时最佳。     

δ型层状MnO2治理碱性染料废水

以层状粉末MnO2为处理剂，对含碱性染料罗丹明B的模拟印染废水进行静态脱色试验。考察了处理时间、体系pH值、MnO2投加量和温度等对脱色的影响，得出了吸附等温方程；采用FT-IR和XPS初步分析了脱色机理。结果表明，投加不同量的MnO2后，10min内基本可达到脱色平衡；较低的pH值和较高的MnO2投加量有助于脱色；温度对脱色影响不大。pH值为3和7时，MnO2对罗丹明B染料的吸附符合Langmuir和Freundlich等温方程；该脱色过程是羟基键合与电性吸附的迭加。     

Acid phosphatases activity in cheese and starters.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5-2 and the enzyme was inhibited by F-minus,Al-3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5-2 gave a Km value for p-nitrophenyl phosphate of 1-2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with Ki values of 1-2 mM, 1-0 mM, 1-0 MM and 1-1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.     

EVIDENCE FOR THE PRESENCE OF MALTASE AND α-GLUCOSIDASE ISOENZYMES IN BARLEY

An examination of the development of α-glucosidase and maltase activities (as measured by the hydrolysis of p-nitrophenyl α-D-glucoside and maltose respectively) indicated that two genotypes of Glacier barley had the same general pattern of enzyme development. However, the development of α-glucosidase activity followed a different course from that of maltase activity suggesting that separate enzyme proteins are involved in hydrolysing these substrates. Further evidence that separate enzyme proteins were responsible for hydrolysis of maltose and p-nitrophenyl α-glucoside was obtained by column chromatography of extracts of germinated barley which indicated the presence of two maltases and two α-glucosidases. The maltases and the α-glucosidases differed in molecular weight, pH of optimum activity and in thermostability. When isomaltose was used as a substrate the optimum pH and behaviour on gel chromatography were coincident with that of maltase activity but different from the α-glucosidases.     

静电纺MnO2/PAN纳米纤维膜的制备及其催化氧化甲醛性能

通过液相沉积法制备MnO2,以PAN/MnO2/N,N-二甲基甲酰胺溶液作为纺丝液进行静电纺丝制备出MnO2/PAN纳米纤维膜。利用扫描电镜、透射电镜、傅里叶红外光谱、热重分析对纤维膜的结构和性能进行分析;并在甲醛废水体系中测试纤维膜催化氧化甲醛性能。结果表明：制备的纳米纤维其平均直径为196.46nm;随MnO2质量分数的增大和纺丝电压的增加,纤维平均直径均下降;pH值为2,温度60℃时,具有最佳甲醛催化氧化性能;纤维吸收12h后,对甲醛去除率达到44.0%;MnO2/PAN纤维膜比MnO2粉末具有更好的催化氧化甲醛性能。     

Effects of depleting ionic strength on 31P nuclear magnetic resonance spectra of micellar casein during membrane separation and diafiltration of skim milk

Membrane separation processes used in the concentration and isolation of micellar casein-based milk proteins from skim milk rely on extensive permeation of its soluble serum constituents, especially lactose and minerals. Whereas extensive literature exists on how these processes influence the gross composition of milk proteins, we have little understanding of the effects of such ionic depletion on the core structural unit of micellar casein [i.e., the casein phosphate nanocluster (CPN)]. The ³¹P nuclear magnetic resonance (NMR) is an analytical technique that is capable of identifying soluble and organic forms of phosphate in milk. Thus, our objective was to investigate changes to the ³¹P NMR spectra of skim milk during microfiltration (MF) and diafiltration (DF) by tracking movements in different species of phosphate. In particular, we examined the peak at 1.11 ppm corresponding to inorganic phosphate in the serum, as well as the low-intensity broad signal between 1.5 and 3.0 ppm attributed to casein-associated phosphate in the retentate. The MF concentration and DF using water caused a shift in the relevant ³¹P NMR peak that could be minimized if orthophosphate was added to the DF water. However, this did not resolve the simultaneous change in retentate pH and increased solubilization of micellar casein protein. The addition of calcium in combination with orthophosphate prevented micellar casein solubilization and simultaneously contributed to preservation of the CPN structure, except for overcorrection of retentate pH in the acidic direction. A more complex DF solution, involving a combination of phosphate, calcium, and citrate, succeeded in both CPN and micellar casein structure preservation while maintaining retentate pH in the region of the original milk pH. The combination of ³¹P NMR as an analytical technique and experimental probe during MF/DF processes provided useful insights into changes occurring to CPN while retaining the micellar state of casein.     

Binding of p-nitrophenyl phosphate and other aromatic compounds by beta-lactoglobulin

Results obtained from gel filtration showed that beta-lactoglobulin binds p-nitrophenyl phosphate with a stoichiometry of 1 mol of ligand per 18,360 monomer. Circular dichroic spectra confirmed the binding and implicated tryptophan and phenylalanine residues in the interaction. Fluorescence of the protein was quenched on binding also supporting complex formation; analysis of these data indicates that p-nitrophenyl phosphate binds to beta-lactoglobulin A with a dissociation constant of 31 microM. The B and C genetic variants of beta-lactoglobulin bind p-nitrophenyl phosphate with dissociation constants of 63 and 70 microM, respectively. In addition, a series of other nitrophenyl compounds and pyridoxal phosphate were also investigated by fluorescence analysis and found to bind to the protein. These results are discussed with respect to a recent hypothesis that beta-lactoglobulin binds retinol and is structurally related to serum retinol binding protein.     

Effects of pH and phosphate status of a silty clay loam on manganese,zinc and copper concentrations in soil fractions and in clover

The field pH of a silty clay loam soil affected manganese, zinc and copper concentrations in the fraction extracted by DTPA; lower pH soils had higher metal concentrations. The soil pH also affected manganese and zinc but not copper concentrations in the soil solution, and in shoots of clover grown in pots. Metal concentrations in soil fractions were not affected by soil phosphate status, but zinc and copper concentrations in clover shoots were significantly lower when plants were grown on soils of high phosphate status. Manganese and zinc levels in shoots were predicted slightly more accurately by using soil solution data than by using concentrations extracted by DTPA.     

A study of the mode of breakdown of some compounds containing P?N bonds which have potential as high analysis multinutrient fertilisers

Three compounds containing high percentages of N, P (and S) were prepared, and their degradation studied in both aqueous and soil environments, with particular reference to the appearance of radicals containing these elements in forms normally used by plants. A model is proposed for the degradation of one of these compounds—hexamethyl-hexaaza-tetraphosphaadamantane (APA)—which eventually releases orthophosphate and methylamine, presumably a precursor of ammonium. The initial degradation reactions involve hydrolysis through amidophosphites, to phosphite, followed by oxidation to phosphate. The latter reaction appears to be dominantly biological, and should therefore proceed in soil systems. Hexamethyl-hexaaza-tetraphosphaadamantane tetrasulphide, a sulphur-containing compound similar to APA, produced phosphite and phosphate on decomposition, and here the phosphate is probably produced directly by hydrolysis reactions. The third compound, hexamethylaminocyclotriphosphazatriene, has a planar structure in contrast to the cage structures of the other two compounds, and decomposed only very slowly in either aqueous medium or in soil.     

Lnsolubilization of Orthophosphates in Fresh or Cooked Ground Pork

The insolubilization of added orthophosphate (1000–10000 ppm, equivalent to 0.1–1 .0% w/w) and of orthophosphate generated through enzymatic hydrolysis of added sodium acid pyrophosphate (SAPP) was studied in fresh and in cooked (65.5°C) ground pork held at 5°C for 6 days. Fresh and cooked pork linearly absorbed or otherwise insolubilized orthophosphate added directly to the meat or generated through enzymatic hydrolysis of added SAPP. Orthophosphate insolubilization was constant throughout the range of addition studied and equivalent to 70–80%. Soluble orthophosphate measurements, therefore, could not be used to estimate the proportion of added pyrophosphate that was hydrolyzed in the meat or, by difference, the proportion of residual, unhydrolyzed SAPP.     

新型皮革渗透剂的制备及其组成与渗透性能的相关性

在不同反应条件下制备系列异辛醇磷酸酯．测定其渗透性和磷酸单、双酯的含量，探讨产物中磷酸单双酯组成比例与渗透性能的相关性，结果表明：双酯与单酯摩尔分数含量的比例为1．5～3．5时，渗透性能最佳。合成异辛醇磷酸酯渗透剂的最佳反应条件是：异辛醇与五氧化二磷的摩尔比在2～3，反应温度70℃，反应时间3h以上。并在制革浴液中将异辛醇磷酸酯渗透剂与JFC渗透剂进行了应用性能比较。     

Sodium Tripolyphosphate Stability and Effect in Ground Turkey Meat

Ground turkey meat, cooked and uncooked, was prepared with and without 0.5% sodium tripolyphosphate (SIP) and stored at 5°C for different periods of time. STP stability was evaluated by determining soluble orthophosphate. Water-holding capacity (WHC), pH, and mi-crobial count were also measured. STP hydrolyzed rapidly in uncooked samples. Refrigerated storage time (up to 6 days) did not affect STP hydrolysis in cooked turkey meat. Heating accelerated the rate of STP hydrolysis. End point temperatures (65, 75, and 85 °C) did not affect the extent of STP hydrolysis. STP increased WHC in both cooked and uncooked samples. STP did not inhibit total microbial growth in cooked or uncooked ground turkey meat.     

Nitrification in premise plumbing: role of phosphate, pH and pipe corrosion

Nitrification in PVC premise plumbing is a weak function of pH over the range 6.5--8.5 and is insensitive to phosphate concentrations 5--1000 ppb. Lead pipe enhanced nitrification relative to PVC, consistent with expectations that nitrifiers could benefit from ammonia recycled from nitrate via lead corrosion. Relatively new copper pipe (< 1.5-years-old) did not allow nitrifiers to establish, but nitrifiers gradually colonized over a period of months in brass pipes when copper concentrations were reduced by pH adjustment or orthophosphate. Nitrifiers were inhibited by trace copper, but not by lead levels up to 8000 ppb. In some systems using chloramines, brass in plastic plumbing systems might be more susceptible to lead/copper leaching, and accelerated dezincification, due to lower pH values resulting from nitrification.     

Hydrolytic Stability at Intermediate pHs of the Common Purine Nucleotides in Food, Inosine-5'-monophosphate, Guanosine-5'-monophosphate and Adenosine-5'-monophosphate

When the purine nucleotides, inosine-5′-monophosphate (IMP), guanosine-5′-monophosphate (GMP) and adenosine-5′-monophosphate (AMP) were subjected to canning temperatures (121°C) at pHs between 3 and 8, extensive phosphate bond hydrolysis to the corresponding nucleoside occurred. The half life for hydrolysis at pH 5 was 63, 41, and 51 min for IMP, GMP, and AMP, respectively. Rates of hydrolysis were even greater at pH 3 because both hydrolysis of the phosphate bond and the glycosidic bond occurred simultaneously. An Arrhenius plot of data collected at different temperatures illustrated that the nucleotides were very stable at room temperature. Half lives for IMP, GMP, and AMP were estimated to be 36, 19, and 40 yr, respectively, at 23°C and a pH of 5.     

Inorganic polyphosphate and exopolyphosphatase in the nuclei of Saccharomyces cerevisiae: dependence on the growth phase and inactivation of the PPX1 and PPN1 genes

Nuclei of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyP) with chain lengths of ca. 10-200 phosphate residues. Subfractionation of the nuclei reveals that the most part of polyP is not associated with DNA. Transition of the yeast cells from stationary phase to active growth at orthophosphate (P(i)) excess in the medium is followed by the synthesis of the shortest polyP (<15 phosphate residues) and hydrolysis of the high-molecular polyP (>45 phosphate residues) in the nuclei. Nuclear exopolyphosphatase (exopolyPase) activity does not depend on the growth phase. The PPX1 gene encoding the major cytosolic exopolyPase does not encode the nuclear one and its inactivation has no effect on polyP metabolism in this compartment. Under inactivation of the PPN1 gene encoding another yeast exopolyPase, elimination of the nuclear exopolyPase is observed. The effect of PPN1 inactivation on the polyP level in the nuclei is insignificant in the stationary phase, while in the exponential phase this level increases 2.3-fold as compared with the parent strain of S. cerevisiae. In the active growth phase, no hydrolysis of high-molecular polyP is detected while the synthesis of short-chain polyP is retained. The data obtained indicate substantial changes in polyP metabolism in nuclei under the renewal of active growth, which only partially depends on the genes of polyP metabolism known to date.     

Effects of mineral salts and calcium chelating agents on the gelation of renneted skim milk

The effects of adding CaCl2, orthophosphate, citrate, EDTA, or a mixture of these, to reconstituted skim milk (90 g of solids/kg solution) on the gelation of renneted milk were mediated by changes in Ca2+ activity and the casein micelle. At pH 6.65, the addition of citrate or EDTA, which removed more than 33% of the original colloidal calcium phosphate with the accompanying release of 20% casein from the micelle, completely inhibited gelation. Reformation of the depleted colloidal calcium phosphate and casein in the micelle, by the addition of CaCl2, removed this inhibition. When the minimum requirements for colloidal calcium phosphate and casein in the micelle were met, the coagulation time decreased with increasing Ca2+ activity, leveling off at high Ca2+ activity. The storage modulus of renneted gels, measured at 3 h, increased with increasing colloidal calcium phosphate content of micelles up to a level at which it was approximately 130% of the original colloidal calcium phosphate in the micelles. Further increases in colloidal calcium phosphate by the addition of CaCl2, orthophosphate, or mixtures of these, which did not change the proportion of casein in the micelle, decreased the storage modulus. The gelation of the renneted milk was influenced by Ca2+ activity, the amounts of colloidal calcium phosphate, and casein within the micelle, with the effects of colloidal calcium phosphate and casein within the micelle clearly dominating the storage modulus. These results are consistent with the model of Horne (Int. Dairy J. 8:171-177, 1998) which postulates that, following cleavage of the stabilizing K-casein hairs by rennet, the properties of the rennet gel are determined by the balance between the electrostatic and hydrophobic forces between casein micelles.     

大米淀粉磷酸酯的制备及其理化性质研究

为合理地利用丰富的大米淀粉资源,以大米淀粉为原料,尿素为催化剂,正磷酸盐为酯化剂,采用响应曲面法确定了制备大米淀粉磷酸酯的最佳工艺条件:磷酸盐用量为淀粉质量的46.61%,催化剂用量为淀粉质量的4.03%,反应pH为5.5,反应时间为4.6 h。在此条件下制得的磷酸酯淀粉的取代度为0.020 34。并用扫描电镜(SEM)对反应前后淀粉颗粒的形貌进行了观察,结果表明,磷酸酯化后,部分淀粉颗粒受到侵蚀。同时研究了大米淀粉磷酸酯的理化性质,包括溶解度、膨胀度、糊化特性、凝沉性、冻融稳定性及黏弹特性。结果表明,相对于原淀粉,磷酸酯淀粉的溶解度、膨胀度、黏度有所提高,糊化温度、凝沉性、凝胶硬度和强度则下降。     

Optimizing the Conditions for Starch Dry Phosphorylation with Sodium Mono‐ and Dihydrogen Orthophosphate under Heat and Vacuum

Starch was phosphorylated by reaction with mono‐ and disodium hydrogen orthophosphate under dry conditions in a vacuum oven at 150‐180 °C (800 mbar). Studying the different factors affecting the reaction showed that the optimal conditions for starch phosphorylation in the monoester form were: 3 h reaction time, 160 °C reaction temperature and pH 6. The different types of starch gave different degrees of substitution; and amylose bound a higher amount of phosphate than amylopectin under similar reaction conditions. Both ash content and acidity of the phosphorylated starch products increased proportionally with the increase in the degree of substitution while the pH of the different modified starch products was nearly in the same range (pH 6.55—6.75).