Preparation and characterization of amino and carboxyl functionalized core-shell Fe3O4/SiO2 for L-asparaginase immobilization: A comparison study

Abstract

Magnetic nanoparticles are well known as facile and effective support for enzyme immobilization since they have a high surface area, large surface-to-volume ratio, easy separation, a fast and high enzyme loading. This study aims to provide insights on whether acidic or basic modified particles are more effective for L-asparaginase (ASNase) immobilization. Therefore, amino (Fe₃O₄/SiO₂/NH₂) and carboxyl-functionalized (Fe₃O₄/SiO₂/COOH) particles were prepared. The functional groups, crystalline structure, magnetic properties, morphology, chemical composition and thermal behaviour of the prepared modified nanoparticles were examined via Fourier-transform infra-red spectroscopy (FTIR), X-ray diffraction (XRD), vibrating-sample magnetometer (VSM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDAX). Under the optimum conditions, the immobilized enzymes were more stable within a certain range of temperatures and pH values in comparison to free enzyme. On the other hand, the immobilized enzymes showed greater stability after incubation for 3?h at 50?°C. The free enzyme maintained only 30% of its initial activity for 4?weeks at 4?°C, while Fe₃O₄/SiO₂/NH₂/ASNase and Fe₃O₄/SiO₂/COOH/ASNase retained more than 78.9% and 56.5% of initial activities under the same conditions, respectively. Moreover, Fe₃O₄/SiO₂/NH₂/ASNase (77.2%) and Fe₃O₄/SiO₂/COOH/ASNase (57.4%) displayed excellent operational stability after 17 repeated cycles. These findings suggested that the Fe₃O₄/SiO₂/NH₂ and Fe₃O₄/SiO₂/COOH may be utilized as efficient and sustainable supports to developed immobilized ASNase in several biotechnological applications.     

Partitioning of mercury in aqueous biphasic systems and on ABEC™ resins

Poly(ethylene glycol)-based aqueous biphasic systems (PEG-ABS) can be utilized to separate and recover metal ions in environmental and hydrometallurgical applications. A concurrent study was conducted comparing the partitioning of mercury between aqueous layers in an ABS [Me-PEG-5000/(NH₄)₂SO₄] and partitioning of mercury from aqueous solutions to aqueous biphasic extraction chromatographic (ABEC-5000) resins. In ammonium sulfate solutions, mercury partitions to the salt-rich phase in ABS, but by using halide ion extractants, mercury will partition to the PEG-rich phase after formation of a chloro, bromo or iodo complex. The efficacy of the extractant increases in the order Cl⁻<Br⁻<I⁻. This behavior is also observed using the ABEC resins where halo complexes of mercury will adsorb to the resin from (NH₄)₂SO₄ solutions with retention following the same order. The onset of mercury extraction or adsorption is different for the three extractants, occurring at the lowest extractant concentration for I⁻, followed by Br⁻, and then Cl⁻. Fluoride does not extract mercury. Extraction or adsorption of mercury is improved at the lowest halide concentrations in the presence of sulfuric acid. The addition of sulfuric acid to (NH₄)₂SO₄ solution results in ABEC retention of mercury even in the absence of halide extractant.     

The Use of a Five-stage Sequential Leaching Procedure for Risk Assessment of Heavy Metals in Waste Rock Utilized in Railway Ballast

A five-stage sequential leaching procedure was used to determine the distribution of 10 metals in three combination samples taken from waste rock material, originating from a Finnish zinc mine, and used as railway ballast in Northern Finland. The leaching procedure consists of the following five sequential fractions: a water-soluble fraction (H₂O), an exchangeable fraction (CH₃COOH), an easily reduced fraction (NH₂OH-HCl), an oxidizable fraction (H₂O₂ + CH₃COONH₄), and a residual fraction (HF + HNO₃ + HCl). The results show, in accordance with all earlier corresponding studies, that a sequential extraction procedure is very suitable for evaluating the effects of external conditions on the solubility of harmful heavy metals, and that external conditions have a large effect on their leachability/solubility, and therefore on their mobility, bioavailability, and environmental risk. In addition, the total concentration of each element is much larger than its solubility in each of the first four fractions (1)–(4) – the potential bioavailability fractions – because the highest concentrations of all metals occurred in the residual fraction (5). The conditions of this phase – three strong acids and strong, long-lasting shaking – are never possible in nature, and therefore the residual fraction is called the inert phase. Thus, the total concentrations of the heavy metals are poor measures of real environmental risk and give no information about the effect of external conditions on their solubility. In addition, the dependency of different elements on the external conditions differs.     

The NH2- and COOH-terminal sequences of the angiotensin-converting enzyme isozymes from rabbit lung and testis

The NH₂-and COOH-terminal sequences of the angiotensin-converting enzymes from rabbit lung and testis have been determined using less than 0.6mg of each protein. They are: (NH₂)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp- and -(Phe, Tyr)-Ser-Leu-Ala(COOH) for the pulmonary enzyme; and (NH₂)Arg-Arg-Val-Ser-Asn-Asn-Gln-Ser-Ser- and -(Phe, Ala)-Glu-Leu-Ser(COOH) for the enzyme from testis.     

Evaluation of recombinant phenylalanine dehydrogenase behavior in aqueous two-phase partitioning

Recombinant Bacillus sphaericus phenylalanine dehydrogenase (PheDH) partitioning was studied in polyethylene glycol (PEG) and ammonium sulfate aqueous two-phase systems (ATPS). The objectives of this work were to investigate influences; varying the molecular mass and concentration of PEG, pH, phase volume ratio (V_R), tie-line length (TLL) and concentration of (NH₄)₂SO₄ on the partition behavior of PheDH. It was revealed that the partitioning was not affected by V_R, while PEG molecular mass and concentration and (NH₄)₂SO₄ concentration had significant effects on enzyme partitioning. Longer TLL and higher pH resulted in better partitioning into the top phase. Under the most favorable partition conditions with 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH₄)₂SO₄ and V_R = 0.25 at pH 8.0, partition coefficient (K_E), recovery (R%), yield (Y%) and TLL were achieved 58.7%, 135%, 94.42% and 39.89% (w/w), respectively. Overall, the promising results obtained in this research indicated that the ATPS partitioning can be provided an efficient and powerful tool for recovery and purification of recombinant PheDH.     

Errors in the determination of low levels of nitrate in lake sediments

Abstract

Error sources associated with the spectrophotometric determination of low levels (e.g. <2 μg g^?1) of nitrate in sediments have been examined and problems identified included incomplete nitrate recovery (attributable in part to anion resorption) and light scattering by colloidal (<0.45 μm) matter in extract solutions (minimised by using uncoloured extract in the reference beam). Optimum retrieval (>90%) of nitrate from the marine lake sediments studied was achieved with 15 min mixing with 0.1 M NH₄CI, using a sediment to extractant ratio of 1:30. The nitrate in the extracts was determined by reducing it to nitrite (using Cd powder), with subsequent colour development based on the addition of sulfanilic acid and N-1-naphthyl-ethylenediamine dihydrochloride. The reduction step was sensitive to the experimental conditions used, but was near quantitative using 0.1 M NH₄CI extracts. (Much lower transformation levels were observed when the nitrate solutions contained KCI or CaSO₄, or when Zn powder was used as the reductant). All the sediments tested sorbed nitrate ion from solution (some very avidly) and this sorbed ion was not readily retrieved by back extraction into NH₄CI solutions.     

Test of soil extractants for their suitability in predicting Ca/Sr ratios in leaves and stems of sugar maple seedlings

Differential uptake and translocation of Ca and Sr in organisms have been reported, calling into question the use of Sr to track Ca cycling in the environment. We investigated the relationship between Ca/Sr ratios in soil extracts of various strengths (H₂O, NH₄Cl, and NH₄EDTA) and seedlings of sugar maple (Acer saccharum Marsh.) grown from natural regeneration on 37 sites. Our objectives were to determine if Ca/Sr ratios in soil extracts are correlated with those in sugar maple tissues, and what soil extractant best duplicate plant tissue Ca/Sr ratios. Leaves had higher Ca/Sr ratios than stems and the extractants did not produce equal Ca/Sr ratios: H₂O had the lowest Ca/Sr, and NH₄EDTA the highest. The relationships between soil extract Ca/Sr ratios and leaf and stem Ca/Sr ratios were significant and linear, but the slopes differed among extractants. The lowest slope (0.45) was observed for the water extract/leaves and the highest (2.15) for the NH₄EDTA extract/stem with discrimination factors ranging from 0.22 with NH₄EDTA to 1.59 for water. Leaf extracts were more strongly correlated with soil Ca/Sr than stem extracts (R ² of 0.57–0.7 vs. R ² of 0.45–0.6, respectively). These findings support the use of Ca/Sr ratios in plants to track their source of soil Ca, but they highlight the need to calibrate the relationships for the plant tissue and soil extractant used.     

材料表面化学刺激对纤连蛋白构象及细胞的影响

生物材料的表面化学性质通过改变吸附蛋白的构象,影响细胞粘附铺展,产生不同的细胞行为.采用金-硫自组装单分子层技术(alkanethiol self-assembled monolayers,SAMs)构建了不同化学基团(-NH2、-COOH、-OH和-CH3)修饰的基底材料表面.运用X射线光能质谱(XPS)和接触角仪表...     

Extraction and three-phase partitioning behavior of proteases from papaya peels

Dried papaya peels exhibited superior proteolytic activity to the fresh peels. An extraction with phosphate buffer pH 7.0 greatly maintained proteolytic activity when compared to water. SDS-PAGE verified that the extracted dried papaya peels held a wide range of proteins. To optimize the three-phase partitioning (TPP) for isolating the papaya peel proteases required a ratio of crude extract to t-butanol, the (NH₄)₂SO₄ concentration and the TPP cycles. The ratio of the crude extract to t-butanol of 1.0:0.5 with the presence of 20% (NH₄)₂SO₄ resulted in the highest proteolytic recovery at 253.5%, and 15.8-fold of purification in the bottom phase. The TPP was then optimized by adding up to 55% (NH₄)₂SO₄ to the bottom phase of the first step. A purification of 10.1-fold with about 89.4% recovery was obtained. This study showed the TPP can be effectively employed for the extraction of proteases from papaya peels.     

Theoretical study on the functionalization of BC2N nanotube with amino groups

Using density functional theory calculations, we investigated properties of a functionalized BC₂N nanotube with NH₃ and five other NH₂-X molecules in which one of the hydrogen atoms of NH₃ is substituted by X = ?CH₃, ?CH₂CH₃, ?COOH, ?CH₂COOH and ?CH₂CN functional groups. It was found that NH₃ can be preferentially adsorbed on top of the boron atom, with adsorption energy of ?12.0 kcal mol^?1. The trend of adsorption-energy change can be correlated with the trend of relative electron-withdrawing or -donating capability of the functional groups. The adsorption energies are calculated to be in the range of ?1.8 to ?14.2 kcal mol^?1, and their relative magnitude order is found as follows: H₂N(CH₂CH₃) > H₂N(CH₃) > NH₃ > H₂N(CH₂COOH) > H₂N(CH₂CN) > H₂N(COOH). Overall, the functionalization of BC₂N nanotube with the amino groups results in little change in its electronic properties. The preservation of electronic properties of BC₂N coupled with the enhancement of solubility renders their chemical modification with either NH₃ or amino functional groups to be a way for the purification of BC₂N nanotubes.     

Shewanella putrefaciens CN32对粘土附着态铵氮释放的影响

【目的】研究产胞外分泌物微生物Shewanella putrefaciens CN32对土壤中常见粘土矿物附着态NH_4~+的释放效果及影响机制。【方法】以吸附NH_4~+的蒙脱石、蛭石、伊蒙混层矿物和黑云母为对象,通过监测S. putrefaciens CN32作用下不同粘土释放的NH_4~+含量及过程,以及监测微生物量及释放的胞外聚合物(Extracellular Polymeric Substances,EPS)的含量变化,研究S. putrefaciens CN32作用下不同粘土矿物类型附着态NH_4~+释放的差异性。【结果】粘土矿物附着态NH_4~+含量从高到低依次为蒙脱石蛭石伊蒙混层矿物黑云母(黑云母NH_4~+吸附量极低,会在非生物作用下几乎完全释放),CN32作用下粘土附着态NH_4~+相对释放量依次为蒙脱石伊蒙混层矿物蛭石;然而,尽管CN32有效促进了粘土附着态NH_4~+释放,但释放的NH_4~+并未在溶液中大量累积,而是多被微生物同化吸收转化为生物有机氮(EPS为主)并吸附在粘土表面,且粘土对EPS的吸附能力表现为蒙脱石伊蒙混层矿物蛭石黑云母;由于粘土吸附NH_4~+及EPS都与矿物中的羟基(结构水或层间水)关系密切,推测EPS对矿物羟基的竞争吸附可能是CN32促进NH_4~+释放的重要原因之一。【结论】以上结果表明,产EPS微生物S. putrefaciens CN32能够促进各类粘土矿物的附着态NH_4~+释放,但释放的NH_4~+可以通过微生物作用转化为有机氮,从而在减少NH_4~+流失的同时增加土壤氮肥的生物可利用性,因此微生物在降低土壤氮肥流失、转化土壤氮肥污染过程中可能起到了重要作用,也揭示了深入系统地分析不同类型土壤(粘土类型不同)中粘土附着态NH_4~+在不同功能微生物作用下的迁移转化过程,是精准评估土壤氮肥施用效率及流失风险的前提之一。     

Arrangement of type IV collagen on NH2 and COOH functionalized surfaces

Apart from the paradigm that cell–biomaterials interaction depends on the adsorption of soluble adhesive proteins we anticipate that upon distinct conditions also other, less soluble ECM proteins such as collagens, associate with the biomaterials interface with consequences for cellular response that might be of significant bioengineering interest. Using atomic force microscopy (AFM) we seek to follow the nanoscale behavior of adsorbed type IV collagen (Col IV)—a unique multifunctional matrix protein involved in the organization of basement membranes (BMs) including vascular ones. We have previously shown that substratum wettability significantly affects Col IV adsorption pattern, and in turn alters endothelial cells interaction. Here we introduce two new model surfaces based on self‐assembled monolayers (SAMs), a positively charged –NH₂, and negatively charged –COOH surface, to learn more about their particular effect on Col IV behavior. AFM studies revealed distinct pattern of Col IV assembly onto the two SAMs resembling different aspects of network‐like structure or aggregates (suggesting altered protein conformation). Moreover, the amount of adsorbed FITC‐labeled Col IV was quantified and showed about twice more protein on NH₂ substrata. Human umbilical vein endothelial cells attached less efficiently to Col IV adsorbed on negatively charged COOH surface judged by altered cell spreading, focal adhesions formation, and actin cytoskeleton development. Immunofluorescence studies also revealed better Col IV recognition by both α₁ and α₂ integrins on positively charged NH₂ substrata resulting in higher phosphorylated focal adhesion kinase recruitment in the focal adhesion complexes. On COOH surface, no integrin clustering was observed. Taken altogether these results, point to the possibility that combined NH₂ and Col IV functionalization may support endothelization of cardiovascular implants. Biotechnol. Bioeng. 2011;108: 3009–3018. © 2011 Wiley Periodicals, Inc.     

不同氮源及其浓度对标志链带藻合成淀粉和油脂的影响北大核心CSCD

【目的】以标志链带藻(Desmodesmus insignis)为实验材料,研究不同氮源及其浓度对该藻生长、总脂和淀粉(碳水化合物)含量的影响,为该藻在生物能源方面的应用提供一定的理论依据。【方法】以硝酸钠、碳酸氢铵或尿素为氮源,5个氮浓度(3、6、9、12和18 mmol/L)的BG-11培养基培养标志链带藻,采用干重法测定生物质浓度、重量法测定总脂、苯酚-硫酸法测定、总碳水化合物和淀粉的含量。【结果】标志链带藻在3种氮源下均能很好的生长。最高油脂含量出现在3 mmol/L硝酸钠实验组,达到32.61%(d.w)。当18 mmol/L碳酸氢铵作为氮源时,总碳水化合物与淀粉的含量以及产率都达到最高,分别为56.54%(d.w)和55.33%(d.w)、0.24和0.23 g/(L·d)。以尿素为氮源时,其生物质浓度和各组分含量与其它氮源实验组差别不大,均有利于该藻的生长及各生化组分含量的积累。【结论】以该藻种生产生物能源的成本等综合考虑,以18 mmol/L碳酸氢铵和尿素为氮源培养标志链带藻最优。     

Synthesis, characterization, and evaluation of a novel 99mTc(CO)3 pyrazolyl conjugate of a peptide nucleic acid sequence

The 16-mer peptide nucleic acid sequence H-A GAT CAT GCC CGG CAT-Lys-NH₂ (1), which is complementary to the translation start region of the N-myc oncogene messenger RNA, was synthesized and conjugated to a pyrazolyl diamine bifunctional chelator (pz). The novel conjugate pz-A GAT CAT GCC CGG CAT-Lys-NH₂ (2) was labeled with technetium tricarbonyl, yielding quantitatively the complex fac-[^99mTc(CO)₃(κ³-pz-A GAT CAT GCC CGG CAT-Lys-NH₂)]²⁺ (4). Complex 4 was obtained with high radiochemical purity and high specific activity, revealing high stability in human serum and in cell culture medium. The identity of 4 was confirmed by comparing its reversed-phase high performance liquid chromatography profile with that of the rhenium analog fac-[Re(CO)₃(κ³-pz-A GAT CAT GCC CGG CAT-Lys-NH₂)]²⁺ (3), prepared by conjugation of fac-[Re(CO)₃(3,5-Me₂pz(CH₂)₂N((CH₂)₃COOH)(CH₂)₂NH₂)]⁺ to 1, using solid-phase techniques. UV melting experiments of 1 and 3 with the complementary DNA sequence led to the formation of stable duplexes, indicating that the conjugation of 1 to the pyrazolyl chelator and to the metal fragment fac-[M(CO)₃]⁺ did not affect the recognition of the complementary sequence as well as the duplex stability. For a first screening, SH-SY5Y human neuroblastoma cells, which express N-myc, were treated with 4. The results show that 4 internalizes (7% of the activity goes into the cells, after 4 h at 37 °C), presenting also a relatively high cellular retention (only 40% of internalized activity is released from the cells after 5 h). An erratum to this article can be found at     

Is the bioavailability index applicable for trace elements in different types of soil?

Abstract

The bioavailability index (BI) is defined as the proportion of reduction in a plant’s accumulation of an element, caused by the removal of the extractable fraction of the element of interest from the soil. The BI and corresponding experimental methods were quantitatively applied to evaluate the bioavailability of trace elements in five Chinese soils. The soil was first extracted with various reagents (DTPA, HCl and NH₂OH.HCI) separately, to remove the extractable elements. The soil, after extraction, was washed with deionised water to eliminate the extractant used in the fractionation analysis. Then the pH of the soil was re-adjusted with CaO. The soil was then fertilised and incubated in a greenhouse for four weeks. Tests showed that after incubation the pH, cation exchange capacity (CEC) and organic matter (OM) of the treated soil were close to their original values. Wheat (Triticum aestivum L.) was planted in both the untreated and treated soil for eight weeks. After harvest the plant concentrations of the elements Cr, Mn, Co, Ni, Zn, Cu, Cd, and Pb were analysed by inductively coupled plasma mass spectrometry (ICPMS). Trace element accumulation by plants grown in the treated soil was reduced significantly compared with that of plants grown in the untreated soil. The results showed that BI values were in the order BI(DTPA)>BI(HCl)>BI(NH₂OH.HCl). This indicated that the DTPA-extractable fraction represented a highly available fraction of the total content. Variations of BI among different trace elements show that Cr, Mn, Zn and Co have a higher BI, in general for the elements tested, whereas, Cu, Cd and Pb have lower values. There are also slight differences in the BI among soils. However, no significant relationship could be found between the BI and the soil characteristics.     

Efficiency of pretreatments for optimal enzymatic saccharification of soybean fiber

The effectiveness of several pretreatments [high-power ultrasound, sulfuric acid (H₂SO₄), sodium hydroxide (NaOH), and ammonium hydroxide (NH₃OH)] to enhance glucose production from insoluble fractions recovered from enzyme-assisted aqueous extraction processing of extruded full-fat soybean flakes (FFSF) was investigated. Sonication of the insoluble fraction at 144 μm_{pp (peak-to-peak)} for 30 and 60 s did not improve the saccharification yield. The solid fractions recovered after pretreatment with H₂SO₄ [1% (w/w), 90 °C, 1.5 h], NaOH [15% (w/w), 65 °C, 17 h], and NH₃OH [15% (w/w), 65 °C, 17 h] showed significant lignin degradation, i.e., 81.9%, 71.2%, and 75.4%, respectively, when compared to the control (7.4%). NH₃OH pretreatment resulted in the highest saccharification yield (63%) after 48 h of enzymatic saccharification. A treatment combining the extraction and saccharification steps and applied directly to the extruded FFSF, where oil extraction yield and saccharification yield reached 98% and 43%, respectively, was identified.     

Evidence for a membrane carbonic anhydrase IV anchored by its C-terminal peptide in normal human pancreatic ductal cells

The high concentration of HCO₃ ^– ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl^–/HCO₃ ^– exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors (n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH₂ antibody directed against the NH₂ terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.The first two authors have contributed equally to this work     

Heterobifunctionalized tetraethylene glycol: A spacer for surface attachment of viral peptide epitopes for ELISA and derivatization of membrane modifying peptides

Summary New hydrophilic linkers of the formula Fmoc-NHCH₂CH₂COO(CH₂CH₂O)₄X (X=COCH₂CH₂COOH Fmoc-Ats (2), X=CONHCH₂COOH Fmoc-Atg (4) and X=CONHCH₂CH₂COOH Fmoc-Ata (5) have been prepared by heterobifunctional modification of tetraethylene glycol as starting material. These linkers represent a useful tool for solid phase peptide synthesis according to Fmoc/tBu strategy. Two examples are presented to illustrate the applicability of these building blocks: (i) spacing between biotin and a peptide epitope of the hepatitis C virus and evaluation in a biotin-streptavidin ELISA, and (ii) coupling of the new linker to the N- and C-terminus of the peptide antibiotic alamethicin to show eventual influences on the peptide's α-helical conformation.     

Contributions to nitrogen leaching and pasture uptake by autumn-applied dairy effluent and ammonium fertilizer labeled with 15N isotope

The objective of this study was to compare the N leaching loss and pasture N uptake from autumn-applied dairy shed effluent and ammonium fertilizer (NH₄Cl) labeled with ¹⁵N, using intact soil lysimeters (80 cm diameter, 120 cm depth). The soil used was a sandy loam, and the pasture was a mixture of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). The DSE and NH₄Cl were applied twice annually in autumn (May) and late spring (November), each at 200 kg N ha^-1. The N applied in May 1996 was labeled with ¹⁵N. The lysimeters were either spray or flood irrigated during the summer. The autumn-applied DSE resulted in lower N leaching losses compared with NH₄Cl. However, the N applied in the autumn had a higher potential for leaching than N applied in late spring. Between 4.5–8.1% of the ¹⁵N-labeled mineral N in the DSE and 15.1–18.8% of the ¹⁵N-labeled NH₄Cl applied in the autumn were leached within a year of application. Of the annual N leaching losses in the DSE treatments (16.0–26.9 kg N ha^-1), a fifth (20.3–22.9%) was from the mineral N fraction of the DSE applied in the autumn, with the remaining larger proportion from the organic fraction of the DSE, soil N and N applied in spring. In the NH₄Cl treatments, more than half (53.8–64.8%) of the annual N leaching loss (55.9–57.6 kg N ha^-1) was derived from the autumn-applied NH₄Cl. DSE was as effective as NH₄Cl in stimulating pasture production. Since only 4.4–4.5% of the annual herbage N uptake in the DSE treatment and 12.3–13.3% in the NH₄Cl treatment were derived from the autumn-applied mineral N, large proportions of the annual herbage N uptake must have been derived from the N applied in spring, the organic N fraction in the DSE, soil N and N fixed by clover. The recoveries of ¹⁵N in the herbage were similar between the DSE and the NH₄Cl treatments, but those in the leachate were over 50% less from the DSE than from the NH₄Cl treatment. The lower leaching loss of ¹⁵N in the DSE treatment was attributed to the stimulated microbial activities and increased immobilization following the application of DSE. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification of plant-esterase in PEG1000/NaH2PO4 aqueous two-phase system by a two-step extraction

Purification of plant-esterase from flour in an aqueous two-phase system (ATPS) was investigated. The effects of various process parameters such as the type of aqueous two-phase systems, the phase-forming salt, the molecular weight and concentration of PEG, the system pH, and the types and concentrations of neutral salts on partitioning of plant-esterase were evaluated. Optimized conditions for the purification of plant-esterase were found in polymer–salt systems, with especially promising results in the PEG1000/NaH₂PO₄ system. Using 27.0% PEG1000/13.0% NaH₂PO₄ (w/w, pH 5.0), and 27.0% PEG1000/13.0% NaH₂PO₄/6.0% (NH₄)₂SO₄ (w/w, pH 5.0), plant-esterase was purified by a two-step extraction. Compared to the results obtained with the conventional salting-out method, this method had a comparable yield (83.16% versus the original yield of 80%), but produced plant-esterase that was 4.8 times as pure (18.46-fold). Integrating dialysis into the aqueous two-phase extraction removed (NH₄)₂SO₄ from the purified plant-esterase. Finally, plant-esterase was freeze-dried to convert the product to powder. This work offers a simple and more efficient process to purify and concentrate plant-esterase. Plant-esterase is used in applications such as organophosphorus compounds (OPs) detection and since our method makes this enzyme easier to isolate, it will enhance researchers’ ability to explore these applications.