重组酸性脲酶对黄酒中尿素和氨基甲酸乙酯的降解应用

氨基甲酸乙酯(Ethyl carbamate,EC)作为一种潜在致癌物质普遍存在于传统发酵食品中。利用酸性脲酶消除EC前体物质尿素是一种具有潜在重要应用价值的策略。本研究在前期成功实现食品级耐乙醇酸性脲酶高效表达制备的基础上,系统研究了重组酸性脲酶对尿素和EC的水解过程。重组酸性脲酶对模拟体系以及黄酒体系中的尿素具有很好的降解能力(60mg/L的尿素在25h内完全被降解),表明该重组酸性脲酶适用于黄酒中尿素的消除。虽然重组酸性脲酶也具有降解EC的催化活性,但在黄酒中添加重组酸性脲酶对EC的浓度无明显影响。进一步研究发现重组酸性脲酶对尿素和EC的Km值分别为0.714 7mmol/L和41.32mmol/L,研究结果为应用定向进化策略改造重组酸性脲酶实现同时水解尿素和EC提供了理论依据。     

Sensitization enhancement of europium in ZnSe/ZnS core/shell quantum dots induced by efficient energy transfer

Eu‐doped ZnSe:/ZnS quantum dots (formed as ZnSe:Eu/ZnS QDs) were successfully synthesized by a two‐step wet chemical method: nucleation doping and epitaxial shell growing. The sensitization characteristics of Eu‐doped ZnSe and ZnSe/ZnS core/shell QD are studied in detail using photoluminescence (PL), PL excitation spectra (PLE) and time‐resolved PL spectroscopy. The emission intensity of Eu ions is enhanced and that of ZnSe QDs is decreased, implying that energy was transferred from the excited ZnSe host materials (the donor) to the doped Eu ions (the acceptor). PLE reveals that the ZnSe QDs act as an antenna for the sensitization of Eu ions through an energy transfer process. The dynamics of ZnSe:Eu/ZnS core/shell quantum dots with different shell thicknesses and doping concentrations are studied via PL spectra and fluorescence lifetime spectra. The maximum phosphorescence efficiency is obtained when the doping concentration of Eu is approximately 6% and the sample showed strong white light under ultraviolet lamp illumination. By surface modification with ZnS shell layer, the intensity of Eu‐related PL emission is increased approximately three times compared with that of pure ZnSe:Eu QDs. The emission intensity and wavelength of ZnSe:Eu/ZnS core/shell quantum dots can be modulated by different shell thickness and doping concentration. The results provide a valuable insight into the doping control for practical applications in laser, light‐emitting diodes and in the field of biotechnology. Copyright © 2014 John Wiley & Sons, Ltd.     

Soybean leaf urease: Comparison with seed urease

Soybeans, Glycine max (L.) Merr., from ureides for transport of nitrogen from the root nodule to the shoot. The most direct routes for ureide utilization include the degradation of ureide-derived urea to NH₃ and CO₂. Ureolytic activity was found in leaf disks of soybean and exhbited optimal activity at pH 7 in the presence of a high concentration of urea (250 m M ). In vitro studies showed neither urea amidolyase nor urea dehydrogenase activity in soybean leaves and the ureolytic activity was characterized as urease. Several biochemical properties of soybean leaf urease were determined and compared to seed urease properties. Soybean leaf urease differed from that of seed in five ways: pH optima (5.25 and 8.75), apparent K_m (0.8 m M ), no inhibition by hydroxyurea, faster electrophoretic mobility and no cross-reactivity with soybean seed urease antibodies. The data suggest that urease is the primary urea metabolizing enzyme present in soybean leaves. The properties of soybean leaf urease support the conclusion that a unique isozyme of urease is present in leaf tissue.     

On-chip detection of protein glycosylation based on energy transfer between nanoparticles

We describe a chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs). Our assay system relies on modulations in the energy transfer between the nanoparticles on a surface. The photoluminescence (PL) of lectin-coated QDs (energy donor) immobilized on a glass slide is quenched by carbohydrate-coated AuNPs (energy acceptor), and the presence of the glycoprotein causes the increase of the PL of QDs. As a proof-of-concept, Concanavalin A-coated QDs (ConA-QDs) and dextran-coated AuNPs (Dex-AuNPs) were used to detect the mannosylated proteins. As a result, the PL intensity of QDs was found to be linearly correlated with the concentration and the number of glycan moiety of the glycoprotein. We anticipated that our simple assay system will find applications for the analysis of glycoproteins with high selectivity and sensitivity in a high-throughput manner.     

Urea potentiometric enzymatic biosensor based on charged biopolymers and electrodeposited polyaniline

A potentiometric biosensor based on urease was developed for the quantitative determination of urea concentration in aqueous solutions for biomedical applications. The urease was either physisorbed onto an electrodeposited polyaniline film (PANI), or immobilized on a layer-by-layer film (LbL) assembled over the PANI film, that was obtained by the alternate deposition of charged polysaccharides (carboxymethylpullulan (CMP) and chitosan (CHI)). In the latter case, the urease (Urs) enzyme was either physically adsorbed or covalently grafted to the LbL film using carbodiimide coupling reaction. Potentiometric responses of the enzymatic biosensors were measured as a function of the urea concentration in aqueous solutions (from 10(-6) to 10(-1) mol L(-1) urea). Very high sensitivity and short response time were observed for the present biosensor. Moreover, a stability study showed a higher stability over time for the potentiometric response of the sensor with the enzyme-grafted LbL film, testifying for the protective nature of the polysaccharide coating and the interest of covalent grafting.     

Photoluminescence properties of hybrid SiO2‐coated CdTe/CdSe quantum dots

Hybrid SiO₂‐coated CdTe/CdSe quantum dots (QDs) were prepared using CdTe/CdSe QDs prepared by hydrothermal synthesis. A CdSe interlayer made CdTe/CdSe cores with unique type II heterostructures. The hybrid SiO₂‐coated CdTe/CdSe QDs revealed excellent photoluminescence (PL) properties compared with hybrid SiO₂‐coated CdTe QDs. Because of the existence of spatial separations of carriers in the type II CdTe/CdSe core/shell QDs, the hybrid QDs had a relatively extended PL lifetime and high stability in phosphate‐buffered saline buffer solutions. This is ascribed to the unique components and stable surface state of hybrid SiO₂‐coated CdTe/CdSe QDs. During the stabilization test in phosphate‐buffered saline buffer solutions, both static and dynamic quenching occurred. The quenching mechanism of the hybrid QDs was not suited with the Stern–Volmer equation. However, the relative stable surface of CdTe/CdSe QDs resulted in lower degradation and relative high PL quantum yields compared with hybrid SiO₂‐coated CdTe QDs. As a result, hybrid SiO₂‐coated CdTe/CdSe QDs can be used in bioapplications. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis of near‐infrared‐emitting CdTeSe and CdZnTeSe quantum dots

We exploited the synthesis of near‐infrared (NIR) emitting ternary‐alloyed CdTeSe and quaternary‐alloyed CdZnTeSe quantum dots (QDs) with rod and tetrapod morphologies, which have tunable emission in the NIR electromagnetic spectrum. The morphologies of the QDs depended strongly on their growth kinetics, probably due to the coordinating ligands used in the preparation. Using oleic acid, stearic acid and hexadecylamine as ligands and keeping the same reaction parameters, QDs with tetrapod and rod morphologies were created. Not only had the capping ligands influenced the morphologies of QDs, but also they influenced the optical properties of QDs. The molar ratios of Cd/Zn and Te/Se upon preparation were adjusted for investigating the effect of composition on the properties of resulting QDs. By varying the composition of QDs, the photoluminescence (PL) wavelength of QDs was tuned from 650 nm to 800 nm. To enhance PL efficiency and stability, QDs were coated with a CdZnS shell. As NIR PL has numerous advantages in biological imaging detection, these QDs hold great potential for application. Copyright © 2012 John Wiley & Sons, Ltd.     

Spontaneous emission of semiconductor quantum dots in inverse opal SiO2 photonic crystals at different temperatures

The photoluminescence (PL) characteristics of CdSe quantum dots (QDs) infiltrated into inverse opal SiO₂ photonic crystals (PCs) are systemically studied. The special porous structure of inverse opal PCs enhanced the thermal exchange rate between the CdSe QDs and their surrounding environment. Finally, inverse opal SiO₂ PCs suppressed the nonlinear PL enhancement of CdSe QDs in PCs excited by a continuum laser and effectively modulated the PL characteristics of CdSe QDs in PCs at high temperatures in comparison with that of CdSe QDs out of PCs. The final results are of benefit in further understanding the role of inverse opal PCs on the PL characteristics of QDs. Copyright © 2015 John Wiley & Sons, Ltd.     

糙皮侧耳脲酶基因的克隆和原核表达分析

尿素是现代农业生产中应用较为广泛的一种氮素化肥,而脲酶（EC 3.5.1.5）是尿素分解利用的关键酶。本文以糙皮侧耳Pleurotus ostreatus栽培菌株New 831为试验材料,探究了糙皮侧耳对尿素的利用情况,结果表明：糙皮侧耳可利用尿素作为唯一氮源,平板培养时尿素最适添加量为20mmol/L;在液体摇瓶培养过程中,培养液中的铵根浓度表现为先急剧升高后缓慢降低。通过对P. ostreatus PC15菌株基因组分析,获得了一个功能注释为脲酶的基因,并克隆获得了其全长基因组DNA（gDNA）和编码区（CDS）片段,命名为Pourease。结果表明：Pourease基因的gDNA和CDS长度分别为3 003bp和2 517bp,由10个外显子和9个内含子组成;Pourease蛋白由838个氨基酸组成,预测分子量为90.03kDa,与SDS-PAGE分析结果相符;Pourease蛋白与细菌、真菌和植物来源的脲酶具有52%-82%的一致性,且含有脲酶保守的镍离子结合位点;与洋刀豆脲酶空间结构类似,Pourease蛋白也以同源三聚体的形式存在。     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

汞、镉对土壤脲酶活性影响的研究Ⅰ.尿素浓度

对不同尿素浓度条件下重金属与土壤脲酶活性关系进行了研究。结果表明,尿素浓度对脲酶活性具有显著的影响。在供试浓度范围内可采用线性和Langmuir模型较好地表征二者关系。并得到脲酶活性的尿浓贡献率,尿浓贡献变化率和最大表观脲酶活性等参数;Hg,Cd明显降低了前述参数值,其中Hg Cd复合污染的抑制作用最强,Hg的生态毒性最大;同时初步获得土壤酶促反应过程中存在吸附机制。     

A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs

A novel urea biosensor based on immobilised recombinant urease as sensitive element and ion sensitive field effect transistor as transducer was developed. Recombinant urease from E. coli with an increased Km was photoimmobilised in PVA/SbQ (poly(vinyl alcohol) containing styrylpyridinium) membrane and has demonstrated quite good performance as biosensitive element. Enzymatic field effect transistors based on such a bioselective element were studied in model buffer solutions. This biosensor demonstrated an extended dynamic range up to 80 mM, a quite good reproducibility (standard deviation of the sensor responses was approximately 2.5%, n= 20 for urea concentration 10 mM) and a high stability. Such characteristics fit with the analytical requirements needed for urea control in plasma and liquids used during renal dialysis.     

Synthesis of glutathione-capped CdS quantum dots and preliminary studies on protein detection and cell fluorescence image.

A series of glutathione (GSH)-capped aqueous CdS quantum dots (QDs) with strong photoluminescence (PL) were prepared by changing the reaction temperatures and times on the basis of optimization of the mole ratio of S to Cd. The reaction time was shortened to about 1/10 compared with that reported previously by increasing the reaction temperature. The absorption and fluorescence spectra indicated good optical properties with PL full width of half-maximum (FWHM) of about 100 nm. The excitation spectrum was broad and continuous in the range 200-480 nm. The PL quantum efficiency (QE) of the prepared QDs was about 36% compared with rhodamine 6G (95%). The shape and size of the CdS QDs were characterized using high-resolution transmission electron microscopy (HRTEM). The prepared QDs were conjugated with bovine serum albumin (BSA) and onion inner pellicle cells and used as fluorescence probes for the first time. The results demonstrated that the fluorescence of CdS can be enhanced by BSA and the enhanced fluorescence intensity is proportional to the concentration of BSA in the range 1.0-10 mg/L. The aggregation of CdS in onion inner pellicle cells and its fluorescence images indicated that the QDs can aggregate around cells soaked for 8 h in CdS solution but enter the interior of cells and become aggregated to the nucleus when they are soaked in CdS solution for longer, e.g. 98 h.     

Exogenous application of urea and a urease inhibitor improves drought stress tolerance in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

Drought is believed to cause many metabolic changes which affect plant growth and development. However, it might be mitigated by various inorganic substances, such as nitrogen. Thus, the study was carried out to investigate the effect of foliar-applied urea with or without urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) on a maize cultivar under drought stress simulated by 15% (w/v) polyethylene glycol 6000. Foliar-applied urea resulted in a significant increase in plant dry weight, relative water content, and photosynthetic pigments under water stress condition. Furthermore, the activities of superoxide dismutase (SOD), peroxidase (POD), and hydrogen peroxidase (CAT), were enhanced with all spraying treatments under drought stress, which led to decreases in accumulation of hydrogen peroxide (H₂O₂), superoxide anion (\({\text{O}}_2^{ \cdot - }\)) and malondialdehyde (MDA). The contents of soluble protein and soluble sugar accumulated remarkably with urea-applied under drought stress condition. Moreover, a further enhancement in above metabolites was observed by spraying a mixture of urea and urease inhibitor as compared to urea sprayed only. Taken together, our findings show that foliar application of urea and a urease inhibitor could significantly enhance drought tolerance of maize through protecting photosynthetic apparatus, activating antioxidant defense system and improving osmoregulation.     

Recycling of urea associated with the host plant urease in the silkworm larvae,Bombyx mori

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.     

CdTe1‐xSex/Cd0.5Zn0.5S core/shell quantum dots: core composition and property

Alloy CdTe_1‐xSe_x quantum dots (QDs) have been fabricated by an organic route using Cd, Te and Se precursors in a mixture of trioctylamine and octadecylphosphonic acid at 280 °C. The variation of photoluminescence (PL) peak wavelength of the CdTe_1‐xSe_x QDs compared with CdTe QDs confirmed the formation of an alloy structure. The Se component drastically affected the stability of CdTe_1‐xSe_x QDs. A Cd_0.5Zn_0.5S shell coating on CdTe_1‐xSe_x cores was carried out using oleic acid as a capping agent. CdTe_1‐xSe_x/Cd_0.5Zn_0.5S core/shell QDs revealed dark red PL while a yellow PL peak was observed for the CdTe_1‐xSe_x cores. The PL efficiency of the core/shell QDs was drastically increased (less than 1% for the cores and up to 65% for the core/shell QDs). The stability of QDs in various buffer solutions was investigated. Core/shell QDs can be used for biological applications because of their high stability, tunable PL and high PL efficiency. Copyright © 2013 John Wiley & Sons, Ltd.     

脲对棒状链霉菌棒酸合成的影响

【目的】棒酸(Clavulanic acid)是棒状链霉菌(Streptomyces clavuligerus)产生的β-内酰胺酶抑制剂,其合成过程中产生副产物脲,旨在探讨脲对棒酸合成的影响。【方法】通过发酵过程中脲和铵盐添加实验、阻断脲酶活性以及pH梯度实验研究脲对棒酸合成影响。【结果】脲添加实验结果表明:低浓度脲降低棒酸产量,当添加脲浓度达到20 mmol/L时,完全抑制棒酸合成。由于脲酶可以把脲水解为铵离子,导致铵离子浓度及pH提高,因此,通过阻断棒状链霉菌脲酶活性,可以更准确地反映脲对棒酸合成的影响。结果发现,脲酶敲除株发酵液中脲大量积累,浓度高达10 mmol/L,但棒酸产量没有明显降低,说明在该浓度下脲自身并不能抑制棒酸合成。添加脲降低野生菌棒酸产量,可能是脲被水解为铵离子或其引起的pH变化所致。而棒酸发酵液添加铵盐的结果显示铵离子对棒酸产量没有抑制作用;另外,pH梯度实验证实不同pH对棒酸产量影响较大。【结论】排除了脲和铵离子对棒酸合成的抑制作用,证实了脲酶水解脲导致pH提高是脲添加导致野生菌棒酸产量降低的真正原因,为进一步阐明棒酸合成调控机制提供了根据。     

Urea: An intermediate of aerobic and anaerobic purine degradation in Rhodopseudomonas capsulata

Abstract A mutant strain ( pur ^-) defective in utilization of purines was isolated from Rhodopseudomonas capsulata . In the mutant, the loss of purine utilization correlated with urease deficiency. In contrast to the wild-type strain, the mutant catalyzed release of urea from purines. The nitrogen of the purine ring was completely liberated as urea indicating that the latter compound is an intermediate of the purine degradation pathway in Rps. capsulata . The degradation pattern was identical under aerobic and anaerobic conditions.     

Catalytic buffers enable positive-response inhibition-based sensing of nerve agents.

We report herein an efficient method to control pH in reactions catalyzed by hydrolytic enzymes, such as the degradation of paraoxon by phosphotriesterase (E.G. 3.1.8.1; OPH), using urease-catalyzed (E.G. 3.5.1.5) urea hydrolysis as a buffering agent. Given the distinct pH profiles of urease and OPH activities, urease produces base on demand in response to pH drop during paraoxon detoxification. As pH changes, the enzyme activities fluctuate to finally stabilize at a pH "set-point," where the rates of acid and base generation are equal. By varying the urease to OPH ratio, various pH "set-points" ranging between 6.5 and 8.5 were achieved within minutes and could be predicted theoretically. This dynamic approach for pH control was successfully applied to the development of a positive-response inhibition-based sensor.     

Genetic tests of the roles of the embryonic ureases of soybean

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in maternal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitro-cultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings.