复合纤维素固定化脲酶对铜离子的吸附作用

脲酶对重金属离子极为敏感。由复合纤维素（CC）固定化脲酸对铜离子的吸附现象得出的结论是：脲酶吸附的Cu~(2+)总量与其活性没有定量关系，而是与固酶重量成比例。从而可以认为，重金属离子不是直接作用于脲酶活性部位使其中毒的，很可能是作用于脲酶大分子其他部位，使分子构象变形后导致脲酶失活的。由于CC固酶对Cu~(2+)有吸附作用，且又不污染系统，所以可用其除去食品、药物及水中的Cu~(2+)离子。     

聚吡咯为基质的脲酶传感器生物电化学响应

用电化学方法在吡咯（Ｐｙ）聚合的同时,将脲酶掺杂进聚吡咯膜（ＰＰｙ）中形成以聚吡咯为基质的脲酶电极．将脲酶电极与ＣＯ２气敏电极结合,组成对脲有电位响应的脲酶生物传感器．测试了传感器对脲的生物电化学响应．研究了聚合条件对酶活性的影响,并测定了掺杂于聚吡咯中的脲酶的活化能等动力学参数．在５０×１０－５～１．０×１０－２ｍｏｌ／Ｌ的浓度范围内,传感器响应的电极电位与脲浓度的对数成正比．     

聚乙烯醇高含水胶固定化脲酶的研究

冷冻-部分脱水法制成的聚乙烯醇高含水胶固定化脲酶活力收率及对脲素的分解能力明显高于聚丙烯酰胺囱定化脲酶,稳定性相近;用低浓度的戊二醛后处理,提高了固定化酶的稳定性。     

外源镧对茶园紫色土酶活性的影响

通过盆栽实验研究了外源稀土元素La对茶园紫色土脲酶、蔗糖酶、纤维素酶及脱氢酶活性的影响。结果表明:低浓度(≤300 mg·kg-1)La3+对茶园紫色土脲酶活性、蔗糖酶活性、纤维素酶活性具有显著促进作用(p0.05),而高浓度(≥800 mg·kg-1)则呈现显著的抑制作用(p0.05)。脲酶、蔗糖酶和纤维素酶最优促进浓度分别为100,300和300 mg·kg-1,抑制浓度分别为800,800和600 mg·kg-1。随培养时间延长,La3+对茶园紫色土脲酶、蔗糖酶、纤维素酶刺激作用有减缓趋势。各浓度La3+处理对茶园紫色土脱氢酶均呈显著抑制作用(p0.05),随浓度升高抑制作用逐步增强,随着培养时间延长,抑制作用有减缓的趋势。相关分析结果表明:脲酶分别与蔗糖酶和纤维素酶呈现极显著正相关(p0.01),蔗糖酶与纤维素酶亦呈现极显著正相关(p0.01),脱氢酶与脲酶、蔗糖酶、纤维素酶相关性不显著。脱氢酶活性可以作为评价稀土元素污染茶园紫色土土壤环境的敏感指标。     

磁性壳聚糖微球用于酶的固定化研究Ⅱ磁性壳聚糖微球对脲酶的吸附及其酶学性质的研究

本文用磁性壳聚糖作为载体用吸附法对脲酶进行固定化研究。结果表明,磁性壳聚糖对脲酶的固载量与磁性壳聚糖微球的粒径、交联度及酶溶液的离子强度成反比;固定化脲酶和自由酶的最适温度分别为80℃和70℃,固定化脲的最适合pH值变化不大,固定化脲酶和自由酶的米氏常数km分别为0.00546mol/L和0.19mol/L。     

电导法及其应用

电导法是以测量欲测溶液的电导为基础的方法。溶液的电导在一定的条件下与存在于溶液中的离子数目、离子所带的电荷数及其淌度有关。     

流动注射分析法测定固定化酶的催化活力

本文介绍了脲酶的固定方法以及用流动注射分析法测定固定化酶的催化活力，测得脲的平均转化率为９５．０％。     

电导实验探讨

由电导法测弱电解质电离常数,是—般高校普遍采用的物理化学实验。     

离子色谱法 第五讲 双柱离子色谱

双柱离子色语法亦称抑制型离子色谱,由于该法检测系统采用通用电导检测器,当洗脱液从分离柱洗脱待测离子时,洗脱液本身有大的背景电导,必须置一个与分离离子电荷相反的离子交换树脂柱或装置于分离柱     

熔盐电导的多维键参数分析

本文用模式识别－人工神经网络结合化学键参数法，总结熔盐电导的半经验规律，在此基础上讨论了熔盐电导的主要影响因素及电导机理。     

Isothermal titration microcalorimetric method for studying the combined ligand binding with application to the binding of ethylurea and (N,N)dimethylurea on urease

A simple, novel method was introduced for determining equilibrium constants and enthalpies of binding of two different competitive ligands on a macromolecule by isothermal titration microcalorimetry technique. This method was applied to the simultaneous binding of ethylurea (I) and (N,N)dimethylurea (X), on jack-bean urease at pH 7.0 (tris-base; 30 mM) at 27°C. The dissociation equilibrium constants measured by this method were markedly consistent with inhibition constants obtained from assay of enzyme activities in the presence of I and X.     

Enzyme coated glass pH-electrode: Its fabrication and applications in the determination of urea in blood samples

A new enzyme coated electrode for the determination of urea in blood samples has been developed. It is based on the encapsulation of urease enzyme in the porous silicate matrix by the sol-gel technique on a glass electrode for the purpose of sensing urea in blood samples. Various parameters like the effect of pH, selection of a suitable buffer of appropriate concentration and interference of common substances in blood samples have been evaluated to optimize the conditions for the determination of urea. The electrode can be used for the determination of urea in the concentration range 0.03-30.0 mM in a solution. The detection limit of the present enzyme-coated electrode is found to be 52 μg/ml of urea. The relative standard deviation for the electrode-to-electrode reproducibility is found to be 2.4% for the determination of 0.1 mM of urea (six replicate electrodes). Sol-gel matrix containing immobilized enzyme was stable for about 25 days at ∼4 °C with 80% urease activity. Urea content in various clinical blood samples has been estimated using this electrode and the results are found to be in good agreement with the standard clinical methods as reported in the literature.     

Gas phase derivatization of ammonia with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to urease assay.

An ammonia-specific and rapid fluorometric method for determination of ammonia and urease activity was developed. The method is designed to assay ammonia levels or urease activity for the rapid diagnosis of Helicobacter pylori infection. 4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole was used to derivatize ammonia and 4-amino-7-nitrobenzo-2-oxa-1,3-diazole was analysed by high performance liquid chromatography at an excitation wavelength of 455 nm and an emission wavelength of 520 nm. Derivatization was designed to react with ammonia gas produced in a strong alkaline pH sample. The fluorescent intensity was linear in the range of 0.1-10 mM ammonia per tube when the reaction was carried out for 15 min at 37 degrees C. Urease activity, judged as the amount of ammonia production from urea, could be measured at 25 ng per tube (S/N = 1.5) with Jack bean meal urease. Because of its rapidity, this assay is potentially superior to the current standard method in use in clinical settings.     

Enzyme and inhibition assay of urease by continuous monitoring of the ammonium formation based on capillary electrophoresis

We present here an easy‐to‐operate and efficient method for enzyme and inhibition assays of urease, which is a widely distributed and important enzyme that catalyzes the hydrolysis of urea to ammonia and CO₂. The assay was achieved by integrating CE technique and rapid on‐line derivatization method, allowing us to continuously drive the sample to the capillary, thus to measure the amount of the product ammonia from the beginning to the end of the reaction. The method exhibits excellent repeatability with RSD as low as 2.5% for the initial reaction rate (n = 5), with the LOD of ammonia of 20 μM (S/N = 5). The enzyme activity as well as the inhibition of urease by Cu²⁺ were investigated using the present method. The results show that Cu²⁺ is a noncompetitive inhibitor on urease, in accordance with the result published in the literature. The enzyme activity and inhibition kinetic constants were obtained and were found to be consistent with the results of traditional off‐line enzyme assays. Our study indicates that the present approach is a reliable and convenient method for analysis of the urease activity and inhibition kinetics by continuous on‐line monitoring of the ammonium formation based on CE.     

Determination of the urease activity and the relative inhibition in the presence of some metal ions: a microcalorimetric study

Batch microcalorimetry has been employed to obtain a calibration curve for the enzymatic activity of urease in solution.This method is simplier, more reliable and easier to handle than the more common techniques (spectrophotometry and potentiometry), because it is based on direct investigation of the enzymatic reaction.By comparison with calorimetric studies employing the thermistor combined with the immobilized, enzyme, this method also allows the catalytic activity to be measured.Variations in the urease activity in the presence of nine metal ions [Hg(II), Ag(I), Cu(II),Zn(II), Cd(II), Ni(II), Co(II), Mn(II) and Mg(II)] are also described.A graphic method has been devised for immediate identification of the minimum inhibitor concentration,determining the start, 50% and complete inhibition of ureasic activity.     

Novel determination of cadmium ions using an enzyme self-assembled monolayer with surface plasmon resonance

The activity of the enzyme urease is known to be inhibited by the heavy metal cadmium. The binding of cadmium to urease and the consequent changes of the enzyme structure are the basis of the surface plasmon resonance (SPR) biosensing system reported herein. To facilitate the formation of a self-assembled monolayer (SAM) of the urease on gold-coated glass SPR sensor disks, the enzyme has been modified with N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP). The urease monolayer was exposed to trace levels of cadmium ions and monitored by SPR. From circular dichroism (CD) data, it is believed that the conformation of the active nickel site of the urease changes upon binding of the cadmium ions. It is this change of the enzyme monolayer, measured by SPR, which has been related to the cadmium ion concentration in the range of 0–10 mg l⁻¹. These data are the first report of a SPR biosensor capable of detecting metal ions.     

Expression,Purification, and Comparative Inhibition of Helicobacter pylori Urease by Regio-Selectively Alkylated Benzimidazole 2-Thione Derivatives

The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC₅₀ values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC₅₀ values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of −9.74 and −13.82 kcal mol⁻¹; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds’ safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.     

Characterization of urease derived from Citrullus lanatus (watermelon) seeds to estimate total Kjeldahl nitrogen in human urine

A urease extract prepared by decanting liquid from a suspension of finely ground Citrullus lanatus (watermelon) seeds was characterized and applied to dilute urine samples to demonstrate a low-cost field method to estimate total Kjeldahl nitrogen (TKN) concentrations in human urine. The extract exhibited a Michaelis-Menten constant, K_m, of 3.00 mM urea and a specific activity of up to 12.2 U/mg protein at an optimum pH of 8.1. A statistical F-test on 54 samples demonstrated that TKN can be estimated as the total ammonium-nitrogen recovered upon addition of urease in dilute fresh and stale urine samples. The total ammonium-nitrogen in urine samples determined after treatment with watermelon seed urease was consistent with that determined using traditional acid digestion techniques. The extract retained 85% of its initial capacity after three months of refrigeration. The effectiveness of this method to assay nitrogen in unbuffered urine samples will be useful in nitrogen analyses in nutrient recovery and urine or slurry storage contexts. Accordingly, this study is useful in understanding the kinetics of a plant-derived urease acting in dilute urine.     

Resonance Rayleigh scattering study on the interaction of gold nanoparticles with berberine hydrochloride and its analytical application

A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC₅₀ (concentration of toxicant giving 50% inhibition) of Hg²⁺, Ag²⁺, Pb², Zn²⁺ is 0.39, 0.40, 2.16, 2.11 mg l⁻¹, respectively. For Cu²⁺ and Cd²⁺ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l⁻¹, respectively. The IC₅₀ and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox™, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.     

Enzyme analysis by means of the air-gap electrode-determination of urease and arginase by monitoring of the initial reaction rate

The air-gap electrode has been used in kinetic chemical analysis, illustrated by the assay of the activities of the enzymes urease and arginase. The determinations are based on monitoring the initial reaction rates of the selective release of ammoniacal nitrogen, which in the arginine—arginase system was ensured by adding excess of urease. The reaction rates measured are in the range 2.5 · 10^-5—1.5 · 10^-3M min^-1; the relative standard deviation is ca. 2.8 %.